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Dandelion prevents liver fibrosis, inflammatory response, and oxidative stress in rats


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Background Liver fibrosis is the main contributor to the chronic liver-associated morbidity and mortality. Purpose The study was conducted to evaluate the effects of whole plant powder of dandelion ( Taraxacum officinale ) on liver fibrosis. Methods Liver fibrosis was induced by the oral administration of 20% carbon tetrachloride (CCL4), twice a week for 8 weeks. Simultaneously, dandelion root extract (500 mg/kg) was daily administered via the same route. Results Dandelion remarkably improved the liver histology as evidenced by histopathological scoring with hematoxylin-eosin staining. Masson staining and hydroxyproline content similarly showed that dandelion decreased collagen deposition. Both mRNA and protein levels of α-smooth muscle actin and collagens 1 and 3 have been decreased after dandelion treatment compared to CCL4 group. Dandelion also downregulated the mRNA expressions of inflammatory factors interleukin-IL-1β, tumor necrosis factor-α, remodeling growth factor-β1, cyclooxygenase-2, and nuclear factor kappa-B and decreased the myeloperoxidase activity. Additionally, the effects of dandelion were associated with the decreased levels of the hepatic oxidative stress markers (malondialdehyde and P. carbonyl) and elevation of the activity of superoxide dismutase activity. Dandelion’s effect to alleviate the fibrosis and inflammation induced by CCL4 treatment in the livers and was more pronounced than with silymarin. The total antioxidant study of dandelion extract revealed that dandelion has notable ferric reducing antioxidant power and high total phenolic content. Conclusion Finally, these results suggest that dandelion prevents the progression of hepatic fibrosis induced by CCL4. The dandelion’s antifibrotic effects could be attributed to its ability to scavenge free radicals and to attenuate inflammatory cells activations.
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R E S E A R C H Open Access
Dandelion prevents liver fibrosis,
inflammatory response, and oxidative stress
in rats
Alaaeldin Ahmed Hamza
, Mona Gamel Mohamed
, Fawzy Mohamed Lashin
and Amr Amin
Background: Liver fibrosis is the main contributor to the chronic liver-associated morbidity and mortality.
Purpose: The study was conducted to evaluate the effects of whole plant powder of dandelion (Taraxacum
officinale) on liver fibrosis.
Methods: Liver fibrosis was induced by the oral administration of 20% carbon tetrachloride (CCL4), twice a week
for 8 weeks. Simultaneously, dandelion root extract (500 mg/kg) was daily administered via the same route.
Results: Dandelion remarkably improved the liver histology as evidenced by histopathological scoring with
hematoxylin-eosin staining. Masson staining and hydroxyproline content similarly showed that dandelion decreased
collagen deposition. Both mRNA and protein levels of α-smooth muscle actin and collagens 1 and 3 have been
decreased after dandelion treatment compared to CCL4 group. Dandelion also downregulated the mRNA
expressions of inflammatory factors interleukin-IL-1β, tumor necrosis factor-α, remodeling growth factor-β1,
cyclooxygenase-2, and nuclear factor kappa-B and decreased the myeloperoxidase activity. Additionally, the effects
of dandelion were associated with the decreased levels of the hepatic oxidative stress markers (malondialdehyde
and P. carbonyl) and elevation of the activity of superoxide dismutase activity. Dandelions effect to alleviate the
fibrosis and inflammation induced by CCL4 treatment in the livers and was more pronounced than with silymarin.
The total antioxidant study of dandelion extract revealed that dandelion has notable ferric reducing antioxidant
power and high total phenolic content.
Conclusion: Finally, these results suggest that dandelion prevents the progression of hepatic fibrosis induced by
CCL4. The dandelions antifibrotic effects could be attributed to its ability to scavenge free radicals and to attenuate
inflammatory cells activations.
Keywords: Dandelion, Hepatic fibrosis, Protection, Oxidative stress, Hepatic stellate cell
Liver fibrosis occurs as a compensatory response to the
process of tissue repair in a wide range of chronic liver in-
jures and inflammations (Cordero-Espinoza & Huch,
2018). Chronic liver injuries and inflammations have been
implicated in the pathogenesis of a number of liver dis-
eases including chronic viral and metabolic disorders.
Dead or dying epithelial cells as well as phagocytes release
inflammatory mediators that initiate inflammatory reac-
tion (Higashi, Friedman, & Hoshida, 2017). Among these
mediators are transforming the growth factor beta 1
(TGFβ1), tumor necrosis factor (TNF α), interleukin IL-
1β, reactive oxygen species (ROS), and cyclooxygenase-2
(COX2) (Higashi et al., 2017; Park, Cha, Youn, Cho, &
Song, 2010; Wahid et al., 2018). The major driver of liver
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* Correspondence:;
Hormone Evaluation Department, National Organization for Drug Control
and Research (NODCAR), 6 Abu Hazem St., Pyramids, Giza, Egypt
Biology Department, College of Science, United Arab Emirates University,
Al-Ain, UAE
Full list of author information is available at the end of the article
The Journal of Basi
and Applied Zoolog
Hamza et al. The Journal of Basic and Applied Zoology (2020) 81:43
fibrogenesis is an activation of hepatic stellate cells (HSCs)
which is the major cellular source of matrix protein-
secreting myofibroblasts (Higashi et al., 2017). Cellular
changes accompanying HSC activation include morpho-
logical changes such as the appearance of the cytoskeletal
protein α-smooth muscle actin (α-SMA) and a dramatic
increase in types I and III collagens (Friedman, 2008). This
excess deposition of ECM disrupts the normal architec-
ture of the liver that gradually degenerates the normal cel-
lular function of the organ and causes liver failure with
significant morbidity and mortality (Friedman, 2008).
Studies have demonstrated that liver fibrosis may be
prevented and even reversed by bioactive food compo-
nents and natural products including silymarin (Bae, Park,
&Lee,2018). Natural product-based drugs recently have
attracted extensive attention in the prevention and treat-
ment of liver disease (Amin et al., 2016;Amin&
Mahmoud-Ghoneim, 2009; Amin & Mahmoud-Ghoneim,
2011; Ashktorab et al., 2019;Hamza,2010; Hamza et al.,
2018;Lietal.,2018). Main reasons for the use of herbal
drugs include their lower cost compared with conven-
tional drugs, minor drug reactions hence reduced side ef-
fects and high safety (Bae et al., 2018). Among myriad of
herbal drugs, silymarin, which is being explored for a wide
variety of disorders such as oxidative stress, inflammatory
disorders, and liver disorders (Ali et al., 2018), the usual
therapeutic dose 200 mg/kg of silymarin administered to
CCL4-induced model of liver fibrosis can inhibit the fibro-
genic mechanism and the progression of initial liver fibro-
sis (Clichici et al., 2015; Neha, Jaggi, & Singh, 2016)
Dandelion (Taraxacum officinale) is a member of the
Asteraceae (Compositae) family and is a common peren-
nial weed distributed in the warmer temperature zones
of the Northern Hemisphere (Schutz, Carle, & Schieber,
2006). As dandelion is an edible plant, it has been used
as traditional herbal medicine in the Arabian zones for
the treatment of liver and spleen ailments (Schutz et al.,
2006). It has been used as folk medicines in China, India,
Russia, Pakistan, and Italy for the treatment of chronic
liver diseases (Devaraj, 2016; Martinez et al., 2015). Sev-
eral health-promoting properties, including diuretic,
anti-inflammatory, anti-carcinogenic, and anti-oxidative
activities, have been attributed to the use different parts
of dandelion (Devaraj, 2016; Martinez et al., 2015). Dan-
delion contains a wide array of phytochemicals whose
biological activities are explored in various human health
care areas (Devaraj, 2016; Martinez et al., 2015). These
include sesquiterpene lactones, terpenoids, polysaccha-
rides, and phenolic compounds (Gonzalez-Castejon, Vis-
ioli, & Rodriguez-Casado, 2012; Schutz et al., 2006;
Williams, Goldstone, & Greenham, 1996). Recently, dan-
delion has garnered attention for its antioxidant and its
anti-inflammatory effects and its possible beneficial ef-
fects against the development of obesity, cancer, and
numerous cardiovascular risk factors (Jeon, Kim, & Kim,
2017; Ovadje, Ammar, Guerrero, Arnason, & Pandey,
2016; Rehman et al., 2017). Dandelion continues to be
commercialized as herbal formulation mainly for its po-
tential to prevent or ameliorate the outcome of several
chronic liver disorder such liver fibrosis. Yet, it received
very limited research attention. Few early studies re-
ported dandelions anti-inflammatory, anti-oxidative, and
the hepatoprotective effects of acute liver damage in-
duced in animals by different chemicals such as by gal-
actosamine (Park, Kim, Purck, Noh, & Song, 2007),
carbon tetrachloride (Clichici et al., 2015; Park et al.,
2010) and acetaminophen (Colle et al., 2012), and
chronic CCL4 liver damage (Al-Malk, Abo-Golayel,
Abo-Elnaga, & Al-Beshri, 2013). Administration of dan-
delion root water-ethanoic extract for 10 days amelio-
rated the CCL4-induced hepatic fibrosis in mice
(Domitrovic, Jakovac, Romic, Rahelic, & Tadic, 2010).
This study suggested that administration of dandelion
root extract promotes the complete regression of fibrosis
and the enchantment of hepatic regenerative capabilities.
This investigation was designed to evaluate the protect-
ive effects of dandelion root extract on hepatic fibrosis
in male rats induced by CCL4 and its relationship with
oxidative stress, inflammation, and HSC activation.
Materials and methods
The herbal preparation of dandelion capsule was manu-
factured by Herbal Factors Company, Ltd. (SKU 4501,
LOT778496, 1550 United Boulevard, Coqutlam, BC,
Canada V3: 6Y2); each capsule contains 800 mg of dan-
delion extracted roots of Taraxacum officinale. Chlora-
min T, types I and III collagens, N-methyl-2-
phenylindol, Folin-Ciocalteu reagent, pyrogallol, super-
oxide dismutase, catalase, 2, 4-dinitophenylhydrazine, o-
dianisidine, p-dimethyl-amino-benzaldehyde, and bovine
albumin were obtained from Sigma Chemical Co. (St.
Louis, MO). Rabbit monoclonal anti-α-smooth muscle
actin (SMA) (ab32575) antibody, rabbit polyclonal anti-
collagen 1 (ab21287), and 3 antibody (ab7778) were pur-
chased from Abcam, and all other chemicals were ob-
tained from local commercial suppliers.
Adult male albino rats (150200 g) of the Wistar strain
were obtained from the Animal House, National
Organization for Drug Control and Research (NODCAR,
Cairo, Egypt). They were maintained on standard pellet
diet and tap water ad libitum and were kept in polycar-
bonate cages with wood chip bedding under 12 h light/
dark cycle and room temperature 2224 °C. Rats were
acclimatized to the environment for 1 week prior to ex-
perimental use.
Hamza et al. The Journal of Basic and Applied Zoology (2020) 81:43 Page 2 of 13
Induction of liver fibrosis
Fibrosis was induced by an oral administration of 20%
CCL4/corn oil, 1 ml/kg body weight, twice a week for 8
weeks to produce slowly reversible cirrhosis, as de-
scribed by Varga, Brenner, and Phan (Varga, Brenner, &
Phan, 2005) and Hamza (Hamza, 2010).
Determination of total phenolic content of dandelion
Total phenolic content was determined by the method
of Singleton (Singleton, Orthofer, & Lamuela-Raventos,
1999) using the Folin-Ciocalteu reagent. Results were
expressed in milligrams of gallic acid equivalent per
grams dry weight of crude plant material.
Determination of total antioxidant capacity of dandelion
The total antioxidant capacity (TAC) in crude extract
was evaluated using ferric reducing antioxidant power
(FRAP) assay (Benzie & Strain, 1996). The FRAP assay
measures the change in absorbance at 593 nm due to the
formation of a blue colored ferrous-tripyridyltriazine
complex from colorless oxidized ferric form by the ac-
tion of electron donating antioxidants. Ascorbic acid was
used as a standard for the calibration curve.
Treatment regime
Twenty-four rats were randomly divided into four
groups (six rats each) and were subjected to the follow-
ing treatments; the first group (fibrotic group) has re-
ceived an oral administration of 20% CCL4/corn oil, 1
ml/kg body weight, twice a week for 8 weeks to produce
a slow reversible cirrhosis; the second group (control
group) has received corn oil (1 ml/kg body weight); the
third group (protected groups) and the fourth group
(standard group) have received CCL4. Following the ad-
ministration of CCL4, dandelion 500 mg/kg body weight
and silymarin 200 mg/kg body weight were adminis-
trated orally and continued daily for 8 weeks. Powder
from dandelion root capsule was suspended in 10 ml of
distilled water before administration. Doses of dandelion
and silymarin were selected based on their previously re-
ported hepatoprotective properties (Park et al., 2010).
The time intervals between the administration of CCL4
and each of Dandelion and silymarin were 5 h to avoid
the disturbance of the absorption of each agent. The
normal control group was treated daily with an equiva-
lent volume of water for 8 weeks. After 8th week, all rats
were anesthetized with 3% sodium pentobarbital (45 mg/
kg, i.p.), and blood samples were collected from the
retro-orbital plexus. After the blood was drown from
rats, the animals were euthanized by cervical dislocation
under 3% sodium pentobarbital anesthesia, and the liver
was quickly taken out and weighted after washed with
cold normal saline.
Sample preparation
The livers from all animals were collected and weighed;
then, harvested liver tissues were fixed in 10% buffered
formalin, for histopathological examination. Other liver
tissues were removed and rinsed with ice-cold isotonic
saline, quickly frozen in liquid nitrogen, and stored at
80 °C for analysis of fibrotic, oxidative stress, and inflam-
matory markers. The serum was collected by centrifu-
ging the blood samples in a refrigerated centrifuge (4 °C)
at 3000 rpm for 20 min (A. A. (Hamza, 2010)) and stored
at 4 °C. For biochemical determination, frozen liver sam-
ples were homogenized in ice-cold Tris-HCL buffer
(150 mM, pH 7.4). The wt/vol ratio of the tissue to the
homogenization buffer was 1:10 wt/vol.
Biochemical assays and histopathology
The level of malondialdehyde (MDA), as marker of lipid
peroxidation, was measured according to the method of
Gerard-Monnier (Gérard-Monnier et al., 1998), where
MDA reacts with N-methyl-2-phenylindol and forms a
blue complex with absorption maximum at 586 nm.
Two hundred microliter of liver sample was added to
650 μl of a solution containing 10 mM N-methyl-2-phe-
nylindol in a mixture of acetonitrile/methanol (3:1),
followed by adding 150 μl of 37% HCl. After 1 h of incu-
bation at 45 °C, the absorbance was measured at 586
nm. The MDA concentration was determined against
MDA standard curve. The results were expressed as
nanomole of MDA per milligram of protein.
The level of liver superoxide dismutase (SOD) enzyme
was assayed according to the method described by
Nandi and Chatterjee (Nandi & Chatterjee, 1988). It is
based on the ability of SOD to inhibit the auto-oxidation
of pyrogallol at alkaline pH. Two 2 ml of reaction mix-
ture contained 1 mM diethylene triaminepenta acetic
acid, 40 μl catalase, 50 mM Tris-cacodylate buffer, pH
8.5 mixed with 15 μl of liver homogenate. The reaction
was started by the addition of 200 μl of freshly prepared
2.6 nM of pyrogallol solution in 10 mM HCl. Change in
absorbance was recorded at 420 nm for 5 min at 1-min
interval. One unit of SOD has been described to cause
50% inhibition of auto-oxidation pyrogallol present in
the assay mixture.
Hepatic protein carbonyl (P. carbonyl) contents were
determined based on the method of Reznick and Packer
(Reznick & Packer, 1994). This method is based on spec-
trophotometric detection of the reaction of 2, 4-
dinitophenylhydrazine with P. carbonyl to form protein
hydrazones at 370 nm. The results were expressed as
nanomole of carbonyl group per milligram of protein
with molar extinction coefficient of 22,000 M/cm. Mye-
loperoxidase (MPO) activity in hepatic homogenate was
assayed based on the method of Hillegass (Hillegass,
Griswold, Brickson, & Albrightson-Winslow, 1990). 2.95
Hamza et al. The Journal of Basic and Applied Zoology (2020) 81:43 Page 3 of 13
ml of substrate contained 50 mM potassium phosphate
buffer containing 0.53 mM o-dianisidine and 20 mM
mixed with 50 μl of liver homogenate. Change in
absorbance was recorded at 460 nm for 5 min at 1-min
interval. One unit of MPO was defined as amount of
MPO present that degrades 1 μM peroxide per a minute
at 25 °C.
Liver collagen concentrations were performed by
measuring hydroxyproline (HP) content in liver samples
using a modification of the method of Edwards and
Brien (Edwards & Brien, 1980). Briefly, 0.5 ml of 20%
liver homogenate was digested in 1 ml of 6 N HCl at 110
°C for 18 h then was dried at 60 °C under vacuum. The
sediment was dissolved in 400 μl of acetate buffer pH
6.5; then, 0.8 ml of 1.41 % chloramin T reagent dissolved
in acetate buffer pH 6.5 was added. After incubation for
25 min at room temperature, 0.8 ml of mixture contain-
ing 15 g p-dimethyl-amino-benzaldehyde and 30% per-
chloric acid in 60 ml n-propanol was added, and mixture
was incubated at 60 °C for 25 min. After cooling, the ab-
sorbance of samples and standards was measured at 550
nm. The results were expressed as microgram of HP per
milligram of protein. The total protein content of liver
was performed according to the Lowry method as modi-
fied by Peterson (Peterson, 1977). Aspartate aminotrans-
ferase (AST) and alanine aminotransferase (ALT)
activities as well as albumin and total protein concentra-
tions were estimated in serum using Randox reagent kits
(Randox Laboratories Ltd., Co. Antrim, UK) and follow-
ing their instruction manual. In all the estimations, ab-
sorbance was recorded using a PerkinElmer, Lambda 25
UV/VIS spectrophotometer.
Histopathological evaluation
Pieces of the livers were fixed in 10% neutral phosphate-
buffered formalin and embedded in paraffin before cut-
ting into 5-μm sections. The hydrated tissue sections
were stained with hematoxylin and eosin (H & E) and
Masson-Trichrome, for the different histological exami-
nations. The sections were examined under an Olympus
DX41-light microscope (Honduras St., London, UK). In
hematoxylin and eosin stain, the presence of necrosis,
inflammation, and necrosis was evaluated and graded as
follows: grade 0, absent; grade 1, present in one third of
the lobules; grade 2, present in two thirds of the lobules;
and grade 3, present in all of the lobules. In Masson
stain of collagen, fibrosis was graded according to the
method of Gui, Wei, Wang, Wu, Sun, and Chen (Gui
et al., 2006): grade 0, normal (no visible fibrosis); grade
1, fibrosis present (collagen fiber present as small septa
in portal area, central area, or peripheral area); grade2,
mild fibrosis (collagen fiber extended as septa from por-
tal tract to central vein forming incomplete septa); grade
3, moderate fibrosis (collagen fibers formed thin
complete septa); and grade 4, severe fibrosis (collagen fi-
bers formed thick septa and pseudo lobe formation).
Immunohistochemical evaluation
Liver tissue sections were mounted onto slides, dewaxed
in xylene, and hydrated and subjected to heat-induced
antigen retrieval according to standard protocols. The
levels of α-smooth muscle actin (α-SMA) and types I
and III collagens were determined by immunohisto-
chemically methods according to the protocols described
by Varga et al. (Varga et al., 2005). The numbers of α-
SMA and types I and III collagen positive cells were
counted in five randomly selected high-power fields (×
400) per liver section for six animals of each group.
Real-time quantitative reverse-transcriptase polymerase
chain reaction (RT-PCR) analysis
Total RNA was isolated from liver frozen tissue by a
RNeasy Mini Kit (QIAGEN, Valencia, CA) and assessed
with a dual spectrophotometer Gene JET Kit (Thermo
Fisher Scientific Inc., Germany, #K0732). RT-PCR was
used for quantitative analysis of gene expression of
TNF-α, NF-kB, IL-1β, COX2 TGF-β,α-SMA, Colla1,
and Colla 3. The PCR reaction was carried out in 48-
well plate Step One real-time PCR systems (Applied Bio-
systems, Foster city, USA), and results were analyzed
using the Applied Biosystems software version 2. Twenty
nanograms of purified RNA from each sample was ap-
plied for reverse transcription with subsequent quantita-
tive PCR amplification with Bioline, amedian lifescience
Company, UK (SensiFASTTMSYBR®Hi-ROX) One-step
Kit (Catalog number PI-50217V). Thermal profile was as
follows: 45 °C for 20 min in one cycle (for cDNA synthe-
sis) followed by 10 min at 95 °C for reverse transcriptase
enzyme inactivation. Forty cycles of PCR amplification
were further carried out as follows: 10 s at 95 °C, 30 s at
58 °C, and 1 min at 72 °C. Changes in the expression of
each target gene were normalized relative to the mean
cycle threshold (CT) values of the housekeeping gene
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) by
ΔCt method. The sequence of primers for all studied
genes was shown in Table 1.
Statistical analysis
Data were expressed as mean ± SEM. Comparisons among
multiply groups were performed by one-way analysis of
variance (ANOVA) followed by Dunnettsttest post-hoc
analysis test for multiple comparisons with P<0.05being
considered as statistically significant. In a nonparametric
analysis, differences between the groups were performed by
the Kruskal-Wallis Htest, and the significant of the differ-
ences between the groups was determined by Mann-
Whitney Utest. All statistical analysis was performed by
SPSS (version 20) statistical program (SPSS Inc., Chicago,
Hamza et al. The Journal of Basic and Applied Zoology (2020) 81:43 Page 4 of 13
IL, USA). Figures were done using GraphPad Prism pro-
gram (version 5) (San Diego, California, USA).
Dandelions overall antioxidant and phenolic contents
The antioxidant and phenolic overall contents are pre-
sented in Fig. 1. The FRAP assay is a direct test of total
antioxidant capacity (TAC). In the present study, dande-
lion had high FRAP value. TAC of dandelion was con-
centration dependent where a concentration of 1000 mg
of dry herb contained the highest TAC which equals to
21.45 μmol ascorbic acid equivalent. Dandelion was
found to contain high total polyphenolic content and
equals to 6.88 mg gallic acid equivalent per 1000 mg of
dry dandelion.
Dandelion improved liver function in CCL4-treated rats
Figure 2shows the effects of dandelion on liver func-
tions biomarkers of in rats treated with CCL4. In rats
treated with CCL4, activities of AST and ALT were
significantly increased in serum whereas serum levels
of albumin and total proteins were significantly de-
creased compared to normal control group. In con-
trast, both serum AST and ALT activities were
significantly decreased while serum levels of albumin
and protein were significantly increased in the dande-
lion treatment group. These effects were comparable
to that of the well-known hepatoprotective agent sily-
marin. The hepatoprotective effects of dandelion were
superior to that of silymarin.
Dandelion attenuated liver damage and fibrosis in CCL4-
treated rats
To further evaluate the effect of dandelion on liver
damage and fibrosis, H & E and Massons trichromic
stained sections were performed (Fig. 3). CCL4 treat-
ment induced severe liver damage, which included
marked fatty degeneration, necrosis of hepatocytes,
and massive intrusion of inflammatory cells to the
liver of CCL4-treated group (Fig. 3a, b). Normal
lobular architecture was observed in the control
group. However, concomitant treatment of dandelion
significantly reduced histological scores of fatty
Table 1 The gene-specific primers used for RT-PCR
Primer sequence 5-3Gene bank accession
Fig. 1 Total phenolic and total antioxidant contents of dandelion
Hamza et al. The Journal of Basic and Applied Zoology (2020) 81:43 Page 5 of 13
degeneration, necrosis, and inflammation in compari-
son with CCL4-treated group. Silymarin treatment
markedly lessened the degrees of liver necrosis and
inflammatory cell intrusions, but the efficacy of dan-
delion in restoring normal architecture of the liver is
more evident. Level of hepatic fibrosis was examined
with Masson stain and HP content as shown in Fig.
3ce. First, liver sections were stained by Masson
stain, which stains collagen fibers blue. After treat-
ment with CCL4, obvious increase in fibrosis was
noted in CCL4-treated group compared with control
group. Treatment with dandelion and silymarin barely
showed signs of liver fibrosis (Fig. 3d). Interestingly,
the level of liver fibrosis and size of fibrous septa
were obviously less in dandelion- and silymarin-
treated groups. Finally, analysis of hepatic HP content,
the major component of collagen protein, was carried
out as a liver fibrosis biomarker (Fig. 3e). In agree-
ment with the results of Masson stain, HP content
was significantly higher in the liver of CCL4-treated
group and further alleviated by the treatment with ei-
ther dandelion or silymarin.
Effect of dandelion on hepatic oxidative stress markers in
CCL4-treated rats
To investigate the effect of dandelion and silymarin on
livers oxidative stress caused by CCL4, hepatic MDA
and P. carbonyl contents as well as SOD activity were
detected. Figure 4shows the effects of dandelion and
silymarin on CCL4-induced oxidative stress damage in
rats. CCL4 treatment caused a significant increase in
hepatic content of MDA and P. carbonyl and a signifi-
cant decrease in hepatic SOD activity compared with
control group. Administration of each of dandelion and
silymarin, normalized hepatic contents of MDA and P.
carbonyl with partly prevented the decrease of SODs ac-
tivity. Compared with dandelion group, there was a sig-
nificant improvement in oxidative stress in silymarin-
Fig. 2 Effect of dandelion and silymarin on serum markers of liver function in CCL4-treated (model). Data are expressed as mean ± SEM for six
animals in each group. Units of ALT and AST are IU/L; units of albumin and T. protein are g/dl. aP< 0.05 vs. control group. bP< 0.05 vs.
model group
Hamza et al. The Journal of Basic and Applied Zoology (2020) 81:43 Page 6 of 13
Fig. 3 Histological results of liver tissues of rats stained with H & E and Massons trichromic. (a&b)The livers of rats treated with CCL4 showing
degenerated and necrotic liver cells associated with inflammation and fatty change. Treatment with dandelion and silymarin reduced these
pathological changes. All pictures are × 100 magnifications. (c&d) In Masson sections, collagens are stained blue. Liver sections of rats treated
with CCL4 (model) and CCL4+ silymarin-treated rats showing massive fibrosis with sizable fibrous septa. The group treated with dandelion and
silymarin showing less formation and accumulation of collagen fibrous. Magnification × 100. eEffect of dandelion and silymarin on hepatic HP
content in CCL4-treated group (model). Each column represents the mean ± SEM for 5 rats per group. Units of HP content are μg/mg protein. a
P< 0.05 vs. control; bP< 0. 05 vs. CCL4 group; cP< 0. 05 vs. dandelion group
Hamza et al. The Journal of Basic and Applied Zoology (2020) 81:43 Page 7 of 13
treated group, but dandelion revealed the best efficacy in
normalization of hepatic SOD activity and P. carbonyl
Effects of dandelion on expression of α-SMA and collagen
types 1 and 3 in CCL4-treated rats
We confirmed stellate cell activation during fibrosis in
liver sections by identifying the expression of the α-SMA
which is a good marker of stellate cell activation during
fibrosis. The expression of α-SMA marker in immuno-
staining sections was evident mostly in the blood vessel
wall of the normal group. As expected, the number of α-
SMA positive cells was significantly increased in fibrous
septa and areas of inflammations in CCL4-treated group
(Fig. 5a). This effect was confirmed by mRNA expression
levels of α-SMA (Fig. 5c) which showed their upregula-
tion of the livers of fibrotic group. Treatment with either
dandelion or silymarin has significantly attenuated the
number of α-SMA positive cells compared to the fibrotic
group. Similarly, dandelion and silymarin significantly
decreased the mRNA expression levels of α-SMA in rats
compared to those of the CCL4-treated group (Fig. 5c).
Dandelion revealed the best effects in mRNA expression
of this marker compared to silymarin-treated group.
Collagen types I and III were immunohistochemically la-
beled and were stained brown. Collagen was weakly de-
posited around centrolobular veins of the normal group.
CCL4 group displayed the depositions of collagen types
I and III, forming fibrous septa surrounding the lobules
(Fig. 5a, b). Treatment with dandelion and silymarin at-
tenuated collagen accumulation and the number of col-
lagen types I and III positive cells in CCL4-treated rats.
In addition, excessive deposition of ECM in the fibrotic
liver was confirmed by real-time PCR (Fig. 5c), and the
amount of collagen 1 and collagen 3 mRNA was signifi-
cantly higher in CCL4-treated rats than in control rats;
however, dandelion treatment markedly alleviated the ef-
fect of CCL4 and reduced the expression of collagen 1
and collagen 3 mRNA (Fig. 5c). Treatment with dande-
lion showing the best effects in those markers of liver fi-
brosis was compared to silymarin-treated group.
Effects of dandelion on expression of inflammatory
markers in CCL4-treated rats
The hepatic MPO activity and mRNA levels of inflam-
matory markers, including IL-1β, TNF-α, TGF β, NF-kB,
and COX-2, in rat livers are shown in Fig. 6. MPO activ-
ity of the liver was adopted as a marker of oxidative
stress and inflammation and tissue neutrophil accumula-
tion and activation. Hepatic MPO activity was signifi-
cantly elevated in CCL4-intoxicated group in
comparison with control group. Dandelion and silymarin
treatment significantly decreased hepatic MPO activity
compared to CCL4-treated group (Fig. 6). CCL4 signifi-
cantly upregulated the mRNA levels of IL-1β, TNF-α,
TGF β, NF-kB, and COX-2 in the livers of rats com-
pared to control group. Dandelion and silymarin signifi-
cantly decreased the mRNA expression level of mRNA
of IL-1β, TNF-α, TGF β, NF-kB, and COX-2 in the livers
of rats compared to CCL4-treated group (Fig. 6). Dande-
lion revealed the best effects in inhibition of inflamma-
tory response of livers compared to silymarin-treated
The current study showed that dandelion treatment re-
duced liver injury, improved liver function, and de-
creased ECM deposition. These protecting consequences
can be attributed, at least in part, to the reduction of
HSC activation, the inhibition of inflammatory signaling
pathway, and the suppression of oxidative stress-induced
damage. Present results showed that repeated doses of
Fig. 4 Effect of dandelion and silymarin on hepatic markers of oxidative stress in CCL4 treated groups (model). Data are presented as mean ±
SEM for 6 animals per group. MDA and P. carbonyl units are nmol/mg protein; SOD enzyme unit is IU/mg protein. aP< 0.05 vs. control group. b
P< 0.05 vs. model group
Hamza et al. The Journal of Basic and Applied Zoology (2020) 81:43 Page 8 of 13
Fig. 5 (See legend on next page.)
Hamza et al. The Journal of Basic and Applied Zoology (2020) 81:43 Page 9 of 13
CCL4 caused a significant elevation in serum AST and
ALT activities and a significant depletion of in serum al-
bumin and total protein levels, indicating the injury of
liver cells and the decrease in liver synthetic function.
However, both dandelion and silymarin treatment attenu-
ated serum AST and ALT activities indicating hepatopro-
tective effects against hepatocellular injury. In additions,
dandelion treatment improved liver synthetic function
confirmed by normalized serum albumin and total protein
levels. Additionally, these hepatoprotective properties of
dandelion were associated with improvements of liver
histopathological changes. The present results are coin-
cided with previous studies that showed the hepatoprotec-
tive effects of dandelion against hepatotoxicity induced by
several chemicals (Colle et al., 2012; Park et al., 2010). Fur-
thermore, hepatotoxicity of CCL4 model in the present
work was accompanied with inflammation and fibrosis.
Fibrogenesis is known to be associated with the necrosis
and inflammation of the liver (Fortea et al., 2018;Wahid
et al., 2018). CCL4 is considered one of the most used
hepatotoxins in the experimental study of liver diseases.
Hepatic responses in rats to chronic CCL4 administration
are shown to be superficially like human cirrhosis (Varga
et al., 2005). In the liver, CCL4 generates methyl trichlor-
ide radicals (CCl3·), which lead to pronounced centrilobu-
lar liver necrosis, induction of the inflammatory response,
activation of HSCs, and increasing of extracellular matrix
production (Ni et al., 2018; Wahid et al., 2018).
(See figure on previous page.)
Fig. 5 The expression of α-SMA collagens I and III positive cells of liver tissues of rats was determined by immunohistochemical satin (aand b)
and by mRNA expression in the liver by quantitative RT-PCR (c). Representative histological pictures (a) and the semi-quantitative analysis (b)
showing the expression of α-SMA and collagens I and III positive cells in different experimental groups. All pictures are × 100 magnifications. In
control section, immunohistochemical staining is only revealed in vascular structures. In CCL4 sections, clear staining for α-SMA and collagens I
and III is manifested along with the fibrous septa. The immunohistochemical expression of cells in each section was calculated by counting
numbers of brown staining, α-SMA positive cells, in five fields per section at × 400 magnification. bColumns represent the means ± SEM for 6
animals per group. aP< 0.05 vs. control group; bP< 0.05 vs. CCL4 group. Analysis of α-SMA and types 1 and 3 collagen mRNA expression in
the liver by quantitative RT-PCR (n= 3 per group). Data were normalized to GAPDH mRNA. Data are presented as means ± SEM. aP< 0.05 vs.
control group; bP< 0.05 vs. CCL4 group; cP< 0.05 vs. dandelion group
Fig. 6 Effects of dandelion and silymarin on inflammatory markers. aMPO activity (n= 6 per group) and mRNA expression of bIL-1β,cTNF-α,d
TGF β,eNF-kB, and fCOX-2 in liver tissues (n= 3 per group). Results of mRNA expression were normalized to GAPDH mRNA. Data are presented
as means ± SEM. aP< 0.05 vs. control group. bP< 0.05 vs. CCL4 group. cP< 0.05 vs. dandelion group
Hamza et al. The Journal of Basic and Applied Zoology (2020) 81:43 Page 10 of 13
Excess deposition of ECM is the characteristic futures
of liver fibrosis (Cordero-Espinoza & Huch, 2018). Our
results showed that CCL4 administration led to sever ac-
cumulation of collagen in the liver tissues as indicated
from the result of the Masson staining, collagens 1 and 2
staining, and the increased level of HP, a considerable
amino acid present in collagen. This was concomitant
with the increased hepatic mRNA expression of collagen
1 and collagen 3. Herein, the treatment with dandelion
has a notable preventive effect against CCL4-induced
liver fibrosis in rats. Besides, its improvement effect in
the histological findings of Masson stain and HP con-
tent, dandelion markedly decreased the expressions of
types 1 and 3 collagens. These findings point out that
dandelion could inhibit the synthesis and deposition of
collagen in liver tissue which may additionally be the
pharmacological basis of its hepatoprotective property.
Moreover, the results of this work confirmed that dan-
delion inhibited liver damage and fibrosis more effect-
ively than silymarin.
Activation of HSCs is a core cellular event responsible
for the production of collagen and the progression of
liver fibrosis (Ni et al., 2018; Shay & Hamilton, 2018). In
CCL4-treated group of the present work, accumulation
of hepatic fibers is associated with increased α-SMA de-
position indicates that activated HSCs are the primary
source for the fibrosis seen in the CCL4-treated rats.
This study showed that dandelion inhibited hepatic fi-
brosis and decreased protein and mRNA expression of
α-SMA; hence, dandelion might inhibit HSCs activation.
Previous studies indicated that the protective effect of
dandelion on liver fibrosis induced by CCL4 in mice was
accompanied with the inactivation of HSCs (Domitrovic
et al., 2010). Therefore, its antifibrotic effect may be due
to the attenuation of HSCs activation.
The possible mechanism underling the hepatotoxic
and fibrotic effect of CCL4 could be the generation of
ROS by its metabolized into the highly reactive tri-
chloromethyl radical (CCl3) (Fortea et al., 2018;Ni
et al., 2018). These radicals are assumed to initiate
oxidative damage hepatocellular membrane via lipid
peroxidation and protein oxidation and induced the
release of inflammatory mediators from activated in-
flammatory cells (Fortea et al., 2018;Shay&
Hamilton, 2018). Furthermore, oxidative stress plays a
vital role in the activation of HSCs during propaga-
tion of fibrogenesis (Higashi et al., 2017). The present
study showed that repeated administration of CCL4 re-
sulted in upregulation of oxidative stress markers MDA,
levels of P. carbonyl, and depletion of SOD activity in liver
dandelion administration, on the other hand, prevented
the increase in MDA and P. carbonyl levels and amelio-
rated the depletion in SOD activity in liver. These findings
suggest that the hepatoprotective antifibrotic effects of
dandelion could be attributed to its antioxidant activity
which coincides with the previous results (Dirleise (Colle
et al., 2012; Davaatseren et al., 2013)). The antioxidant ac-
tivity of dandelion was confirmed in this study by FRAP
assay. Thus, the antioxidant property of dandelion could
be attributed to its high phenolic contents that was con-
firmed in this study. These phenolic contents can act in
several ways, including direct free radical scavenging, che-
lating of metal ions, and regeneration of membrane-
bound antioxidants. This finding is consistent with previ-
ous findings that were demonstrated the polyphenolic
compounds and antioxidant activity of dandelion root
(Domitrovic et al., 2010) and leaf extracts (Davaatseren
et al., 2013;Parketal.,2010). Dandelion was shown to
prevent the toxicity of several other models via inhibiting
oxidative damage (Park et al., 2010;Schutzetal.,2006).
Several polyphenolic compounds were isolated from dan-
delion of fruits, leaves, flowers, and roots such as chicoric
chlorogenic, caffeic, p-coumaric, ferulic, vanillic, and pro-
tocatechuic acids (Gonzalez-Castejon et al., 2012;Schutz
et al., 2006).
Prolong hepatic damage triggers the progression of in-
flammatory responses and inflammatory cells infiltration
including neutrophils and lymphocytes (Higashi et al.,
2017). One of the important neutrophil specific enzymes
released after neutrophil infiltrations is MPO (Hillegass
et al., 1990). Our study showed increased hepatic MPO
activity demonstrating that tissue injury, oxidative stress,
and then fibrosis involve the contribution of neutrophil
infiltrations. In the current work, elevation of inflam-
matory cell infiltrations and expressions of different
inflammatory markers including IL-1β,TNF-α,TGF-
β1, COX-2, and NF-κB expression collectively with
excess deposition of ECM revealed that the propaga-
tion of inflammation during repeated administration
of CCL4 and chronic hepatic damage are strongly in-
volved in activation of HSCs and promotion of fibro-
sis. The anti-inflammatory effect of dandelion was
herein confirmed by the decrease in the MPO activity
in the liver and by the inhibiting of the expressions
of pro-inflammatory markers in liver tissues. We
propose that the anti-inflammatory effect of dandelion
could be involved in pharmacological mechanisms
that lead to antifibrotic effect in the liver. A previous
animal study has revealed the hepatoprotective effect
of two polysaccharides isolated from dandelion, as
shown by the alleviations of inflammatory responses
and the amelioration of oxidative stress. These poly-
saccharides (administered at 304.92 mg/kg body
weight, for 7 days) attenuated CCL4-induced hepatic
damage in albino rats through the attenuated of in-
flammatory markers including NF-kB, iNOS, COX-2,
TNF-a, and IL-1 (Gonzalez-Castejon et al., 2012;Park
et al., 2010).
Hamza et al. The Journal of Basic and Applied Zoology (2020) 81:43 Page 11 of 13
Given its hepatoprotective and antifibrotic properties,
silymarin is a commonly prescribed drug for patients
with liver diseases (Clichici et al., 2015). In the present
work, the hepatoprotective capacities of dandelion were
shown to be more potent than our positive control, sily-
marin in inhibition of liver fibrosis and inflammation.
This could attribute to the differences in polyphenolic
compound classes and contents. Dandelion contains a
wide array of phytochemicals including sesquiterpene
lactones, terpenoids, polysaccharides, and phenolic com-
pounds ((Gonzalez-Castejon et al., 2012; Martinez et al.,
2015; Williams et al., 1996).
Dandelion was effective in the prevention of CCL4-
induced liver fibrosis in rats. The primary mechanism of
this antifibrotic effect could be attributed to its antioxi-
dant and anti-inflammatory properties.
ALT: alanine aminotransferase; AST: Aspartate aminotransferase; CCL4: Carbon
tetrachloride; COX2: Cyclooxygenase-2; FRAP: Ferric reducing antioxidant
power; HP: Hydroxyproline; α-SMA: Smooth muscle α-actin; HSC: Hepatic
stellate cells; TGFβ1: Growth factor beta 1; ROS: Reactive oxygen species; TNF
α: Tumor necrosis factor; SOD: Superoxide dismutase;
MDA: Malondialdehyde; P. carbonyl: Protein carbonyl; NF-Kb: Nuclear factor
The authors would like to thank Hanan Mohamed Mehany and Mohamed
Mustafa, at animal house, NODCAR, Egypt, for their excellent technical help.
A.A.H., F.M.L., M.G., and A.A. designed the study. A.A.H., F.M.L., M.G., and A.A.
performed the experiments and did the statistical analysis. A.A.H., F.M.L., M.G.,
and A.A. assisted with methodology and contributed resources. A.A.H and
A.A. wrote the first draft of the manuscript, and all authors contributed to
the editing of the revised manuscript and approved the manuscript.
This work has been partially supported by grant Al Jalila Foundation fund #
21S098 for Amr Amin.
Availability of data and materials
The datasets supporting the conclusions of this article are available from the
corresponding author on reasonable request.
Ethics approval and consent to participate
The protocol was conducted in accordance with standard guide to the care
and use of experimental animals (Canadian Council of Animal Care 1993)
and according to the ethical standards approved by the Animal Ethics
Committee for animal care and use of NODCAR (NODCAR/II/45/19).
Consent for publication
Not applicable
Competing interests
The authors declared no potential conflicts of interest with respect to the
research, authorship, and/or publication of this article.
Author details
Hormone Evaluation Department, National Organization for Drug Control
and Research (NODCAR), 6 Abu Hazem St., Pyramids, Giza, Egypt.
Department, College of Science, United Arab Emirates University, Al-Ain, UAE.
The University of Chicago, Chicago, IL, USA.
Received: 25 March 2020 Accepted: 10 June 2020
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Hamza et al. The Journal of Basic and Applied Zoology (2020) 81:43 Page 13 of 13
... As an example, Chlorella algae, a blue-green microalgae, may restore the function of pancreatic insulinsecreting cells in diabetic rats treated with streptozotocin [8]. Also, dandelion flowers have antioxidant properties that prevent chronic inflammation and development of obesity, cancer, and numerous cardiovascular risk factors [9]. Saponins, a diverse class of natural compounds present in some plant species, have the ability to inhibit various cancers in vitro and in vivo [10]. ...
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The renal system is engaged in metabolic syndrome (MS) and metabolites of arachidonic acid (AA) participate in renal homeostasis and disruption of functionality. Hibiscus sabdariffa L (HSL) is used as a diuretic and could improve renal function. The aim of this study was to assess if treatment with HSL at 2% improves renal function in MS through the metabolites of AA. A total of 24 male Wistar rats were divided into four groups: Group 1, control (C); Group 2, MS with 30% sucrose in drinking water, Group 3, MS plus HSL infusion at 2% (MS+HSL); and Group 4, C+HSL. We evaluated the perfusion pressure changes (∆-PP), the activities of cyclooxygenases (COXs), the percentage of AA, the expressions of PLA2, COX2, COX1, 5-LOX, TAXS and CYP450, and the concentrations of prostaglandins in the kidney from rats with MS. There was a decrease in the ∆-PP, in the activities of COXs, and the expressions of COX2 and CYP450 (p ≤ 0.03, respectively)as well asPGE2, TxB2, and LKB4 (p ≤ 0.01, respectively). However, the percentage of AA and expressions of PLA2 and PGE1 (p = 0.01, respectively) were increased in C and MS+HSL. The HSL treatment improved the function and anatomical structure of the kidneys in the MS rats, through antioxidant molecules, and inhibited the pathways that metabolize the AA including that of PLA2, COX2, 5-LOX, TAXS, and CYP450 while favoring the COX1 pathway. This improves the vascular resistance of renal arterioles.
... A growing number of studies have demonstrated that natural drugs can be used to treat liver diseases, such as liver cancer, liver injury, and fatty liver disease, owing to their superior anti-inflammatory, anti-oxidant, regulation of fat metabolism, and anti-liver cancer properties. Specifically, aescin (El-Dakhly et al., 2020), hawthorn , and dandelion (Alaaeldin et al., 2020) significantly reduced CCl4-induced liver toxicity and fibrosis in rodents. Safranal (Ali et al., 2022) and Salvadora persica (Bayan et al., 2018) have significant anti-angiogenesis and anti-hepatocellular carcinoma effects. ...
Ethnopharmacological relevance: Nonalcoholic fatty liver disease (NAFLD) is a manifestation of metabolic syndrome in the liver and the leading cause of chronic liver disease worldwide. Digeda-4 decoction (DGD-4) is a commonly prescribed Mongolian herbal drug for treating acute and chronic liver injury and fatty liver. However, the mechanisms underlying the improvement of dislipidemia and liver injury via treatment with DGD-4 remain unclear. Disassembling a prescription is an effective approach to studying the effects and mechanisms underlying Mongolian medicine prescriptions. By disassembling a prescription, it is feasible to discover effective combinations of individual herbs to optimize a given prescription. Accordingly, we disassembled DGD-4 into two groups: the single Lomatogonium rotatum (L.) Fries ex Nym (LR) (DGD-1) and non-LR (DGD-3). Aim of this study: To study whether DGD-4 and its disassembled prescriptions have protective effects against tyloxapol (TY)-induced NAFLD and to explore the underlying mechanisms of action and compatibility of prescriptions. Material and methods: NAFLD mice were developed by TY induction. Biochemical horizontal analyses, enzyme-linked immunosorbent assay, and liver histological staining were performed to explore the protective effects of DGD-4 and its disassembled prescriptions DGD-3 and DGD-1. Furthermore, we performed immunohistochemical analyses and Western blotting to further explore the expression of target proteins. Results: DGD-4 and its disassembled prescriptions could inhibit TY-induced dislipidemia and liver injury. In addition, DGD-4 and its disassembled prescriptions increased the levels of p-AMPKα and p-ACC, but decreased the levels of SREBP1c, SCD-1, SREBP-2, and HMGCS1 proteins. The activation of lipid metabolic pathways SIRT1, PGC-1α, and PPARα improved lipid accumulation in the liver. Moreover, DGD-4 could inhibit hepatocyte apoptosis and treat TY-induced liver injury by upregulating the Bcl-2 expression, downregulating the expression of Bax, caspase-3, caspase-8, and the ratio of Bax/Bcl-2, and positively regulating the imbalance of oxidative stress (OxS) markers (such as superoxide dismutase [SOD], catalase [CAT], malondialdehyde [MDA], and myeloperoxidase [MPO]). DGD-1 was superior to DGD-3 in regulating lipid synthesis-related proteins such as SREBP1c, SCD-1, SREBP-2, and HMGCS1. DGD-3 significantly affected the expression of lipid metabolic proteins SIRT1, PGC-1α, PPARα, apoptotic proteins Bcl-2, Bax, caspase-3, caspase-8, and the regulation of Bax/Bcl-2 ratio. However, DGD-1 showed no regulatory effects on Bax and Bcl-2 proteins. Conclusion: This study demonstrates the protective effects of DGD-4 in the TY-induced NAFLD mice through a mechanism involving improvement of dyslipidemia and apoptosis by regulating the AMPK/SIRT1 pathway. Although the Monarch drug DGD-1 reduces lipid accumulation and DGD-3 inhibits apoptosis and protects the liver from injury, DGD-4 can be more effective overall as a therapy when compared to DGD-1 and DGD-3.
... Liver tissue was deposited into 4% paraformaldehyde, dehydrated, and then embedded in paraffin. Then, for different histological examinations, the hydrated tissue sections were either stained with hematoxylin and eosin (H&E) or Masson-Trichrome, as previously described [16,30]. ...
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This study aimed to explore the effects of the incremental injection of lipopolysaccharide (LPS) on liver histopathology, inflammation, oxidative status, and mitochondrial function in piglets. Forty healthy Duroc × Landrace × Yorkshire castrated boars (21 ± 2 days old, weight 6.84 ± 0.11 kg) were randomly assigned to five groups (n = 8) and then slaughtered on days 0 (group 0, without LPS injection), 1 (group 1), 5 (group 5), 9 (group 9), and 15 (group 15) of LPS injection, respectively. The results showed that, compared to the piglets without LPS injection, LPS injection caused liver injury in the early phase, as manifested by the increased activities of serum liver injury-related parameters (aspartate amino transferase, alanine aminotransferase, alkaline phosphatase, cholinesterase, and total bile acid) on day 1, and impaired liver morphology (disordered hepatic cell cord arrangement, dissolved and vacuolized hepatocytes, karyopycnosis, and inflammatory cell infiltration and congestion) on days 1 and 5. Meanwhile, LPS injection caused liver inflammation, oxidative stress, and mitochondrial dysfunction on days 1 and 5, as reflected by the upregulated mRNA expression of TNF-α, IL-6, IL-1β, TLR4, MyD88, and NF-κB; increased MPO and MDA content; and impaired mitochondrial morphology. However, these parameters were ameliorated in the later phase (days 9~15). Taken together, our data indicate that the incremental injection of the LPS-induced liver injury of piglets could be self-repaired.
The plant parts of Pleurolobus gangeticus (L.) J.St.-Hil. (synonym: Desmodium gangeticum), Fabaceae, were extracted and evaluated for its antitumor potential using in vitro and in vivo lymphoma models. Cytotoxic potentials and apoptosis induction was evaluated using in vitro systems. Ethanol extract, among others, had the lowest IC50 value of 17.5 μg/ml in cytotoxicity assay conducted in DLA (Dalton lymphoma ascites) cells. Cytotoxicity assays performed on YAC-1 cells showed reduction in the number of live cells from 10 × 104 to 2.2 × 104 after 48 h of treatment with ethanol extract (15 μg/ml) at which time point the untreated cells multiplied to reach 17.5 × 104 in number. The flow cytometric analysis revealed that at this concentration, 44.7% cells are already in the early apoptotic phase by 24 h of treatment. Considering its bioactivity, ethanol extract was further used for in vivo toxicology profiling and antitumor studies in mice models. Treatment with nontoxic doses of ethanol extract (200 and 400 mg/kg b.w.) significantly reduced the tumor burden in mice. The biopsy analysis of tumor tissue of ethanol extract treated animals also showed a considerable number of apoptotic and necrotic cells. Ethanol extract was also subjected to chromatographic analysis (GC–MS and LC–MS), which revealed presence of several pharmacologically important molecules, but the absence of salicin was also noticeable. This highlights the role of other compounds detected in giving the extract its tumor reduction property. Further investigation to identify the active components and to obtain a deeper knowledge on their mechanism of action is worthwhile to acquire novel safer and effective anticancer drugs.
Ethnopharmacological relevance: Jianpi Shengqing Huazhuo Formula (JSH) is a modified prescription based on traditional Chinese medicine theory and classic prescriptions (Buzhong Yiqi Decoction and Yuye Decoction). It has been found that JSH has a good effect on obese patients with early abnormal glucose and lipid metabolism. Therefore, this experiment was conducted to study its clinical efficacy and pharmacological effect. Aim of the study: To observe the clinical efficacy of JSH and explore the mechanism of the formula to improve glucose and lipid metabolism in obese rats. Materials and methods: 1. Clinical observation: 10 overweight/obese patients with abnormal glucose and lipid metabolism were selected to observe the indicators of serum glucose, serum lipids and liver damage of the patients before and after treatment with JSH. 2. Animal experiment: Fifty SD rats were randomly divided into control group, model group, Metformin group (120 mg/kg/day), JSH-L group (5 g/kg/day) and JSH-H group (20 g/kg/day), with 10 rats in each group. The obese Sprague-Dawley (SD) rat model was produced by feeding 60% high-fat diet for 8 weeks, and the drug group was given prophylactic administration for 8 weeks. At the end of the experiment, body weight, abdominal fat, plasma glucose, plasma lipids, plasma alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured. The levels of interleukin-6 (IL-6), interleukin 1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) in plasma were detected by Elisa, and the changes of malondialdehyde (MDA), glutathione (GSH) and catalase (CAT) in plasma and liver tissue were detected by kits. The pathological changes and lipid deposition in liver were observed by HE staining and oil red O staining, and the changes in the number of mitochondria in liver cells were observed by transmission electron microscopy. RT-qPCR and Western Blot (WB) were used to detect the mitochondrial regulation-related indicators PGC-1α, NRF1, TFAM, MFN2, DRP1 and apoptosis-related indicators Bcl-2, Bax, caspase 8 in liver tissue. Results: 1. Clinical observation: after one month administration, the patient's body weight, BMI, 2 h oral glucose tolerance test (2hOGTT), glycated hemoglobin (HbA1c), triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) decreased significantly, and the indicators of liver damage AST and ALT also decreased significantly. 2. Animal experiments: JSH can significantly reduce body weight and abdominal fat area, improve glucose and lipid metabolism, and also reduce plasma IL-6, IL-1β and TNF-α content in obese rats, and improve oxidative stress; HE staining and oil red O staining also showed that JSH can alleviate liver damage and lipid deposition in the liver. Further observations of liver cell ultrastructure showed that JSH can ameliorate the reduction of liver mitochondria caused by a high-fat diet and promote the expression of indicators of mitochondrial biogenesis related to PGC-1α, NRF1, and TFAM. Moreover, JSH could promote the expression of MFN2 and DRP1, decrease Bcl-2 and increase Bax in the liver. Conclusions: 1. Clinical observation: JSH can reduce body weight, serum glucose, serum lipid, and liver injury in overweight/obese patients. 2. Animal experiment: JSH regulates PGC-1α/NRF1/TFAM signaling pathway promotes liver mitochondrial biogenesis, improves glucose and lipid metabolism in obese rats, and regulates mitochondrial dependent apoptosis indicators Bcl-2/Bax to reduce liver injury.
Ethnopharmacological relevance: In Traditional Chinese Medicine (TCM), cholestasis liver disease belongs to jaundice. Yinchenzhufu decoction (YCZFD) is a classic formula used for treating jaundice. Aim of the study: This study was aimed to investigate the potential mechanism and effective components of YCZFD in chronic cholestatic liver injury (CCLI). Materials and methods: A chronic cholestatic mouse model induced by 3, 5-diethoxycarbonyl-1, 4-dihydroxychollidine was used to investigate the effect of YCZFD. Then, metabolomics was used to investigate the metabolites influenced by YCZFD. Serum and liver bile acid (BA) levels were measured using liquid chromatography coupled with triple quadruple mass spectrometry (LC-MS/MS), and the gene and protein expressions of BA transporters and metabolic enzymes were detected. Additionally, the pharmacokinetics of multiple components of YCZFD was explored to clarify the potential effective components. The effects of absorbed components of YCZFD on BA metabolism and transporter function, inflammation, and farnesoid X receptor (FXR) and pregnane X receptor (PXR) activation were analyzed using sandwich cultured rat hepatocytes, AML12 cells, and dual-luciferase receptor systems, respectively. Results: YCZFD decreased the liver damage in chronic cholestatic mice. Serum metabolomics results indicated that the main pathways influenced by YCZFD involved primary BA biosynthesis and arachidonic acid metabolism. YCZFD upregulated the expression of FXR, PXR, and BA efflux transporters and the metabolic enzymes of liver tissues, promoting BA excretion and metabolism in cholestatic mice. Additionally, YCZFD downregulated the expression of genes and proteins of the toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) pathway and decreased liver inflammation. The pharmacokinetic study indicated that multiple components showed different pharmacokinetic properties. Among the absorbed components of YCZFD, multiple components activated the transcription of FXR and PXR, regulated BA transporters and metabolic enzyme function, and reduced the gene expression of TLR4 and NF-κB1. Conclusion: YCZFD can ameliorate CCLI by promoting the excretion and metabolism of BAs and inhibiting inflammation via the TLR4/NF-κB signaling pathway. The multiple components of YCZFD could act on BA homeostasis regulation and anti-inflammation, exhibiting a combined effect against CCLI.
Lectins are proteins of non-immunological origin with the ability to bind to carbohydrates reversibly. They emerge as an alternative to conventional antifungals, given the ability to interact with carbohydrates in the fungal cell wall inhibiting fungal growth. The lectin from D. violacea (DVL) already has its activity described as anti-candida in some species. Here, we observed the anti-candida effect of DVL on C. albicans, C. krusei and C. parapsilosis and its multiple mechanisms of action toward the yeasts. Additionally, it was observed that DVL induces membrane and cell wall damage and ROS overproduction. DVL was also able to cause an imbalance in the redox system of the cells, interact with ergosterol, inhibit ergosterol biosynthesis, and induce cytochrome c release from the mitochondrial membrane. These results endorse the potential application of DVL in developing a new antifungal drug to fight back against fungal resistance.
The polypeptide antibiotic Polymyxin B (PMB) can cause acute kidney injury (AKI), we found that ferroptosis is one of the main mechanisms of renal injury caused by PMB. It was reported that baicalein can inhibit ferroptosis. Therefore, in this study we examined whether baicalein could attenuate PMB-induced renal injury by inhibiting ferroptosis. We confirmed that baicalein could reduce PMB-induced renal injury in vivo and in vitro studies. In the in vitro study, baicalein significantly increased the survival rate of human HK2 tubular epithelial cells. The results of HE staining and electron microscopy in mice also showed that baicalein reduced PMB-induced renal injury, and significantly decreased the levels of BUN and Scr. By detecting ferroptosis-related indicators, we found that pre-incubation of baicalein in HK2 cells down-regulated Fe2+ level, lipid peroxidation, MDA and HO-1 which had been increased by PMB. Furthermore, baicalein up-regulated the levels of SCL7A11, GPX4 and GSH that were decreased by PMB. Moreover, intraperitoneal injection of baicalein in the animal model down-regulated kidney iron level, PTGS2 and 4HNE, and increased the GSH level, which suggested that baicalein could inhibit PMB-induced ferroptosis. Finally, by detecting changes in levels of p53 and p53 K382 acetylation, baicalein was observed to decrease elevated p53 K382 acetylation after PMB treatment, further confirming that baicalein inhibits ferroptosis by reducing p53 K382 acetylation via upregulation of SIRT1 expression. In conclusion, these results suggest that baicalein decreases p53 acetylation level by elevating SIRT1, which can then inhibit PMB-induced ferroptosis and ultimately attenuates AKI.
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Saffron is a natural compound that has been used for centuries in many parts of the world as a food colorant and additive. It was shown to have the ability to mitigate various disorders through its known anti-inflammatory and anti-oxidant properties. Several studies have shown the effectiveness of saffron in the treatment of various chronic diseases like inflammatory bowel diseases, Alzheimer’s, rheumatoid arthritis as well as common malignancies of the colon, stomach, lung, breast, and skin. Modern day drugs generally have unwanted side effects, which led to the current trend to use naturally occurring products with therapeutic properties. In the present review, the objective is to systematically analyze the wealth of information regarding the potential mechanisms of action and the medical use of saffron, the “golden spice”, especially in digestive diseases. We summarized saffron influence on microbiome, molecular pathways, and inflammation in gastric, colon, liver cancers, and associated inflammations.
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Despite being a cardinal experimental model, the induction of cirrhosis in rats by repeated exposure to carbon tetrachloride (CCl4) has low reproducibility. Here, we compared two models of cirrhosis induced by orogastric administration of CCl4 once (CCl4-1xWk) or twice a week (CCl4-2xWk) for 12 weeks in male Sprague-Dawley rats. Control rats received water instead of CCl4. Both CCl4 protocols similarly attenuated body weight gain (p < 0.01 vs. Control). Although both CCl4 protocols increased hepatic fibrosis, portal hypertension and splenomegaly, the magnitude of these alterations was higher and more consistent in CCl4-2xWk rats. Importantly, two CCl4-1xWk rats did not develop cirrhosis versus a 100% yield of cirrhosis in CCl4-2xWk rats. The CCl4-2xWk protocol consistently induced liver atrophy together with hematological, biochemical and coagulation abnormalities characteristic of advanced cirrhosis that were absent in CCl4-1xWk rats. Ascites occurred in 20% and 80% of rats in theCCl4-1xWk and CCl4-2xWk groups (p < 0.01). All rats showed normal renal function, arterial blood gases and stable systemic hemodynamics. The total dose of CCl4 and mortality rate were similar in both protocols. The CCl4-2xWk protocol, therefore, was highly reproducible and effective for the induction of experimental cirrhosis within a confined time, representing a valuable advance for liver research.
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Liver disease is one of the most risk factors threatening human health. It is of great significance to find drugs that can treat liver diseases, especially for acute and chronic hepatitis, non-alcoholic fatty liver disease, and liver cancer. The search for drugs with good efficacy from traditional natural medicines has attracted more and more attention. Tibetan medicine, one of the China's traditional medical systems, has been widely used by the Tibetan people for the prevention and treatment of liver diseases for hundreds of years. The present paper summarized the natural Tibetan medicines that have been used in Tibetan traditional system of medicine to treat liver diseases by bibliographic investigation of 22 Tibetan medicine monographs and drug standards. One hundred and ninety three species including 181 plants, 7 animals, and 5 minerals were found to treat liver diseases in traditional Tibetan medicine system. The most frequently used species are Carthamus tinctorius, Brag-zhun, Swertia chirayita, Swertia mussotii, Halenia elliptica, Herpetospermum pedunculosum, and Phyllanthus emblica. Their names, families, medicinal parts, traditional uses, phytochemicals information, and pharmacological activities were described in detail. These natural medicines might be a valuable gift from the old Tibetan medicine to the world, and would be potential drug candidates for the treatment of liver diseases. Further studies are needed to prove their medicinal values in liver diseases treatment, identify bioactive compounds, elucidate the underlying mechanism of action, and clarify their side effects or toxicity with the help of modern phytochemical, pharmacological, metabonomics, and/or clinical trial methods.
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The purpose of this study was to investigate the anti-cancer property of grape seed extract (GSE) during early stages of developing liver cancer using a two-stage carcinogenic model combining diethylnitrosamine (DEN) and acetylaminofluorene. Administration of GSE at doses 25, 50 and 100 mg/kg per day started at the beginning of promotion periods and continued for 14 weeks. GSE dramatically inhibited pre-neoplastic foci formation as well as significantly decreased the number and the area of placental glutathione-S-transferase in livers of DEN-treated rats by approximately 4 & 10 fold deductions, respectively. GSE’s effects were associated with induced apoptosis, reduced cell proliferation, decreased oxidative stress and down regulation of histone deacetylase activity and inflammation makers, such as cyclooxygenase 2, inducible nitric oxide synthase, nuclear factor-kappa B-p65 and p- phosphorylated tumor necrosis factor receptor expressions in liver. GSE treatment also decreased the viability of HepG2 cells and induced early and late apoptosis through activating caspase-3 and Bax. Furthermore, GSE induced G2/M and G1/S cell cycle arrest. The present study provides evidence that the GSE’s anticancer effect is mediated through the inhibition of cell proliferation, induction of apoptosis, modulating oxidative damage and suppressing inflammatory response.
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Background Atherosclerosis is a chronic vascular inflammatory disease. Since even low-level endotoxemia constitutes a powerful and independent risk factor for the development of atherosclerosis, it is important to find therapies directed against the vascular effects of endotoxin to prevent atherosclerosis. Taraxacum officinale (TO) is used for medicinal purposes because of its choleretic, diuretic, antioxidative, anti-inflammatory, and anti-carcinogenic properties, but its anti-inflammatory effect on endothelial cells has not been established. Methods We evaluated the anti-inflammatory activity of TO filtered methanol extracts in LPS-stimulated human umbilical vein endothelial cells (HUVECs) by monocyte adhesion and western blot assays. HUVECs were pretreated with 100 μg/ml TO for 1 h and then incubated with 1 μg/ml LPS for 24 h. The mRNA and protein expression levels of the targets (pro-inflammatory cytokines and adhesion molecules) were analyzed by real-time PCR and western blot assays. We also preformed HPLC analysis to identify the components of the TO methanol extract. Results The TO filtered methanol extracts dramatically inhibited LPS-induced endothelial cell–monocyte interactions by reducing vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1, and pro-inflammatory cytokine expression. TO suppressed the LPS-induced nuclear translocation of NF-κB, whereas it did not affect MAPK activation. Conclusions Our findings demonstrated that methanol extracts of TO could attenuate LPS-induced endothelial cell activation by inhibiting the NF-κB pathway. These results indicate the potential clinical benefits and applications of TO for the prevention of vascular inflammation and atherosclerosis. Electronic supplementary material The online version of this article (10.1186/s12906-017-2022-7) contains supplementary material, which is available to authorized users.
Immune cells play an important role in controlling liver tumorigenesis, viral hepatitis, liver fibrosis and contribute to pathogenesis of liver inflammation and injury. Accumulating evidence suggests the effectiveness of natural killer (NK) cells and Kupffer cells (KCs) against viral hepatitis, hepatocellular damage, liver fibrosis, and carcinogenesis. Activation of natural killer cells provides a novel therapeutic strategy to cure liver related diseases. This review discusses the emerging roles of immune cells in liver disorders and it will provide baseline data to scientists to design better therapies for treatment.
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Liver fibrosis is characterized by an increased and altered deposition of extracellular matrix (ECM) proteins that make up excessive tissue scarring and promote chronic liver injury. Activation of hepatic stellate cells (HSCs) is a pivotal cellular event in the progression of liver fibrosis. However, the mechanisms involved in the development of liver fibrosis are only now beginning to be unveiled. The Notch pathway is a fundamental and highly conserved pathway able to control cell-fate, including cell proliferation, differentiation, apoptosis, regeneration and other cellular activities. Recently, the deregulation of Notch cascade has been found involved in many pathological processes, including liver fibrosis. These data give evidence for a role for Notch signaling in liver fibrosis. In addition,more and more date are available on the role of Notch pathways in the process. Therefore, this review focuses on the current knowledge about the Notch signaling pathway, which dramatically takes part in HSC activation and liver fibrosis, and look ahead on new perspectives of Notch signaling pathway research. Furthermore, we will summarize this new evidence on the different interactions in Notch signaling pathway-regulated liver fibrosis, and support the potentiality of putative biomarkers and unique therapeutic targets.
Epithelial cell loss alters a tissue's optimal function and awakens evolutionarily adapted healing mechanisms to reestablish homeostasis. Although adult mammalian organs have a limited regeneration potential, the liver stands out as one remarkable exception. Following injury, the liver mounts a dynamic multicellular response wherein stromal cells are activated in situ and/or recruited from the bloodstream, the extracellular matrix (ECM) is remodeled, and epithelial cells expand to replenish their lost numbers. Chronic damage makes this response persistent instead of transient, tipping the system into an abnormal steady state known as fibrosis, in which ECM accumulates excessively and tissue function degenerates. Here we explore the cellular and molecular switches that balance hepatic regeneration and fibrosis, with a focus on uncovering avenues of disease modeling and therapeutic intervention.