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Effect of bergenin on the kidney of C57BL/6J mice with high fat-diet
induced oxidative stress
Sagadevan Ambika and Ramalingam Saravanan*
Department of Biochemistry and Biotechnology, Annamalai University,
Annamalai Nagar- 608 002, Tamil Nadu, India
*Assistant professor: Email : jayamsaranbio@gmail.com
Keywords: high fat diet; bergenin; nephritic markers; lipid peroxidation; antioxidant
ABSTRACT. The present study evaluated the protective effect of bergenin on high fat diet (HFD)
induced diabetic mice. C57BL/6J mice were segregated in two groups, one fed standard diet (NC)
and the other fed HFD for 16 weeks. Mice were fed continuously with high fat diet for 16 weeks
and subjected to intragastric administration of bergenin (10, 20 and 40 mg/kg body weight (BW)),
metformin (25 mg/kg BW) 9 to 16 weeks. At the end of the treatment nephritic markers, lipid
peroxidation product, antioxidant and histopathological examination were carried out to assess the
efficacy of the treatment. HFD fed mice showed increased plasma glucose, insulin, altered nephritic
markers, antioxidant and histopathological abnormalities. Oral Treatment with bergenin (40 mg/kg
BW) showed near normalized levels of plasma glucose, lipid peroxidation product, antioxidants,
improved insulin and reduced kidney damage. The effects of bergenin were comparable with
standard drug, metformin. These data suggest that bergenin protect kidney from deleterious effect
of glucose.
1. INTRODUCTION
Diabetes mellitus (DM) is a complex metabolic disease characterized by high blood glucose
levels and a disorder of carbohydrate, fat and protein metabolism. The abnormal increase of blood
glucose in diabetes will result in long-term damage and dysfunction of various organs including the
eyes, kidneys, nerves and blood vessels [1]. Diabetic nephropathy (DN) is one of the most
devastating diabetes complications and the leading cause of end stage renal disease [2]. DN is
caused by both metabolic alterations (hyperglycaemia and possibly hyperlipidaemia) and
haemodynamic alterations (systemic and glomerular hypertension). Oxidative stress consumes nitric
oxide, which prevents flow-mediated dilation (FMD) of blood vessels (endothelial dysfunction),
subjecting the endothelium to injury. This leads to production of cytokines, acceleration of
inflammation, worsening of blood vessel rigidity due to atherosclerosis, further impairment of FMD
and susceptibility to oxidative stress. Inflammation, endothelial dysfunction and oxidative stress can
be thought of as a “vicious cycle” that leads to significant kidney damage and cardiovascular
events. The injurious effects of hyperglycemia induce vascular damage through complex
overlapping pathways; formation of advanced glycation end products (AGE), activation of protein
kinase C and generation of reactive oxygen species (ROS) [3]. Growing evidence suggests that
ROS may play important role in the initiation and progression of DN. The effect of antioxidant
therapy is well-documented in cell and animal studies [4]. Among the various therapeutic strategies,
combination of antihyperglycemic, antihyperlipidemic, antihypertensive, and antioxidants may be
beneficial for the prevention of diabetes complication.
Growing prevalence in unfastened-radical biology and lack of effective therapies for
maximum chronic diseases, the usefulness of the nutritional inclusion of herbal antioxidants found
in plant foods is a crucial fitness-defensive and sickness-preventing component in people. Plants are
rich source of bioactive components; the most important of these are polyphenols and flavonoids
compounds. They show off high antioxidant homes that terminate free-radical mediated reactions
with the aid of donating hydrogen atom or an electron to the radicals [5,6]. Bergenin is (C-glucoside
of 4-O-methylgallic acid) polyphenol compound. It is dihydroisocoumarin derivative isolated from
International Letters of Natural Sciences Submitted: 2016-02-24
ISSN: 2300-9675, Vol. 54, pp 58-65 Revised: 2016-03-28
doi:10.18052/www.scipress.com/ILNS.54.58 Accepted: 2016-04-05
© 2016 SciPress Ltd., Switzerland Online: 2016-05-11
SciPress applies the CC-BY 4.0 license to works we publish: https://creativecommons.org/licenses/by/4.0/
several medicinal plants such as Ficuus racemosa [7], Mallotus japonicas[8], Bergenia crassifolia,
Caesalpinia digyna, Astibe thunbergii, Ardisia japonica plant and also other genera [9]. Bergenin
contains five hydroxyl groups which are consider to be potentially active and it exhibit various
biological activities such as antioxidant [10], hepatoprotective [11], antiarrhythmic [12],
antimicrobial, antiviral and antiulcerogenic activities [13], anti-inflammatory [14]. Bergenin is
reported as insulin sensitisers [7] and has potential antidiabetic effect [15]. Hence, the present study
was designed to evaluate the protective effect of bergenin in early DN in HFD induced diabetic
mice.
2. MATERIALS AND METHODS
2.1. Chemicals
Bergenin was purchased from Carbo synth (Compton, Berkshire, UK). All other chemicals
used in this study were of analytical grade obtained from HIMEDIA and S.D. Fine chemicals
Research laboratories Pvt. Ltd (Mumbai, Maharashtra, India).
2.2. Experimental animals
Male C57BL/6J mice 3 weeks of age were obtained from NIN Hyderabad and housed in
polypropylene cages. Animals were maintained under standard conditions with a 12h light/dark
cycle. The animals received a standard pellet diet (Karnataka State Agro Corporation Ltd., Agro
feeds division, Bangalore, India) and water ad libitum. After acclimatization for period of 1 week,
mice were randomly divided into six groups. The animals used in the present study were cared and
maintained as per the principles and guidelines of the Institutional Animal Ethical Committee
(IAEC), Annamalai Nagar, in accordance with the Indian National law on animal care and use. The
study protocols were approved by the Institutional Animal Ethics Committee of Rajah Muthiah
Medical College and Hospital, Annamalainagar (Reg No. 160/1999/CPCSEA, Proposal number:
913).
2.3. Experimental induction of diabetes
The type 2 diabetes was induced through HFD. The standard diet which is commercially
obtained from Sai Enterprises, Chennai, had a fat composition of 4.2%. The beef tallow based high
fat diet was composed of protein -17.7 g, fat-35.2 g, carbohydrate-34.5 g, fibre-3.4 g, minerals - 6.8
g and vitamins- 1.8g. Mice (6 nos.) from normal control group (group I) were fed standard diet for a
period of 16 weeks. Mice from rest of the groups (group II-VI) were fed high fat diet for a period of
16 weeks. At the end of 8
th
week, the mice from all the groups were tested for blood glucose levels.
Mice with blood glucose level of 220 mg/dL and above were considered to have developed insulin
resistance and were subjected to intragastric administration of various doses of bergenin and
metformin (as mentioned in the experimental design) during 8 to 16 weeks.
2.4. Diet and experimental design
The experimental design consisted of six groups of mice. Group I: Normal control (NC)
mice fed with a standard diet for 16 weeks. Group II: HFD diabetic mice fed with high fat diet for a
period of 16 weeks. Group III: NC mice fed with standard diet for 16weeks and administered with
bergenin (10 mg/kg BW) by gavage for the last 8 weeks. Group IV: HFD diabetic mice
administered with bergenin (20 mg/kg BW) by gavage for the last 8 weeks. Group V: HFD diabetic
mice administered with bergenin (40 mg/kg BW) by gavage for the last 8 weeks. Group VI: HFD
diabetic mice administered with metfomin (25mg/kg BW) by gavage for the last 8weeks. At the end
of experimental period mice were fasted overnight. The mice were sacrificed by cervical
dislocation. Blood was collected by cutting the jugular vein into heparinized glass tubes. Plasma
was obtained from blood samples after centrifugation (1500g for 10 min) and stored at 4°C. Kidney
tissue was excised immediately from the mice and washed in ice-cold isotonic saline and blotted
with a filter paper. The dosage of bergenin was selected from the dose-fixation study using three
different doses (10 mg, 20 mg and 40 mg /kg BW). From the results of the study (Table 1), we
International Letters of Natural Sciences Vol. 54 59
found that 40 mg/kg BW to be effective in lowering glucose and insulin levels when compared to
10 mg and 20 mg /kg BW. Thus, the 40 mg /kg BW was taken as the optimum dose for treatment.
2.5. Biochemical analysis
Plasma glucose was estimated by the method of Trinder (1969) [16]. Plasma insulin was
measured by the method of Burgi et al. (1988) [17]. Serum urea was estimated by the method of
Fawcett and Scott (1960) [18], uric acid by the enzymatic method described by Caraway (1955)
[19] and creatinine was estimated using the method of Tietz, 1987. Superoxide dismutase (SOD) in
the kidney tissues was assayed by the method of Kakkar et al. (1984) [20]. The activity of catalase
(CAT) in the kidney tissues was determined by the method of Sinha (1972) [21]. The activity of
glutathione peroxidase (GPx) in the kidney tissues was measured by the method of Rotruck et al.,
(1973) [22]. Reduced glutathione (GSH) in the kidney tissues was estimated by the method of
Ellman (1959) [23]. Vitamin C in the kidney tissues was estimated by the method of Roe and
Kuether (1943) [24]. Vitamin E in the kidney tissues was estimated by the method of Baker et al.,
(1980) [25]. TBARS and LHP (Lipid hydroperoxide) in kidney tissues were estimated by the
methods of Niehaus and Samuelsson (1968) [26] and Jiang et al., (1992) [27], respectively.
2.6. Histological examination
For histological analysis, sections from the kidney of mice from NC, HFD, HFD + bergenin
and HFD + metformin were fixed in 10 % buffered formalin for 24 h and embedded in paraffin.
Tissue sections were deparaffinized, stained with haematoxylin-eosin (H&E) and examined under
microscope at 400x magnification.
2.7. Statistical analysis
The results were expressed as mean ± standard deviation (SD) for 6 mice in each group.
Data were analysed by one- way analysis of variance followed by Duncan’s multiple range test
(DMRT) using SPSS version 16 (SPSS, Chicago,IL). Post hoc testing was performed for inter-
group comparisons using the least significance difference (LSD) test; p values\0.05 were considered
as significant.
3. RESULTS
Figure 1 delineate significant increase in plasma glucose and insulin levels were noted in
HFD induced diabetic mice group than in the control group. Bergenin treatment to HFD mice
reduced glucose and insulin level to near normal. These values were compared with metformin
activity.
Table 1 shows increased serum activities of urea, uric acid and creatinine were found in
HFD induced diabetic mice, indicating damage to kidney cells. Treatment of HFD induced diabetic
mice with bergenin resulted in significantly lower urea, uric acid and creatinine activity. These
values were compared with metformin activity. Table 2 lists TBARS and LHP levels in the different
groups. Significantly higher TBARS and LHP levels were found in HFD induced diabetic mice
compared with control mice. In bergenin treated HFD induced diabetic mice, TBARS and LHP
levels were significantly lower compared with metformin.
Table 3 lists the activities of enzymatic and non-enzymatic antioxidants in the kidney of the
different groups. The activities of enzymatic antioxidants SOD, CAT, GPx were significantly
lowered in HFD induced diabetic mice. The GSH, as well as vitamin C and E levels, were
significantly decreased in HFD-induced diabetic mice compared with controls. In bergenin treated
HFD-induced diabetic mice, these parameters returned to normal levels.
Histopathology sections of kidney tissues were given in figure 2 HFD fed mice showed
focal proliferative glomerulo nephritis with mild increase in cellularity in cortex and closely packed
tubules with normal medulla in diabetic mice. Treatment with bergenin and metformin groups
showed normal glomeruli and medulla histology comparable with untreated control mice.
60 Volume 54
4. DISCUSSION
Immoderate power consumption from HFD results in a boom in energy as fats, accompanied
through an elevation in mitochondrial macronutrient oxidation, favoring an excessive production of
ROS generation, thereby mounted oxidative stress [28]. HFD feeding has been documented to result
in dyslipidemia, hyperglycemia and hyperinsuliunemia in mice [29]. Our results are consistent with
previous studies which found that consumption of HFD markedly induces an increase in glucose
and insulin. In the present study bergenin treatment significantly reduced the glucose and insulin
level. Kumar et al (2012) also reported that bergenin has antidiabetic activity in streptozotocin –
nicotinamide induced diabetic rats [15].
Oxidative stress plays an important role within the pathogenesis and progression of renal
sickness [30]. HFD induces alteration of renal lipid metabolism via an imbalance between
lipogenesis and lipolysis within the kidney, as well as systemic metabolic abnormalities and next
renal lipid accumulation and lipid peroxidation leading to renal harm [31]. The accumulated adipose
tissue around the kidneys penetrates into the medullary sinuses, increases intrarenal pressures
causing damage to the renal tissue. Damaged renal tissue acts as a source of ROS generation and
develops lipid peroxidation. An accelerated lipid peroxidation within the kidney tissue is probably
concerned in the onset of kidney lesions within the rodent models of obesity [32]. Elevated ranges
of TBARS and LHP inside the kidney of HFD precipitated diabetic mice is regular with a preceding
record that HFD induces oxidative damage [33]. The antioxidant enzymes along with SOD, CAT
and GPX are themselves liable to reactive oxygen species (ROS) assault [34]. The decreased SOD
activity in HFD induced diabetic mice can be attributed to inactivation of the enzyme because of
glycation of Lys122 and Lys128 residues [35]. Reduced GSH, vitamins E and C may be because of
elevated utilization to fight free radicals and/or decreased regeneration from their oxidized forms.
The biochemical evaluation exhibited significant lower in enzymatic and nonenzymatic antioxidants
with great increase in lipid peroxidation markers in HFD caused diabetic mice. In our examine,
intragastric administration of bergenin to HFD mice confirmed recovery of the degrees of
enzymatic and nonenzymatic antioxidants with reduced lipid peroxidation byproducts. Accordingly,
our look at suggests that bergenin ought to definitely impair the generation of oxidative stress
thereby lowering the risk of HFD-precipitated kidney injury. Nazir et al., reported that bergenin has
strong antioxidant properties by scavenging the free radicals [36].
In general, creatinine level is considered to assess kidney function [37]. Elevated levels of
serum creatinine, urea and uric acid were observed in diabetic kidney. Blood urea is produced as a
result of protein breakdown and formed in the liver which carried via the bloodstream to the
kidneys to be eliminated. Urea is hydrolysed in the presence of water and urease to produce
ammonia and carbon dioxide. Uric acid inside the sample is oxidized by using uricase to allantoin.
The assay of creatinine turned into based totally on the response of creatinine with alkaline picrate.
Creatinine is a breakdown product formed in certain muscular tissues and carried thru the
bloodstream and eliminated by way of the kidneys. If the kidneys dysfunction, they are unable to
dispose of the usual amount of these materials and as an end result, the blood urea and creatinine
stages will enhance [38]. Moreover, these elevations were found to be associated with interstitial
atrophy, epithelial necrosis as well as atrophic changes in glomeruli, and thus DN [39]. Diabetic
mice showed significant increase in serum creatinine, urea and uric acid levels which were
significantly reduced after treatment with bergenin indicating recovery toward normal level.
Histopathological observations in the kidney sections showed focal proliferative glomerulonephritis
with mild increase in cellularity and closely packed tubules with normal medulla of HFD induced
diabetic mice. Kidney sections from HFD + bergenin and HFD + Metformin groups showed normal
glomeruli with closely packed tubules comparable with untreated control mice
International Letters of Natural Sciences Vol. 54 61
5. CONCLUSION
According to the studies on HFD induced diabetic mice administration of bergenin reduced
the hyperglycemia, hyperinsulinemia, oxidative stress, improved antioxidant status and slowing the
progession of early DN. Our histological studies reveal the protective effect of bergenin against
kidney damage. Our study suggests that bergenin can serve as a beneficial compound in
management of type 2 diabetes. Further studies will be in progess to elicit the exact mechanism of
bergenin for its antidiabetogenic effect.
Acknowledgements
The authors thoughtfully wish to place on record with gratitude to university grant
commission (UGC), New Delhi, which rendered financial support by granting SRF to the scholar
Ambika. S.
Table 1. Effect of Bergenin on urea, uric acid, creatinine of serum in experimental mice
Group
Urea (mg/dL)
Uric acid (mg/dL)
Creatinine (mg/dL)
Normal control
23.19 ± 1.32
a
1.44 ± 0.08
a
0.96 ± 0.05
a
HFD
49.42 ± 2.87
b
3.90 ± 0.19
b
3.09 ± 0.22
b
HFD + BGN(10 mg/kg BW)
40.18 ± 2.65
c
3.20 ± 0.17
c
2.11 ± 0.19
c
HFD + BGN(20 mg/kg BW)
35.33 ± 2.43
d
2.99 ± 0.16
d
1.87 ± 0.14
d
HFD + BGN(40 mg/kg BW)
27.25 ± 2.21
e
2.85 ± 0.16
e
1.20 ± 0.07
e
HFD + metformin (25 mg/kg BW)
25.28 ± 1.56
a
1.98 ± 0.10
f
0.99 ± 0.06
a
Values that have a different superscript letter (a, b, c,d,e,f) differ significantly with each other
(p < 0.05,DMRT). BGN-bergenin, MET-metformin
Table 2. Effect of Bergenin on TBARS and LOOH in the kidney of HFD-fed C57BL/6J mice
Group TBARS LOOH
Normal control
1.43 ± 0.07
a
69.72 ± 3.89
a
HFD
3.85 ± 0.18
b
153.31 ± 9.32
b
HFD + BGN (40 mg/kg BW)
1.60 ± 0.08
cd
85.54 ± 5.67
c
HFD + MET (25 mg/kg BW)
1.51 ± 0.07
da
74.09 ± 4.60
a
Values that have a different superscript letter (a, b, c,d) differ significantly with each other
(p < 0.05,DMRT).
Table 3. Effect of Bergenin on the activities of enzymatic and non enzymatic antioxidant in the
kindey of diabetic and normal mice
Group Normal control HFD HFD+BGN
(40 mg/kg BW)
HFD+MET
(25 mg/kg BW)
SOD (U*/mg protein) 12.42±1.01
a
7.98±0.56
b
9.54±0.77
c
11.29±0.70
d
CAT (U
#
/mg protein) 34.21±2.32
a
18.72±1.23
b
29.11±2.10
c
31.74±1.70
d
GPX (U
♣
/mg protein)
10.21±0.69
a
4.97±0.31
b
7.42±0.45
c
8.28±0.63
d
GSH (µg/mg protein) 12.09 ± 0.78
a
6.34 ± 0.38
b
11.89 ± 0.89
a
10.64 ± 0.62
d
Vitamin C (µg/mg protein) 1.58 ± 0.08
a
0.39 ± 0.0.01
b
0.92 ± 0.04
c
1.21 ± 0.07
d
Vitamin E (µg/mg protein) 5.49 ± 0.35
a
2.05 ± 0.0.18
b
4.54 ± 0.29
c
4.94 ± 0.37
d
U* = enzyme concentration required to inhibit the NBT to 50% reduction in one minute.
U
#
= µmole of H
2
O
2
consumed/minute. U
♣
= µg of GSH utilized/minute. Values that have a
different superscript letter (a, b, c, d) differ significantly with each other (p < 0.05,DMRT).
62 Volume 54
Figure 1. Effect of Bergenin on plasma glucose and insulin levels in HFD-fed C57BL/6J
mice.Values that have a different superscript letter (a, b, c,d,e,f) differ significantly with each other
(p < 0.05,DMRT). BGN-bergenin, MET-metformin
Figure 2. 2(a) normal control - normal glomeruli with closely packed tubules and normal medulla.
2(b) HFD- focal proliferative glomerulonephritis with mild increase in cellularity. 2(c) HFD+
Bergenin- normal glomeruli with closely packed tubules. 2(d) HFD+metformin- normal glomeruli
and medulla.
International Letters of Natural Sciences Vol. 54 63
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