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ORIGINAL RESEARCH
published: 02 November 2017
doi: 10.3389/fmicb.2017.02142
Frontiers in Microbiology | www.frontiersin.org 1November 2017 | Volume 8 | Article 2142
Edited by:
Luciana Balboa,
Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET),
Argentina
Reviewed by:
Anca Dorhoi,
Friedrich Loeffler Institute Greifswald,
Germany
Elsa Anes,
Universidade de Lisboa, Portugal
*Correspondence:
Ying Zhang
yzhang5@jhu.edu
Xin Jiang
jiangxingao@163.com
†These authors have contributed
equally to this work.
Specialty section:
This article was submitted to
Microbial Immunology,
a section of the journal
Frontiers in Microbiology
Received: 24 June 2017
Accepted: 19 October 2017
Published: 02 November 2017
Citation:
Zhang Q, Sun J, Wang Y, He W,
Wang L, Zheng Y, Wu J, Zhang Y and
Jiang X (2017) Antimycobacterial and
Anti-inflammatory Mechanisms of
Baicalin via Induced Autophagy in
Macrophages Infected with
Mycobacterium tuberculosis.
Front. Microbiol. 8:2142.
doi: 10.3389/fmicb.2017.02142
Antimycobacterial and
Anti-inflammatory Mechanisms of
Baicalin via Induced Autophagy in
Macrophages Infected with
Mycobacterium tuberculosis
Qingwen Zhang 1†, Jinxia Sun 1† , Yuli Wang1, Weigang He 1, Lixin Wang 1, Yuejuan Zheng 1,
Jing Wu 2, Ying Zhang 3
*and Xin Jiang 1
*
1Department of Immunology and Microbiology, School of Basic Medical Sciences, Shanghai University of Traditional Chinese
Medicine, Shanghai, China, 2Department of Infectious Diseases, Institute of Infectious Diseases, Huashan Hospital, Fudan
University, Shanghai, China, 3Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health,
Johns Hopkins University, Baltimore, MD, United States
Tuberculosis (TB) remains a leading killer worldwide among infectious diseases and the
effective control of TB is still challenging. Autophagy is an intracellular self-digestion
process which has been increasingly recognized as a major host immune defense
mechanism against intracellular microorganisms like Mycobacterium tuberculosis (Mtb)
and serves as a key negative regulator of inflammation. Clinically, chronic inflammation
surrounding Mtb can persist for decades leading to lung injury that can remain
even after successful treatment. Adjunct host-directed therapy (HDT) based on both
antimycobacterial and anti-inflammatory interventions could be exploited to improve
treatment efficacy and outcome. Autophagy occurring in the host macrophages
represents a logical host target. Here, we show that herbal medicine, baicalin, could
induce autophagy in macrophage cell line Raw264.7 and caused increased killing of
intracellular Mtb. Further, baicalin inhibited Mtb-induced NLRP3 inflammasome activation
and subsequent inflammasome-derived IL-1β. To investigate the molecular mechanisms
of baicalin, the signaling pathways associated with autophagy were examined. Results
indicated that baicalin decreased the levels of phosphorylated protein kinase B (p-Akt)
and phosphorylated mammalian target of rapamycin (p-mTOR) at Ser473 and Ser2448,
respectively, but did not alter the phosphorylation of p38, JNK, or ERK both in
Raw264.7 and primary peritoneal macrophages. Moreover, baicalin exerted an obvious
inhibitory effect on nuclear factor-kappa B (NF-κB) activity. Finally, immunofluorescence
studies demonstrated that baicalin promoted the co-localization of inflammasome with
autophagosome may serve as the underlying mechanism of autophagic degradative
effect on reducing inflammasome activation. Together, baicalin definitely induces the
activation of autophagy on the Mtb-infected macrophages through PI3K/Akt/mTOR
pathway instead of MAPK pathway. Furthermore, baicalin inhibited the PI3K/Akt/NF-κB
signal pathway, and both autophagy induction and NF-κB inhibition contribute to
Zhang et al. Baicalin-Inducing Autophagy against Mycobacterium tuberculosis
limiting the NLRP3 inflammasome as well as subsequent production of pro-inflammatory
cytokine IL-1β. Based on these results, we conclude that baicalin is a promising
antimycobacterial and anti-inflammatory agent which can be a novel candidate for the
development of new adjunct drugs targeting HDT for possible improved treatment.
Keywords: baicalin, Mycobacterium tuberculosis, host-directed therapy, autophagy, inflammasome
INTRODUCTION
Tuberculosis (TB) continues to be a major cause of significant
morbidity and mortality world-wide. The latest World Health
Organization (WHO) report indicates that TB remains a global
emergency (WHO, 2016). New available anti-TB drugs are
limited in number and activity and they mostly direct microbial
targets and have faced many obstacles (Zumla et al., 2015)
such as increasing drug resistance, complex drug regimens,
lengthy, and toxic treatment durations. Recent work on
host immunity, host-pathogen interactions and host-directed
interventions have shown that supplementation anti-TB therapy
with host modulators, such as imatinib and nitazoxanide may
shorten the treatment times, reduce the lung damage caused by
inflammation, and lower the risk of relapse or reinfection (Hawn
et al., 2013). Host-directed therapy (HDT) is a new strategy
for adjuvant therapy in fighting against TB, which focuses on
potentiating key components of host antimycobacterial effector
mechanisms, while restricting inflammation and pathological
damage in the lung (Hawn et al., 2013; Zumla and Maeurer, 2016;
Machelart et al., 2017; Yang, 2017).
Autophagy is a highly conserved and fundamental biological
process in eukaryotic cells (Mizushima et al., 2008). Most of
autophagic physiological effects, such as maintaining cell, tissue,
and organism homeostasis, are the result of its degradative
activities (Boya et al., 2013), while the unconventional function
of autophagy such as biogenesis and secretory roles in protein
processing are beginning to be recognized (Deretic et al., 2012;
Boya et al., 2013). A cardinal structural and functional feature of
autophagy is the formation of bilayer membrane organelles called
autophagosomes. The generation of LC3-II is an emblematic
event associated with autophagy and the reduction of p62,
a specific substrate protein of autophagosome, signifies the
generation of highly lytic degradative organelles, autolysosome,
resulting from the fusion of autophagosome with lysosome.
Increasing evidences have shown antimicrobial role of autophagy
against Mycobacterium tuberculosis (Mtb) (Gutierrez et al., 2004;
Singh et al., 2006; Bradfute et al., 2013). The mechanism of
killing of intracellular mycobacteria by autophagy is based
on the strong degradative and other antimicrobial properties
unique to autolysosome (Ponpuak et al., 2010). Autophagy
Abbreviations: TB, tuberculosis; Mtb, Mycobacterium tuberculosis; WHO,
World Health Organization; HDT, Host-directed therapy; ASC, apoptosis-
associated speck-like protein containing a caspase recruitment domain; PI3K,
phosphatidylinositol 3 kinase; Akt, protein kinase B; mTOR, mammalian target
of rapamycin; MAPK, mitogen activated protein kinases; NF-κB, nuclear factor-
kappa B; NLR, NOD-like receptor; DMEM, Dulbecco’s Modified Eagle’s Medium;
CFU, Colony forming unit; BCA, bicinchoninic acid; PBS, phosphate-buffered
saline; BSA, bovine serum albumin; CQ, chloroquine; Con, control; Lys, lysate;
Sup, supernatant; rapa, rapamycin.
eliminates mycobacteria through several mechanisms. First,
induction of autophagy indirectly promotes maturation of Mtb
phagosomes into degradative organelles (Harris et al., 2007;
Fabri et al., 2011), conquering the well-known Mtb-mediated
phagosome maturation arrest. Second, autophagosomes directly
capture a subset of intracellular Mtb that then progress into
degradative autolysosomes (Gutierrez et al., 2004; Watson et al.,
2012). Third, autophagy has a bactericidal effect relying on
the classical antimicrobial peptides such as cathelicidin through
fusion with lysosomes where cathelicidin is stored or neo-
antimicrobial peptides produced through autophagic proteolysis
of innocuous cytosolic proteins such as ubiquitin (Alonso et al.,
2007) and ribosomal proteins (Yuk et al., 2009; Ponpuak et al.,
2010; Fabri et al., 2011). Thus, autophagy can be triggered by
immunological and physiological stimuli enabling macrophages
to kill intracellular Mtb.
The activation of autophagy can be regulated by a wide variety
of signals (He and Klionsky, 2009; Yang and Klionsky, 2009; Yin
et al., 2016). The kinase mTOR is a major modulator of autophagy
and it receives inputs from different signaling pathways, and
is a downstream target of the phosphatidylinositol 3 kinase
(PI3K)/protein kinase B (Akt) pathway. The PI3K/Akt/mTOR
signaling pathway has been recognized to negatively regulate the
activation of autophagy (Heras-Sandoval et al., 2014). Moreover,
the activation of mitogen activated protein kinases (MAPK)
pathway can induce autophagy (Krishna and Narang, 2008;
Zhou et al., 2015). MAPK is a well-known serine/threonine
protein kinase, the associated signal pathway is one of the most
important regulatory mechanisms in eukaryotic cells, with p38,
JNK, ERK1/2 being the key members of MAPK sub-families
(Krishna and Narang, 2008). Additionally, PI3K/Akt pathway
also contributes to the activation of NF-κB by inducing the
phosphorylation level of IKKα/βand IκBα(Kang et al., 2012; Guo
et al., 2016).
Macrophages infected with Mtb secrete proinflammatory
cytokines, including IL-1βand IL-18 (Koo et al., 2008;
Kleinnijenhuis et al., 2009). The increase of IL-1βcould trigger
other immunological and inflammatory cells to synthesize
proinflammatory cytokines including TNF-α, IL-6 et al,
causing subsequently inflammatory and immunological damage
(Dinarello, 1996; Dorhoi and Kaufmann, 2016). Although the
production of IL-1 and TNF-α, as well as other proinflammatory
cytokines is designed to be protective, if left unchecked, their
excessive or inappropriate production may lead to severe
inflammatory diseases (Beutler and Cerami, 1988; Dorhoi and
Kaufmann, 2014). Relevant to Mtb infection, IL-1-coated beads
are capable of inducing large granulomas in lung tissue (Kasahara
et al., 1988). Furthermore, production of elevated TNF-αcould
cause severe inflammation in vital organs (such as lungs and
spleen) and leading to early death (Bekker et al., 2000). It has
Frontiers in Microbiology | www.frontiersin.org 2November 2017 | Volume 8 | Article 2142
Zhang et al. Baicalin-Inducing Autophagy against Mycobacterium tuberculosis
been shown that Mtb activates NLRP3 inflammasome and results
in the production of mature IL-1βin infected macrophages
(Mishra et al., 2010; Wong and Jacobs, 2011; Dorhoi et al.,
2012). The inflammasome is a macromolecular protein complex
consisting of at least three components: a NLR (NOD-like
receptor) protein such as NLRP3, apoptosis-associated speck-
like protein containing a caspase recruitment domain (ASC), and
pro-caspase-1. Upon activation by agonists, the inflammasome
processes pro-IL-1βinto a mature, biologically active IL-1βfor
secretion extracellularly (Lamkanfi and Dixit, 2014; He et al.,
2016). The NLRP3 inflammasome can be regulated by NF-κB
pathway where synthesis of NLRP3 and pro-IL-1βprovides the
first signals for inflammasome activation (Ghonime et al., 2014;
Patel et al., 2017). Interestingly, a number of consistent reports
have unequivocally indicated that autophagy plays a negative
role in the process of inflammation by inhibiting the releasing
of IL-1β(Lupfer et al., 2013; Martins et al., 2015; Saitoh and
Akira, 2016). Loss of autophagy (ATG16L1 deficiency) increases
IL-1βlevels which aggravates the degree of inflammation in a
mouse gut inflammation model (Saitoh et al., 2008). Autophagy
inhibits the production of IL-1βindirectly, by lowering the
endogenous stimuli of inflammasome activation (Nakahira et al.,
2011; Zhou et al., 2011) and may also directly, via autophagic
degradation of inflammasome components (Harris et al., 2011;
Shi et al., 2012). Consistently, there are studies which have
shown that autophagy protects from excessive inflammation
during Mtb infection (Castillo et al., 2012) and protects against
Mtb pathogenesis in vivo (Castillo et al., 2012; Watson et al.,
2012). Thus, autophagy plays an important role in fighting
against TB by direct killing the pathogen while preventing
excessive inflammatory injury as well. In fact, as an adjunctive
therapy, it has been demonstrated that autophagy contributes
to the efficacy of frontline anti-tuberculosis chemotherapeutics,
such as isoniazid and pyrazinamide (Kim et al., 2012). Thus,
pharmaceutical manipulation on autophagy could be potentially
useful as new strategies in anti-tuberculosis chemotherapeutics.
Baicalin is a flavonoid isolated from the extracts of dried
roots of Scutellaria baicalensis Georgi (Huang Qin), a plant that
belongs to the labiatae family, and its chemical structure has
been verified (de Oliveira et al., 2015). Baicalin possesses many
biological activities such as antibacterial, anti-inflammatory, anti-
allergic, anti-spasmodic, and anti-cancer (Srinivas, 2010; Yu
et al., 2013). Furthermore, baicalin could induce autophay in
cancer (Zhang et al., 2012; Lin et al., 2013) causing subsequent
autophagic tumor cell death. The molecular mechanisms of
bacalin-induced autophagy in cancer cells involves blocking of
the Akt signaling (Lin et al., 2013) and downregulation of CD147
(Zhang et al., 2012). The present study was carried out to
investigate the mechanism of the immunological protective effect
of baicalin in macrophages infected with Mtb.
MATERIALS AND METHODS
Mice and Reagents
Female C57BL/6 J mice (4–8 weeks of age, weight 20 ±3 g)
were obtained from Vital River Laboratory Animal Technology
Co., Ltd. (Beijing, China). All mice were acclimated for at least
1 week before the experiments and housed in a pathogen-
free facility. Animal experiments were carried out in strict
accordance with the National Institute of Health Guide for
the Care and Use of Laboratory Animals, with the approval
of the Scientific Investigation Board of Shanghai University of
Traditional Chinese Medicine (Shanghai, China). DMSO, bovine
serum albumin (BSA) and OPTI-MEM medium were purchased
from Sigma (St. Louis, MO). RIPA lysis buffer, BCA Protein Assay
Kit and Protein A/G agarose/sepharose beads were obtained
from the Beyotime Institute of Biotechnology (Shanghai, China).
The following antibodies were used: anti-NLRP3 (cat. #15101),
anti-IL-1β(cat. #12507), anti-ASC (cat. #67824), anti-LC3
(cat. #2775), anti-p62 (cat. #5114), anti-Akt (cat. #4691), anti-
phosphorylated Akt (Ser473) (cat. #4060), anti-mTOR (cat.
#2972), anti-phosphorylated mTOR (Ser2448) (cat. #5536), anti-
phosphorylated p38 (cat. #4511), anti-phosphorylated JNK (cat.
#4668), and anti-phosphorylated ERK1/2 (cat. #4370) were
purchased from Cell Signaling Technology, Inc. (CST, Danvers,
MA, USA); rabbit anti-caspase-1 (cat. #sc-514), goat anti-rabbit
(cat. #sc-2012), donkey anti-goat (cat. #sc-2094), and goat
anti-mouse LC3 (cat. #sc-16755) were purchased from Santa
Cruz Biotechnology, Inc. (Santa Cruz CA, USA); donkey anti-
rabbit IgG H&L antibody (conjugated with Alexa Fluor R
488;
ab150073) and donkey anti- goat IgG H&L antibody (conjugated
with Alexa Fluor R
647; ab150131) were acquired from Abcam
(Cambridge, UK); anti-β-actin (cat. #66009-1-lg) monoclonal
antibody was from ProteinTech Group (Chicago, IL). Baicalin
(Molecular Weight: 446.36, purity >98%) was purchased from
shanghai tauto biotech co., LTD. (shanghai, China). Dulbecco’s
Modified Eagle’s Medium (DMEM) was obtained from HyClone
FIGURE 1 | Effect of baicalin on the viability of Raw264.7. (A) The chemical
structure of baicalin; (B) Proliferation assay was conducted to assess the
cytotoxic effect of baicalin on Raw264.7. Data are shown as means ±SD of
three independent experiments.
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Zhang et al. Baicalin-Inducing Autophagy against Mycobacterium tuberculosis
Laboratories, Inc (Logan, UT, USA). Middlebrook 7H9 and 7H10
media were obtained from Difco (Detroit, MI, USA) and oleic
acid-albumin-dextrose-catalase (OADC) supplements were from
BD Biosciences (BD, Sparks, MD, USA).
Cell Culture
The Raw264.7 murine macrophage cell line was cultured in
DMEM supplemented with 10% fetal bovine serum (FBS) in 5%
CO2 at 37◦C. Thioglycolate-elicited mouse primary peritoneal
macrophages were prepared from female C57BL/6 J mice as
described previously (Jiang et al., 2017). After 2 h, non-adherent
cells were removed and the adherent cells were used as primary
peritoneal macrophages.
CCK-8 Assay for Cell Viability
Raw264.7 (1 ×104cells/100 µl) cells were seeded into 96-well
culture plates overnight at 37◦C and atmospheric conditions of
5% CO2. The culture medium was then replaced with medium
containing different concentrations of baicalin (0, 12.5, 25, 50,
100 µM) for 24, 48, 72 h. At the end of the culture, 10 µl of the
CCK-8 reagent was added to each well. After 1–2 h of incubation
at 37◦C, the absorbance was determined at 450 nm using a
Synergy 2 Microplate Reader (Bio-Tek, USA).
Bacterial Strains
The Mtb H37Ra was used in this study. H37Ra strain was
grown in Middlebrook 7H9 or 7H10 broth supplemented with
0.2% glycerol, 0.05% Tween-80, and 10% Middlebrook OADC
supplement.
Mtb Infection
The Raw264.7 or primary peritoneal macrophage cells were
seeded at various specifications of the cell culture plates and
grown at 37◦C overnight. The cells (1 ×106, 1 ×105, or
5×105) were infected with Mtb H37Ra (MOI =10). After
4 h of co-incubation at 37◦C, cells were washed three times
FIGURE 2 | Baicalin can promote the activation of autophagy in Mtb-infected macrophages. (A) Western blot analysis of LC3 I/II and p62 expression in Mtb-infected
Raw264.7 cells after treatment with different concentrations of baicalin (0, 12.5, 25, 50, 100 µM) or rapamycin (1 µg/ml) for 12 h. The right bar graphs show the
statistical results for the relative quantitative expression of LC3 II and p62. (B) Western blot analysis of LC3 I/II and p62 expression in Mtb-infected Raw264.7 cells.
After different times of Mtb infection (0, 4, 12, 24h), cells were treatment with or without baicalin (100 µM). The right bar graphs showed the statistical results for the
relative quantitative expression of LC3 II and p62. (C) Western blot analysis of LC3 I/II and p62 expression in Mtb-infected Raw264.7 cells after treatment with baicalin
(100 µM) or CQ (10, 20 µM) for 12 h. The right bar graphs showed the statistical results for the relative quantitative expression of LC3 II and p62 expression. Data are
shown with the means ±SD of at least three independent experiments. *p<0.05.
Frontiers in Microbiology | www.frontiersin.org 4November 2017 | Volume 8 | Article 2142
Zhang et al. Baicalin-Inducing Autophagy against Mycobacterium tuberculosis
with sterile phosphate-buffered saline (PBS) and cultured with
DMEM containing 10% FBS in the presence and absence of
different concentrations of baicalin (0, 12.5, 25, 50, or 100 µM)
for different times.
Colony Forming Unit (CFU)
Raw264.7 cells (5 ×105) were seeded in six-well plates, after
infection for 4 h (MOI =10), cells were washed with sterile PBS
and added new complete medium (10% FBS) into the plates in
the presence or absence of baicalin (100 µM). After 48 h, the
cells were ruptured (0.1% Triton X-100) to release intracellular
bacteria and diluted to appropriate dilutions with sterile PBS for
CFU count on 7H10 agar plates.
Western Blot
Cells were collected and lysed in lysis buffer, and then the
whole cell lysate was separated by SDS-PAGE and further
transferred onto nitrocellulose membranes. After blocking with
TBST (0.5% Tween-20) containing 5% (w/v) non-fat milk, the
membranes were incubated with specific primary antibodies
against NLRP3, IL-1β, caspase-1, ASC, mTOR, p-mTOR, Akt, p-
Akt, p-p38, p-JNK, p-ERK, or p-p65 at 4◦C overnight in blocking
solution, all antibodies were diluted at 1:1,000. Following three
times of washed with TBST, the membranes were incubated
with HRP-conjugated secondary antibodies at room temperature
for 1 h. The chemiluminescence was detected using the ECL-
chemiluminescent kit (Thermo Scientific) with Protein Simple
(USA).
Co-immunoprecipitation
Raw264.7 cells were lysed at 4◦C in ice-cold cell lysis buffer
and cell lysates were cleared by centrifugation (12,000 g, 10 min).
Concentrations of proteins in the supernatant were determined
FIGURE 3 | Inhibitory effect of baicalin on bacillary loads. Bacterial CFUs were
counted after baicalin treatment for 48 h. **p<0.01, Data are shown as mean
±SD of three independent experiments.
by bicinchoninic acid (BCA) assay. Before immunoprecipitation,
samples containing equal amounts of proteins were pre-cleared
with various irrelevant IgG or specific antibodies (2–5 mg/ml)
overnight at 4◦C with gentle rotation and subsequently incubated
with Protein A/G agarose/sepharose beads at 4◦C with gentle
rotation. Following 3 h incubation, agarose/sepharose beads were
washed extensively with PBS for four times and proteins were
eluted by boiling in 1×SDS sample buffer before SDS-PAGE
electrophoresis.
Measurement of Mature IL-1β
Raw264.7 cells (1 ×106) were cultured in six-well plates which
infected with H37Ra for 4 h. Then washed twice with sterile
PBS and replaced with 1 ml OPTI-MEM medium containing
different concentration of baicalin (0, 12.5, 25, 50, or 100 µM).
After 12 h, the supernatant of each well were concentrated
according to the literature and with slightly modification (Shi
et al., 2012). Removing 0.8 ml of the medium collected from each
well and mixed with 0.8 ml methanol and 0.2 ml chloroform,
FIGURE 4 | Baicalin inhibits the aggregation of ASC. (A) Immunofluorescence
assay was performed and rabbit anti-ASC (green) and DAPI (blue) were used
for immunostaining. Images were obtained with laser scanning confocal
microscopy. (B) Quantification of the confocal images about ASC. The
confocal images about ASC were quantified with ZEN 2011 Blue Version. Data
are shown with means ±SD. *p<0.05.
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Zhang et al. Baicalin-Inducing Autophagy against Mycobacterium tuberculosis
vortexed, and centrifuged at 12,000 g for 5 min. The upper phase
from each sample was removed and 0.8 ml methanol added.
The samples were centrifuged again for 5 min at 12,000 g to
remove the supernatant, and the pellet was placed in room
temperature for 10 min to volatilize the methanol. Seventy
milliliters of 1×loading buffer was added to each sample followed
by boiling for 10 min prior to SDS-PAGE and immunoblotting
with antibodies to detect mature IL-1β(AB-400-NA, R&D
Systems). The adherent cells from each well were lysed with the
above-mentioned lysis buffer and quantified before immunoblot
to determine the cellular content of the different proteins.
Immunofluorescence
Following the appropriate treatment, the cells were washed twice
with PBS, fixed with 4% paraformaldehyde at room temperature
for 10 min, and washed again with PBS. The cells were treated
with penetrating reagents (0.2% of BSA, 2% of Triton X-100)
for 10 min at 4◦C and washed again with PBS, and then the
cells were blocked with 5% bovine serum albumin for 30 min
at room temperature. Rabbit anti-ASC, anti-LC3, anti-caspase-
1, and anti-pp65 antibodies were used for immunofluorescence.
Donkey anti-mouse IgG and goat anti-rabbit FITC conjugated
antibodies were used as secondary antibodies. The nuclei
were stained with DAPI at the concentration of 1 µg/ml for
10 min. In this experiment, confocal microscopy (LSM 880,
Zeiss optics international trading co., LTD) was used for
examination.
Statistical Analysis
Statistical analysis was performed by using SPSS 18.0 software
(SPSS, Inc., Chicago, IL, USA). P-values were assessed by one-
way analysis of variance (ANOVA), results were given as means
±standard deviations (SD). Data shown are representative of at
least triplicate experiments. A value of p<0.05 was considered
to be statistically significant.
RESULTS
Effect of Baicalin on the Viability of
Raw264.7
To optimize the concentration of baicalin, the cell viability assay
was conducted to evaluate potential drug-induced toxicity. The
proliferation of Raw264.7 cells was tested using the CCK-8 kit.
As shown in Figure 1, baicalin (within 100 µM) did not affect
the viability of Raw264.7 during observation periods (24, 48, or
72 h). Thus, the concentrations of baicalin within 100 µM were
considered as safe for cells and could be used for the subsequent
studies.
Baicalin Induces Autophagy in Mtb
Infected Macrophages
Mtb is an intracellular pathogen which can proliferate within
infected macrophages by preventing the maturation of the
phagosome where the bacteria reside. Autophagy represents
a recognized cell-autonomous defense against intracellular
pathogens that can employ various mechanisms for elimination
of invasive Mtb (Gutierrez et al., 2004; Singh et al., 2006;
Ponpuak et al., 2010). To assess the effect of baicalin on
autophagy induction, we examined the expression of LC3 II
and p62 (the biomarker of autophagosome) after treatment
FIGURE 5 | Baicalin evidently inhibits the Mtb-induced NLRP3 inflammasome
activation. (A) Levels of NLRP3, ASC and pro-caspase-1 expression in cell
lysates and the mature IL-1βin the supernatant were determined by
immunoblot. (B) ASC or NLRP3 immunoprecipitates from Raw264.7 cells
were immunoblotted for NLRP3 or ASC respectively, and re-blotted for ASC or
NLRP3 respectively. Experiment performed three times.
FIGURE 6 | Baicalin inhibits the expression of Mtb-induced NLRP3 in primary
peritoneal macrophage cells and chloroquine (CQ) prevented this effect. The
expression of NLRP3, LC3 I/II and p62 in Mtb-infected primary peritoneal
macrophage cells after treatment with different concentrations of baicalin or
CQ (10, 20 µM) was detected by Western blot. The concentration of baicalin
when used in combination with CQ was 100 µM.
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Zhang et al. Baicalin-Inducing Autophagy against Mycobacterium tuberculosis
with different concentrations of baicalin (0, 12.5, 25, 50,
or 100 µM). As shown in Figure 2A, baicalin induced the
activation of autophagy in a dose-dependent manner and the
concentration of 100 µM triggered most potent autophagy. The
autophagy activation induced by baicalin (100 µM) was time-
dependent (Figure 2B). To further validate the effect of baicalin
on autophagy, we next evaluated the autophagic flux utilized
with an autophagy inhibitor, chloroquine (CQ). As shown
in Figure 2C, either 10 or 20 µM of CQ caused remarkable
accumulation of LC3 and p62 compared to baicalin treatment.
These data demonstrated that baicalin indeed induced the
activation of autophagy in a concentration- and time-dependent
manner.
Baicalin Has a Significant Killing Effect on
Mtb in Macrophages
Mtb, as a well-known intracellular pathogen, has developed
several schemes (e.g., dampening the antimicrobial activity of
ROS and RNS, preventing the maturation of early phagosomes,
inhibiting the fusion of phagosome with lysosome, interrupting
autophagy process) to escape from the antimicrobial mechanisms
of macrophages and thus survive intracellularly (Awuh and Flo,
2017). Studies have shown that autophagy possesses the ability
to eliminate intracellular bacteria. Since we have confirmed the
induction effect of baicalin on autophagy (Figure 2), to assess
the antibacterial effect of baicalin on Mtb, we performed the
colony forming unit (CFU) counting assay. As shown in Figure 3,
FIGURE 7 | Bacalin suppresses the PI3K/Akt/mTOR pathway but has no effect on the MAPK signaling in Raw264.7 cells. (A) Western blot analysis of mTOR,
p-mTOR, Akt, and p-Akt expression in Raw264.7 cells. β-actin was used as a control. The right bar graphs show the statistical results for the relative quantitative
expression of p-Akt and p-mTOR. (B) Western blot analysis of p-JNK, p-ERK, and p-p38 expression in Raw264.7 cells. The right and below bar graphs show the
statistical results for the relative quantitative expression of p-p38, p-ERK, and p-JNK. Data are shown with the means ±SD of at least three independent experiments.
*p<0.05.
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Zhang et al. Baicalin-Inducing Autophagy against Mycobacterium tuberculosis
baicalin exerted an obvious antibacterial effect (the bactericide
rate of baicalin reached 86.7% compared to untreated control)
and this ability most likely relied on the induction of autophagy
that further facilitated the killing of Mtb, as baicalin had no
bactericidal effect on within 2.24 mM (data not shown).
Baicalin Suppresses the Activation of
NLRP3 Inflammasome
Activation of inflammasome is an important post-transcriptional
event to facilitate IL-1βrelease. Uncontrolled activation of
inflammasome is associated with several inflammatory diseases,
including TB (Wong and Jacobs, 2011; Mishra et al., 2013).
NLRP3 inflammasome has been reported to contribute to the
inflammatory tissue damage during mycobacterial infection
(Kasahara et al., 1988; Mishra et al., 2013). Consistently,
our immunofluorescence assay (Figure 4) indicated that Mtb
triggered the accumulation of ASC which is essential for the
activation of NLRP3 inflammasome or AIM2 inflammasome
(Bryan et al., 2009; Mishra et al., 2010). We found that AIM2 was
unchanged in Mtb-infected Raw264.7 regardless of the presence
or absence of baicalin (data not shown). Moreover, Western blot
showed that Mtb induced the increase of NLRP3, ASC, pro-
caspase-1 which are the components of NLRP3 inflammasome
FIGURE 8 | Bacalin suppresses the PI3K/Akt/mTOR pathway but has no effect on the MAPK signaling in primary peritoneal macrophage cells. (A) Western blot
analysis of mTOR, p-mTOR, Akt, and p-Akt expression in primary peritoneal macrophage cells. β-actin was used as a control. The right bar graphs show the
statistical results for the relative quantitative expression of p-Akt and p-mTOR. (B) Western blot analysis of p-JNK, p-ERK, and p-p38 expression in peritoneal
macrophage cells. The right and below bar graphs show the statistical results for the relative quantitative expression of p-p38, p-ERK, and p-JNK. Data are shown
with the means ±SD of at least three independent experiments. *p<0.05.
Frontiers in Microbiology | www.frontiersin.org 8November 2017 | Volume 8 | Article 2142
Zhang et al. Baicalin-Inducing Autophagy against Mycobacterium tuberculosis
(Figure 5A). On the contrary, baicalin treatment could either
inhibit the formation of ASC specks in immunofluorescence
(Figure 4) or decrease the NLRP3, ASC, pro-caspase-1 in a dose-
dependent manner (Figure 5A). In addition, the inflammasome-
derived IL-1βin the supernatant in Mtb infected macrophages
was inhibited by baicalin. Furthermore, co-immunoprecipitation
(Co-IP) showed that baicalin treatment restrained the interaction
of NLRP3 with ASC (Figure 5B). Besides, in primary peritoneal
macrophages, baicalin also showed inhibitory effect on the
expression of NLRP3 and this inhibition ability was remarkably
weakened when autophagy activity was blocked by CQ
(Figure 6). ESAT-6 has been demonstrated the essential element
to activate NLRP3 inflammasome (Mishra et al., 2010), although
intracellular concentrations of ESAT-6 are similar in both H37Rv
and H37Ra, the H37Ra remains defective for ESAT-6 secretion as
the phoP gene mutation (Fortune et al., 2005). We speculate the
potential mechanism on the activation of H37Ra-induced NLRP3
inflammasome that may be the case that in vitro cultures of the
strain with some lysed bacteria may release ESAT-6 and therefore
activate the inflammasome as observed in Figures 4–6. Together,
our data indicated that baicalin could suppress the Mtb-induced
NLRP3 inflammasome activation.
Baicalin Activates Autophagy by Inhibiting
PI3K/Akt/mTOR Signaling Pathway Instead
of MAPK Pathway
The activation of autophagy can be modulated by a wide
range of signals. Inhibition of PI3K/Akt/mTOR and activation
of MAPK pathway are known to induce autophagy activation.
Then, we analyzed the effect of baicalin on these two signaling
pathways. Results indicated that baicalin treatment inhibited
the phosphorylation of Akt (Ser473) and mTOR (Ser2448)
in a time-dependent manner both in Raw264.7 (Figure 7A)
and peritoneal macrophages (Figure 8A). In addition, we also
assessed the MAPK pathway and our results showed that
baicalin had no effect on the phosphorylation of p38, JNK
or ERK (Figures 7B,8B). This is different with the reports
that H37Rv could cause the activation of MAPK signaling
pathway triggering secretion of inflammatory cytokines (Fietta
et al., 2002; Jung et al., 2006). This difference is probably
caused by the differences between the H37Ra and H37Rv
strains (Jena et al., 2013). Although the H37Ra we used
here is an attenuated strain which may cause lower toxic
inflammatory reaction to host, but in terms of basic mechanism
research, it is an irreplaceable good model for the study of
intracellular TB as reported (Hart and Armstrong, 1974). Thus,
our data revealed that baicalin inhibited the PI3K/Akt/mTOR
signaling pathway instead of MAPK pathway to activate
autophagy.
Baicalin Inhibits Mtb-Induced NF-κB
Activation
NF-κB plays essential roles in the activation of NLRP3
inflammasome (Ghonime et al., 2014; Lamkanfi and Dixit, 2014;
He et al., 2016) and the transcriptional induction of various
genes involved in inflammation. As shown in Figure 9, Mtb
FIGURE 9 | Baicalin inhibits Mtb-induced NF-κB activation. (A) Western blot
analysis of p-p65 expression in Raw264.7 cells. β-actin was used as a loading
control. The below bar graph shows the statistical result for the relative
quantitative expression of p-p65. (B) Western blot analysis of p-p65
expression in primary peritoneal macrophage cells. β-actin was used as a
loading control. The below bar graph shows the statistical result for the relative
quantitative expression of p-p65. Data are shown as the means ±SD of at
least three independent experiments. *p<0.05.
infection caused the activation of NF-κB signaling as evidenced
by the elevated p65 phosphorylation levels both in Raw264.7
(Figure 9A) and primary peritoneal macrophages (Figure 9B).
Confocal images indicated that Mtb increased nuclear entry of
phosphorylated p65, while baicalin treatment markedly reduced
the production of phosphorylated protein and prevented it from
entering into the nucleus (Figure 10).
Baicalin Can Induce Co-localization of
Autophagosome and Inflammasome
Autophagy has been shown to play an important role in
regulating inflammasome activation through the removal
of inflammasome-activating endogenous signals or the
sequestration and degradation of inflammasome components
(Harris et al., 2011; Shi et al., 2012). We evaluated the
relationship between them by means of immunofluorescence
using confocal laser scanning microscope. As shown in
Frontiers in Microbiology | www.frontiersin.org 9November 2017 | Volume 8 | Article 2142
Zhang et al. Baicalin-Inducing Autophagy against Mycobacterium tuberculosis
FIGURE 10 | Baicalin prevents the nuclear translocation of Mtb-induced NF-κB. Confocal microscopy of Raw264.7 cells with different treatments immunostained with
anti-pp65 (green) and DAPI (blue). Scale bars shown are 20 µm.
Figure 11, Mtb induced the accumulation of ASC protein
which represented the activation of NLRP3 inflammasome,
and baicalin treatment decreased the ASC specks. Meanwhile,
baicalin elevated the production of LC3 and induced the
co-localization of LC3 and ASC, which is representative of
autophagosome and inflammasome respectively. Thus, we
conclude that baicalin induced autophagy activation, inhibited
the activity of NLRP3 inflammasome through autophagic
degradative mechanism, and restrained the secretion of
IL-1β.
DISCUSSION
This study first reveals the roles of baicalin-mediated autophagy
inducing effect in protection against Mtb infection. Our work
demonstrated that baicalin inhibited the phosphorylation of
Akt/mTOR thereby inducing autophagy to kill intracellular Mtb
and showed suppressing effect on the activation of NLRP3
inflammasome and NF-κB signaling pathway triggered by Mtb
(as shown in Figure 12). Baicalin is a safe, effective and widely
available herb monomer which can be extracted from plants of
genus Scutellaria (grown in Asian countries including China)
or Oroxylum indicum (grown in other countries) (Dinda et al.,
2017). The extracts from the roots of Scutellaria baicalensis
are widely used in traditional Chinese medicines to treat
various diseases such as hepatitis, atherosclerosis, dysentery,
as well as common cold and other respiratory disorders (Li-
Weber, 2009). Consistent with our work, previous studies have
reported that baicalin was capable of inducing autophagy to
cause cancer cell autophagic death (Zhang et al., 2012; Lin
et al., 2013). Unlike the results of these publications and our
research, there a study reported that baicalin inhibited influenza
A virus induced autophagy (Zhu et al., 2015). As for TB, to
our knowledge, this is the first demonstration that baicalin has
antimycobacterial activity via induction of autophagy of host
macrophages.
Frontiers in Microbiology | www.frontiersin.org 10 November 2017 | Volume 8 | Article 2142
Zhang et al. Baicalin-Inducing Autophagy against Mycobacterium tuberculosis
FIGURE 11 | Baicalin promotes co-localization of inflammasome with autophagosome. Confocal microscopy of Raw264.7 cells with different treatments
immunostained with anti-LC3 antibody (pink), anti-ASC antibody (green), and DAPI (blue). Scale bars shown are 5 µm.
Induction of autophagy in Mtb-infected macrophages
by several means (physiologically, immunologically, or
pharmacologically) has been shown to kill Mtb (Gutierrez
et al., 2004). Subsequent studies have extended the initial
observation and established the key role of autophagy in
defense against TB (Deretic, 2008; Harris et al., 2009; Jo, 2013).
Consistent with these data, our results demonstrated that
baicalin stimulated the activation of autophagy as evidenced
by the upregulation of LC3II and downregulation of p62
and the unobstructed autophagic flux process after baicalin
treatment. In addition, baicalin caused inhibition of intracellular
Mtb replication. We attributed this killing effect to baicalin-
stimulated autophagy effect because baicalin showed no direct
impact on Mtb proliferation at 300 µM in our in vitro study.
Autophagy has anti-inflammatory activity and protects the host
from tissue necrosis and lung pathology (Castillo et al., 2012).
The existing reports agree that autophagy negatively regulating
inflammasome activation through a variety of mechanisms
(Saitoh et al., 2008; Harris et al., 2011; Nakahira et al., 2011;
Zhou et al., 2011; Shi et al., 2012; Lupfer et al., 2013; Martins
et al., 2015; Saitoh and Akira, 2016). Inflammasome has been
recognized to be involved in the pathological progress of
Mtb infection (Carlsson et al., 2010; Wong and Jacobs, 2011;
Castillo et al., 2012; Dorhoi et al., 2012; Mishra et al., 2013)
causing granulomatous lung lesions and systemic inflammatory
responses (Bekker et al., 2000). Although granulomas have long
been considered to benefit the host by containing and restricting
mycobacteria, recent studies have demonstrated that tuberculous
granuloma provides a safety shelter for bacterial growth,
persistence, and proliferation (Davis and Ramakrishnan, 2009;
Silva Miranda et al., 2012; Cambier et al., 2014) and even caused
lung damage (Bekker et al., 2000; Philips and Ernst, 2012).
Our data indicated that baicalin inhibited the Mtb-induced
inflammation process by restraining the NLRP3 inflammasome
activation and subsequent production of inflammatory mediators
triggered by Mtb infection. Complementing anti-TB drugs with
anti-inflammation interventions could improve treatment
efficiency and outcome evidenced by reports that patients
treated with corticosteroids in conjunction with TB drugs
contribute to a modest decrease in mortality and is helpful
in extrapulmonary tuberculosis including meningitis and
pleural disease (Critchley et al., 2013; Prasad et al., 2016).
Frontiers in Microbiology | www.frontiersin.org 11 November 2017 | Volume 8 | Article 2142
Zhang et al. Baicalin-Inducing Autophagy against Mycobacterium tuberculosis
FIGURE 12 | Schematic summary of baicalin-driven regulation of autophagy and inflammation during Mtb infection. Baicalin inhibits the phosphorylation of Akt, thus
inducing the activation of autophagy through PI3K/Akt/mTOR signaling pathways leading to bactericidal and inflammasome-inhibitory effect. Furthermore, baicalin
inhibits the PI3K/Akt/NF-κB pathway contributing to the inhibitory effect on NLRP3 inflammasome activation.
Nevertheless, considering the strong immunosuppressive effects
and many other side effects, caution is needed when applying
corticosteroids in pulmonary TB. In contrast, baicalin possess
the advantage over corticosteroids that has no side effects such as
immunosuppression. Moreover, baicalin has been demonstrated
to inhibit sterile (Wang et al., 2016) or bacterial inflammation
(Guo et al., 2013; Liu et al., 2017) and showed immunoprotective
effect in the sepsis model (Hu et al., 2015). Hence, baicalin could
be a new candidate for the development of adjunctive anti-TB
therapies.
Having confirmed the antimycobacterial and anti-
inflammatory effects of baicalin, we next explored the functional
mechanisms. Two classical autophagy related signaling pathways
were assessed, the PI3K/Akt/mTOR signaling pathway which
has been recognized as negatively regulating the activation of
autophagy (Heras-Sandoval et al., 2014), and the MAPK pathway
which acts as a positive regulator of autophagy (Krishna and
Narang, 2008; Zhou et al., 2015). Data indicated that baicalin
inhibited the phosphorylated Akt (Ser473) and mTOR (Ser2448)
but without influencing phosphorylated JNK, ERK, or p38.
Previous studies have suggested that baicalin showed evident
inhibitory effect on NF-κB activation (Guo et al., 2013; Fu
et al., 2016). Here, our data showed that baicalin significantly
inhibited the phosphorylation of NF-κB and prevented its entry
to the nucleus. Based on our findings, we conclude that baicalin
actually targeted PI3K/Akt to restrain the NF-κB activity, which
is consistent with previous studies (Kang et al., 2012; Guo et al.,
2016).
Taken together, our data demonstrated that baicalin has
both anti-inflammatory and antimycobacterial effects on Mtb-
infected macrophages and this endows baicalin the great
potential as a candidate of HDT for new anti-TB adjuvant
therapy. Additionally, better understanding the basic biology
of mycobacterial pathogenesis may guide the search for more
effective and specific HDT targets. Drugs like baicalin that
manipulate host cellular defense mechanisms such as autophagy
could achieve bactericidal and anti-inflammatory effects may
provide new opportunities to combat intracellular pathogens
Frontiers in Microbiology | www.frontiersin.org 12 November 2017 | Volume 8 | Article 2142
Zhang et al. Baicalin-Inducing Autophagy against Mycobacterium tuberculosis
like Mtb. Future studies are needed to evaluate baicalin in
conjunction with TB drugs for improved treatment of TB in
animal models and if promising in humans.
AUTHOR CONTRIBUTIONS
XJ and YiZ conceived and designed the experiments. QZ, JS, YW,
WH, and JW performed the experiments. XJ, YiZ, QZ, JS, YuZ,
and LW analyzed the data. QZ, JS, XJ, and YiZ wrote the paper.
All authors have read and approved the final manuscript.
ACKNOWLEDGMENTS
This work was supported by Innovation Program of Shanghai
Municipal Education Commission (15ZZ065), the National
Natural Science Foundation of China (81102869).
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Conflict of Interest Statement: The authors declare that the research was
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Copyright © 2017 Zhang, Sun, Wang, He, Wang, Zheng, Wu, Zhang and Jiang.
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