International Immunopharmacology 24 (2015) 72–79
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Baicalein pretreatment protects against liver ischemia/reperfusion injury via inhibition of NF-κB pathway in mice Anding Liu a,b, Liang Huang c, Hua Fan d, Haoshu Fang e, Yan Yang a, Shenpei Liu a, Jifa Hu a, Qi Hu f,⁎, Olaf Dirsch b, Uta Dahmen b a
Experimental Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Experimental Transplantation Surgery, Department of General, Visceral and Vascular Surgery, Friedrich-Schiller-University Jena, Jena, Germany Department of transfusion, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China d Department of Hepatobiliary Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing, China e Department of Pathophysiology, Anhui Medical University, Hefei, China f Department of Geriatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan,China b c
a r t i c l e
i n f o
Article history: Received 27 August 2014 Received in revised form 30 October 2014 Accepted 12 November 2014 Available online 22 November 2014 Keywords: Baicalein Ischemia/reperfusion Liver Inflammation Apoptosis
a b s t r a c t Ischemia/reperfusion (I/R) is a pathophysiologic process that occurs during hemorrhagic shock, liver resection and liver transplantation. Baicalein, the main active ingredient of the Scutellaria root, exerts anti-inflammatory and anti-apoptotic properties in the setting of I/R injury in the heart and brain. However, the role of baicalein in liver I/R injury and its regulatory mechanisms remain poorly understood. This study was designed to evaluate the effects of baicalein in a model of liver I/R in mice and to explore the possible mechanisms. Baicalein (100 mg/kg) was intraperitoneally injected 1 h before warm ischemia. Pretreatment with baicalein protected against liver I/R injury, as indicated by the decreased serum aminotransferase levels and the reduced histopathologic abnormalities. Baicalein also significantly reduced cellular hepatic apoptosis in response to I/R injury. Moreover, pretreatment with baicalein significantly inhibited nuclear factor-kappa B (NF-κB) activation and the subsequent proinflammatory cytokine production, and decreased leukocyte infiltration. In vitro studies, baicalein treatment inhibited the proinflammatory cytokine production via the modulation of NF-κB signaling pathway in lipopolysaccharide-stimulated macrophages. Taken together, these results suggest that baicalein could protect against liver I/R injury via inhibition of inflammation by down-regulating NF-κB activity, and suppression of cellular hepatic apoptosis. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Ischemia/reperfusion (I/R) injury is triggered when the liver is transiently deprived of oxygen and reoxygenated. It is a leading cause of liver injury after liver transplantation, liver resection and trauma [1,2]. I/R injury is a complex phenomenon leading to an injurious inflammatory response, which is characterized by the activation of resident Kupffer cells and the infiltration of leukocytes, as well as the production of inflammatory cytokines [3]. Activated immune cells (e.g., Kupffer cells) release a large amount of inflammatory mediators, including reactive oxygen species (ROS), tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-1. These inflammatory mediators result in a direct cellular damage, and indirectly lead to the migration and accumulation of neutrophils in the liver via inducing the expression of chemokines and ⁎ Corresponding author at: Department of Geriatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, No.1095 Jiefang Avenue, Wuhan 430030, China. Tel./fax: +86 27 8366 3306/3659. E-mail address: [email protected] (Q. Hu).
http://dx.doi.org/10.1016/j.intimp.2014.11.014 1567-5769/© 2014 Elsevier B.V. All rights reserved.
adhesion molecules [4,5]. Activated neutrophils result in additional, prolonged injury via the release of oxidants and proteases [6]. Baicalein (5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one) is a main active ingredient derived from the dried root of Scutellaria, which is a popular herb in traditional Chinese medicine. Baicalein shows a variety of biological activities, including anti-inflammatory [7] , anti-apoptotic [8], anti-oxidant [9], anti-thrombotic [10], and anticancer [11] properties. Previous investigations have shown that baicalein acts as an anti-inflammation agent, inhibits the lipopolysaccharide (LPS)-induced inflammation, improves the vasoreactivity and the survival rate, as well as reverses the organ injury in septic animals [12,13]. Baicalein also exerts a cytoprotective role in H2O2-induced apoptosis by inhibiting the mitochondria-dependent caspase activation [8]. Recent studies have demonstrated that baicalein is able to decrease I/R injury in the brain and heart [14,15]. Moreover, baicalein protects animals from D-galactosamine(GaIN)/LPS induced acute liver failure [16] and carbon tetrachloride (CCl4)-induced liver damage [17] via inhibition of inflammation and apoptosis in murine models. However, its impact on liver I/R injury and its molecular mechanisms remain unclear.
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Prompted by the close associations between the pathological features of liver I/R and the protective properties of baicalein, we hypothesized that baicalein could protect against hepatic I/R injury. To test this hypothesis, we investigated the beneficial effects of baicalein, as well as its plausible signaling mechanisms in a mouse model of liver I/R.
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2.4. Liver and kidney damage assessment Serum alanine transaminase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine levels were measured using an automated chemical analyzer (Hitachi Co, Tokyo, Japan). 2.5. Histopathology
2. Materials and methods 2.1. Experimental design Experimental design was described in Fig. 1. To investigate whether baicalein could protect against liver I/R injury, mice were pretreated with either baicalein (100 mg/kg, i.p. Sigma-Aldrich, St. Louis, MO) or vehicle (dimethyl sulfoxide, DMSO, Sigma-Aldrich) 1 h prior to warm ischemia. Mice were killed at 1, 6 and 24 h of reperfusion. Liver injury, inflammatory cytokine, neutrophil infiltration, apoptosis and nuclear factor-kappa B (NF-κB) activation were analyzed. To investigate the possible mechanism of the anti-inflammatory effects of baicalein, murine macrophage cell line RAW264.7 and primary murine peritoneal macrophages were used in vitro cell culture studies. RAW264.7 cells and peritoneal macrophages were pretreated with various doses of baicalein (1–10 μm) or vehicle (DMSO) for 1 h and then exposed to LPS (Escherichia coli serotype O55:B05 type, Sigma-Aldrich, 10 ng/mL) for different periods. The production of inflammatory cytokine and the activation of NF-κB were analyzed.
Liver tissue was fixed in 4.5% buffered formalin for at least 24 h. Paraffin embedding was performed using standard techniques. Sections (4 μm) were stained with Hematoxylin–Eosin and assessed for tissue damage. 2.6. Myeloperoxidase (MPO) immunohistochemistry The presence of neutrophils in the liver was assessed by myeloperoxidase (MPO) staining. Briefly, after de-paraffinization, rehydration and antigen retrieval, the sections were incubated with MPO antibody (1:50; Abcam, Cambridge, UK) for 1 h at room temperature. After washing, the sections were incubated with goat anti-rabbit secondary antibody and visualized with diaminobenzidine. MPO-positive neutrophils were counted in 5 high-power fields (HPFs) per section at a magnification of 400 ×. The results are expressed as the mean number of MPO-positive neutrophils per HPF. 2.7. Caspase-3 activity assay
2.2. Animals Male inbred C57BL/6 mice (8–10 weeks old, weighing within 20– 22 g) were purchased from Wuhan university Center for Animal Experiment (Wuhan, China). All animals were housed under standard animal care conditions and had free access to water and food. All procedures were carried out according to the ethical guidelines of the Animal Care and Use Committee of Huazhong University of Science and Technology. 2.3. Partial hepatic warm I/R The partial hepatic warm I/R model was generated as described previously [18]. In brief, mice were completely anesthetized with pentobarbital (60 mg/kg, i.p.). After opening the abdomen and dissecting the interlobular ligaments, a microvascular clamp was used to interrupt the arterial and portal venous blood supply to the left lateral and median liver lobes for 60 min.
Relative caspase-3 activity in the liver tissues was detected with a caspase-3 colorimetric assay kit (Abcam) according to the manufacturer's instructions. 2.8. RAW264.7 and peritoneal macrophage cell culture The murine macrophage cell line RAW264.7 was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Peritoneal macrophages were isolated as described previously [19]. Cells were plated in 24-well plates at a density of 5 × 105 cells/ well for cytokine assay or in 6-well plates at a density of 2 × 106 cells/ well for Western blotting. 2.9. Assessment of cell viability RAW264.7 cells were cultured in a 96-well plate at a density of 5 × 103 cells/well. Following cultured with baicalein at different doses
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Fig. 1. Schematic diagram of the experimental protocol.
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Fig. 2. In vivo toxicity profile of baicalein. Mice were treated with either baicalein at doses of 10, 50 or 100 mg/kg or vehicle (DMSO), liver and renal functions were assessed after the injections. (A) The serum ALT and AST levels were analyzed as a measure of hepatocellular injury. (B) The serum BUN and creatinine levels were analyzed as a measure of renal damage. The data are shown as mean ± SD. n = 4 per group.
for 3 h, cells were treated with 20 mg/mL) and incubated for 4 h. The medium were then removed and the remaining MTT-formazan crystals were dissolved in 200 nm, using 200 μL of DMSO as blank.
2.13. Enzyme-linked immunosorbent assay (ELISA) The levels of TNF-α and IL-6 in serum and cultured medium were analyzed using commercially available ELISA kits (R&D Systems, Minneapolis, MN).
2.10. Isolation of nuclear and cytoplasmic protein Nuclear and cytoplasmic extraction kit was bought from Pierce (Thermo Pierce, Rockford, IL). Cytoplasmic and nuclear protein from liver tissues or RAW264.7 cells was isolated according to the instructions of the manufacturer.
2.11. NF-κB activity assay NF-κB activity was measured using nuclear extracts from liver tissues or RAW264.7 cells. Activation of NF-κB was quantified using the TransAMNF-κB assay kits (Active Motif, Carlsbad, CA) according to the producer's instructions.
2.12. Gel electrophoresis and Western blotting Western blotting was performed as described previously [19]. Membranes were incubated overnight with primary rabbit anti-cleaved caspase-3 antibody (1:1000, Cell Signaling Technology, Beverly, MA), rabbit anti-cleaved caspase-7 antibody (1:1000, Cell Signaling Technology), rabbit anti-NF-κB (p65) antibody (1:1000, Cell Signaling Technology), rabbit anti-IκBα antibody (1:1000, Cell Signaling Technology), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:20,000; Sigma-Aldrich), and rabbit lamin B1 antibody (1:1000, Abcam). The membranes were developed with ECL Western blotting substrate (GE Healthcare, Buckinghamshire, UK) and Western blots were visualized on the Kodak Image Station (Carestream Health Inc, Rochester, NY).
2.14. Quantitative polymerase chain reaction (PCR) Real time PCR for TNF-α and IL-6 was performed as described previously [20]. The sequences of primers are as follows: TNF-α forward: 5′TGCTGGGAAGCCTAAAAGG-3′; reverse: 5′-CGAATTTTGAGAAGATGATC CTG-3′, IL-6 forward: 5′-TCAATTCCAGAAACCGCTATGA-3′ reverse: 5′CACCAGCATCAGTCCCAAGA-3, and ß-actin forward: 5′-AGAGGGAAAT CGTGCGTGAC-3′; reverse: 5′-CAATAGTGATGACCTGGCCGT-3′. 2.15. Statistical analysis The data are expressed as means ± SD. Differences between groups were evaluated for significance using a one-way ANOVA combined with Bonferroni's post hoc test. All tests were performed using SigmaStat v3.5 (Systat-Software, Erkrath, Germany). A p-value below 0.05 was considered to indicate statistical significance. 3. Results 3.1. Baicalein pretreatment protects against liver I/R injury The serum ALT, AST, BUN and creatinine levels were measured at 24 h and 7 day after baicalein injection to evaluate liver and kidney function, respectively. As shown in Fig. 2, baicalein doses of up to 100 mg/kg did not alter liver and kidney function compared to vehicle. The histologic examination of liver and kidney sections did not show histopathologic abnormalities after baicalein injection (data not shown).
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Cleaved Caspase3 Cleaved Caspase7 GAPDH Fig. 3. Baicalein attenuates liver injury and hepatic apoptosis. Mice were treated with either baicalein (100 mg/kg) or vehicle (DMSO) 1 h prior to ischemia. (A) The ALT and AST levels were analyzed as a measure of hepatocellular injury. (B) Routine histopathology was performed on formalin-fixed liver sections obtained from mice that had undergone 60 min of ischemia followed by 6 h of reperfusion (Original magnification ×400). (C) The caspase-3 activity was measured after 60 min of ischemia and 6 h of reperfusion. (D) The cleaved caspase-3 and cleaved caspase-7 cleavage were measured by Western blotting analysis. The data are shown as mean ± SD. n = 6 per group. *p b 0.05 compared to the vehicle-treated I/R group.
As shown in Fig. 3A, liver warm I/R resulted in significant increases in serum levels of ALT and AST. However, baicalein treatment significantly decreased the serum ALT and AST levels when compared to the vehicle control group. The liver histologic examination showed tissue injuries after I/R injury, including severe hepatocellular necrosis and cytoplasmic vacuolization of hepatocytes. In contrast, minimal damage was noted in baicalein-treated mice (Fig. 3B).
proinflammatory cytokines. Similarly, the serum levels of TNF-α and IL-6 were significantly lower in the baicalein-treated animals compared to the vehicle controls (Fig. 4A). The presence of neutrophils in the livers was assessed by MPO staining. After 60 min of warm ischemia and 6 h of reperfusion, the numbers of MPO-positive stained neutrophils were increased following I/R injury. The neutrophil infiltration was significantly less in the baicalein-treated animals than in the vehicle-injected animals (Fig. 4B).
3.2. Baicalein pretreatment decreases hepatic apoptosis following I/R injury
3.4. Baicalein modulates the NF-κB signaling pathway following I/R injury
As shown in Fig. 3C, after 60 min of warm ischemia and 6 h of reperfusion, the caspase-3 activity was significantly increased in the vehicletreated animals. However the caspase-3 activity in the baicalein-treated mice was markedly decreased compared to the vehicle control group. Furthermore, baicalein treatment significantly decreased the expression of cleaved caspase-3 and cleaved caspase-7 following I/R injury compared to the vehicle control group (Fig. 3D).
We then investigated whether baicalein pretreatment could affect the activation of NF-κB in the livers following I/R injury. As shown in Fig. 5A, compared to the sham operated animals, liver I/R in the vehicle-treated animals resulted in an increase of NF-κB in the nucleus and a decrease of IκBα in the cytoplasm. However, baicalein pretreatment significantly inhibited the increased levels of nuclear NK-κB and the decreased levels of cytoplasmic IκBα. The NF-κB activity was significantly inhibited in the baicalein-treated animals in response to I/R injury compared to the vehicle-injected animals (Fig. 5B).
3.3. Baicalein pretreatment decreases the production of inflammatory mediators and the infiltration of neutrophils following I/R injury Liver I/R in the vehicle-treated animals resulted in significant increases in the expression levels of TNF-α and IL-6 after reperfusion when compared to the sham operated animals. However, the baicalein-treated animals inhibited the increased expression of these
3.5. Baicalein pretreatment suppresses the LPS-induced proinflammatory cytokine production in macrophages As shown in Fig. 6A, the production of TNF-α and IL-6 was significantly increased in cultured medium of RAW264.7 cells after
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Fig. 4. Baicalein attenuates I/R induced-proinflammatory cytokine production and neutrophil infiltration. (A) The hepatic TNF-α and IL-6 mRNA expression levels were measured by quantitative PCR after 60 min of ischemia and 6 h of reperfusion. (B) The serum TNF-α and IL-6 levels were assessed by ELISA. (C) MPO staining was performed on the liver sections from mice at 6 h after reperfusion (Original magnification ×400). The numbers of MPO-positive neutrophils that infiltrated the livers were determined. Representative images from 6 mice/group were selected. The data are shown as mean ± SD. n = 6 per group. *p b 0.01 compared to the vehicle-treated I/R group.
stimulated with LPS. However, baicalein pretreatment showed a dose-dependent reduction of LPS-stimulated TNF-α and IL-6 production. To further investigate the anti-inflammatory effects of baicalein, peritoneal macrophages were isolated and pretreated with baicalein prior to LPS stimulation. The levels of TNF-α and IL-6 in the cultured medium were increased dramatically after treatment with LPS for 3 h. However, baicalein pretreatment significantly blunted the production of TNF-α and IL-6 (Fig. 6B). To exclude any possible interference on cell viability, the RAW264.7 cells were treated with the
escalating concentration of baicalein. As shown in Fig. 6C, baicalein concentrations of up to 10 μM did not affect the cell viability. 3.6. Baicalein pretreatment suppresses the activation of NK-κB by LPS in macrophages To investigate whether the anti-inflammatory effects of baicalein was mediated via inhibition of the NF-κB signaling pathway, we examined the effects of baicalein on the expression levels of IκBα and NF-κB.
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Fig. 5. Baicalein inhibits the activation of NF-κB following I/R injury. Mice were subjected to 60 min of ischemia after pretreatment with either baicalein (100 mg/kg) or vehicle (DMSO). Liver samples were harvested at 1 h of reperfusion. (A) The cytoplasmic IκBα and the nuclear NF-κB (p65) were measured by Western blotting analysis. Representative blots are presented and GAPDH and lamin B1 are served as a loading control for the cytoplasm and nuclear fractions, respectively. (B) The NF-κB activation during I/R injury was assessed by ELISA. The data are shown as mean ± SD. n = 6 per group. *p b 0.01 compared to the vehicle-treated I/R group.
As shown in Fig. 6D, Western blotting analysis showed that the levels of cytoplasmic IκBα were decreased, and the levels of nuclear NF-κB were increased in cells stimulated with LPS. However, baicalein pretreatment significantly prevented the degradation of cytoplasmic IκBα and the increase of nuclear NF-κB. Furthermore, baicalein pretreatment significantly blocked the NF-κB activity induced by LPS (Fig. 6E). 4. Discussion In the current study, we documented that baicalein treatment attenuated liver I/R injury via inhibition of the NF-κB activation and the subsequent inflammatory response, and suppression of the cellular apoptosis. Additionally, we confirmed that baicalein inhibited the expression of inflammatory cytokines and the activation of NF-κB in LPSstimulated macrophages. To our knowledge, this study is the first to provide in vivo evidence of the potential therapeutic value of baicalein in liver I/R injury. Several pharmacologic chemicals exhibit protective effects on liver I/R injury in animal models. However, none of them have shown any benefits in clinical trials of liver transplantation and liver resection in part due to the lack of safety and effectivity [21]. We demonstrated that baicalein doses of up to 100 mg/kg did not alter liver and kidney function in vivo. In addition, the anti-inflammatory effects of baicalein were not resulted from the increased cell death of macrophages for reducing the proinflammatory cytokine production. Our results were supported by Zong et al. [22], who demonstrated that mice received feed with 0.05% baicalein (dose: 100 mg/kg/day baicalein) for 12 weeks didn't affect their body weight. Hsieh et al. showed that baicalein concentrations of up to 30 μM did not affect the viability of HMC-1 cell in vitro [23]. In fact, baicalein is one of the major flavonoids from the root of Scutellaria, which has been used extensively in traditional Asian medicine to treat a variety of medical conditions for hundreds of years. The inflammatory response following reperfusion plays an important role in the pathophysiology of hepatic I/R injury [4,24,25]. The higher levels of circulation proinflammatory cytokines, such as TNFα and IL-6, have been shown to correlate with the early postoperative rejections and the infections during human liver transplantation [26]. Moreover, the neutralization of proinflammatory cytokines, such as TNF-α and IL-1, reduces the liver injury and mortality in a rat model of hepatic I/R [25,27]. Thus, an anti-inflammatory strategy via modulating the exaggerated inflammatory response to protect against liver I/R has been demonstrated in numerous studies [3,28]. It has demonstrated that baicalein reduces the production of TNF-α, inducible nitric oxide synthase (iNOS) nitric oxide in LPS-stimulated macrophages, and effectively protects animals from endotoxin shock
[12,13]. Furthermore, pretreatment with baicalein prevents D-GalN/ LPS-induced liver damage [16] and CCl₄-induced acute liver injury [17] in part by reducing inflammatory cytokine production. Additionally, baicalein preconditioning attenuates the myocardial I/R injury via inhibition of inflammation [15], as demonstrated recently. In agreement with these studies, we demonstrated that baicalein treatment significantly inhibited the production of inflammatory cytokines and the infiltration of neutrophils, and led to the reduction of the liver injury after I/R. These data indicate that the protective effects conferred by baicalein may be partially via the inhibition of inflammation. The mechanism of the anti-inflammatory effects of baicalein involves its ability to inhibit NF-κB signaling pathway, which plays an important role in liver I/R injury [28]. NF-κB is activated upon I/R injury and regulates the production of proinflammatory cytokines/ chemokines [28]. The inhibition of NF-κB activation leads to a decrease of proinflammatory cytokine production and an amelioration of I/R-induced tissue injuries [29]. It has been reported that the inhibitory effects of baicalein on LPS-induced pro-inflammatory cytokine expression both in vitro and in vivo are mediated by blocking NF-κB activation [12,13,16]. In this study, we identified that baicalein could effectively disrupt the activation of the NF-κB singling pathway by showing its activity on inhibiting the degradation of IκBα and the nuclear translocation of NF-κB in the livers following I/R. To investigate the possible mechanism of the anti-inflammatory effects of baicalein, the murine macrophage cell line RAW264.7 and the primary murine peritoneal macrophages were used in vitro cell culture studies. Kupffer cells, the hepatic resident macrophages, are important for triggering the inflammatory response in liver I/R injury [3,30]. Here, we clearly demonstrated that baicalein pretreatment inhibited the production of proinflammatory cytokines in LPS-stimulated macrophages. Consistent with the finding in vivo, the LPS-induced IκBα degradation and the NF-κB nuclear translocation were inhibited by baicalein administration in LPS-stimulated RAW264.7 cells. These findings suggest that the modulation of the IκBα/NF-κB signaling pathway in macrophages accounts, at least in part, for the antiinflammatory and the protective effects of baicalein. Since recent studies suggest that baicalein also exhibits protective effects via the suppression of apoptosis [8,16,17], we observed apoptotic cell death following liver I/R injury. Our results demonstrated that baicalein treatment inhibited apoptotic cell death as indicated by the decreases of I/R-induced caspase protein cleavage and caspase-3 activity. This finding was partly supported by baicalein reducing plasma cytokines levels, such as TNF-α, ROS and nitric oxide, which can initiate the apoptotic cascade via the death receptor/caspase pathway [31]. Baicalein pretreatment exhibits significant anti-
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Fig. 6. Baicalein inhibits the activation of NF-κB in RAW264.7 cells activated by LPS. (A) RAW264.7 cells and (B) peritoneal macrophages were pretreated with various doses of baicalein (1–10 μM) or vehicle (DMSO) for 1 h and then exposed to LPS (10 ng/mL) for 3 h. The TNF-α and IL-6 concentrations in the cultured medium were measured by ELISA. (C) The cell viability was determined by MTT assay in RAW264.7 cells. (D) The cytoplasmic IκB and the nuclear NF-κB (p65) were measured in RAW264.7 cells stimulated with LPS (10 ng/mL) for 30 min after 1 h of pretreatment with baicalein (10 μM) or DMSO. Representative blots are presented and GAPDH and lamin B1 are served as a loading control for the cytoplasm and nuclear fractions, respectively. (E) The NF-κB activation was assessed by ELISA. The data are shown as mean ± SD. n = 3 per group. *p b 0.01 compared to the vehicle-treated group.
apoptotic protection against cardiomyocyte I/R injury via mitochondrial oxidant signaling [32]. Additionally, the molecular mechanisms underlying baicalein inhibition of D-GalN/LPS-induced apoptosis are associated with the protection of mitochondria, increasing the Bcl-2/ Bax ratio and blocking the release of cytochrome c [16]. In conclusion, we found that baicalein possessed strong beneficial effects against liver I/R injury in a mouse model. Mechanistically, the action of baicalein involves the attenuation of NF-κB activation and the
subsequent expression of inflammatory cytokine, and suppression of apoptosis.
Acknowledgments This project was supported by grants from the National Natural Science Fund of China (NSFC) (81102689, 81300343) and the Specialized
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[16] Wu YL, Lian LH, Wan Y, Nan JX. Baicalein inhibits nuclear factor-kappaB and apoptosis via c-FLIP and MAPK in D-GalN/LPS induced acute liver failure in murine models. Chem Biol Interact 2010;188:526–34. [17] Huang HL, Wang YJ, Zhang QY, Liu B, Wang FY, Li JJ, et al. Hepatoprotective effects of baicalein against CCl(4)-induced acute liver injury in mice. World J Gastroenterol 2012;18:6605–13. [18] Liu A, Dirsch O, Fang H, Sun J, Jin H, Dong W, et al. HMGB1 in ischemic and nonischemic liver after selective warm ischemia/reperfusion in rat. Histochem Cell Biol 2011;135:443–52. [19] Liu A, Fang H, Dirsch O, Jin H, Dahmen U. Oxidation of HMGB1 causes attenuation of its pro-inflammatory activity and occurs during liver ischemia and reperfusion. PLoS One 2012;7:e35379. [20] Liu A, Fang H, Yang Y, Sun J, Fan H, Liu S, et al. The fibrin-derived peptide bbeta15-42 attenuates liver damage in a rat model of liver ischemia/reperfusion injury. Shock 2013;39:397–403. [21] Gurusamy KS, Gonzalez HD, Davidson BR. Current protective strategies in liver surgery. World J Gastroenterol 2010;16:6098–103. [22] Zong J, Zhang DP, Zhou H, Bian ZY, Deng W, Dai J, et al. Baicalein protects against cardiac hypertrophy through blocking MEK-ERK1/2 signaling. J Cell Biochem 2013;114: 1058–65. [23] Hsieh CJ, Hall K, Ha T, Li C, Krishnaswamy G, Chi DS. Baicalein inhibits IL-1beta- and TNF-alpha-induced inflammatory cytokine production from human mast cells via regulation of the NF-kappaB pathway. Clin Mol Allergy 2007;5:5. [24] Cardinal J, Pan P, Dhupar R, Ross M, Nakao A, Lotze M, et al. Cisplatin prevents high mobility group box 1 release and is protective in a murine model of hepatic ischemia/reperfusion injury. Hepatology 2009;50:565–74. [25] Wanner GA, Muller PE, Ertel W, Bauer M, Menger MD, Messmer K. Differential effect of anti-TNF-alpha antibody on proinflammatory cytokine release by Kupffer cells following liver ischemia and reperfusion. Shock 1999;11:391–5. [26] Hamilton G, Vogel S, Fuegger R, Gnant FX. Mechanisms of tumor necrosis factoralpha and interleukin-6 induction during human liver transplantation. Mediators Inflamm 1993;2:303–7. [27] Shito M, Wakabayashi G, Ueda M, Shimazu M, Shirasugi N, Endo M, et al. Interleukin 1 receptor blockade reduces tumor necrosis factor production, tissue injury, and mortality after hepatic ischemia–reperfusion in the rat. Transplantation 1997;63: 143–8. [28] Lentsch AB, Kato A, Yoshidome H, McMasters KM, Edwards MJ. Inflammatory mechanisms and therapeutic strategies for warm hepatic ischemia/reperfusion injury. Hepatology 2000;32:169–73. [29] Suetsugu H, Iimuro Y, Uehara T, Nishio T, Harada N, Yoshida M, et al. Nuclear factor {kappa}B inactivation in the rat liver ameliorates short term total warm ischaemia/ reperfusion injury. Gut 2005;54:835–42. [30] Caban A, Oczkowicz G, Abdel-Samad O, Cierpka L. Influence of Kupffer cells on the early phase of liver reperfusion. Transplant Proc 2002;34:694–7. [31] Malhi H, Gores GJ. Cellular and molecular mechanisms of liver injury. Gastroenterology 2008;134:1641–54. [32] Chang WT, Li J, Vanden Hoek MS, Zhu X, Li CQ, Huang HH, et al. Baicalein preconditioning protects cardiomyocytes from ischemia–reperfusion injury via mitochondrial oxidant signaling. Am J Chin Med 2013;41:315–31.
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Baicalein pretreatment protects against liver ischemia/reperfusion injury via inhibition of NF-κB pathway in mice Anding Liu a,b, Liang Huang c, Hua Fan d, Haoshu Fang e, Yan Yang a, Shenpei Liu a, Jifa Hu a, Qi Hu f,⁎, Olaf Dirsch b, Uta Dahmen b a
Experimental Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Experimental Transplantation Surgery, Department of General, Visceral and Vascular Surgery, Friedrich-Schiller-University Jena, Jena, Germany Department of transfusion, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China d Department of Hepatobiliary Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing, China e Department of Pathophysiology, Anhui Medical University, Hefei, China f Department of Geriatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan,China b c
a r t i c l e
i n f o
Article history: Received 27 August 2014 Received in revised form 30 October 2014 Accepted 12 November 2014 Available online 22 November 2014 Keywords: Baicalein Ischemia/reperfusion Liver Inflammation Apoptosis
a b s t r a c t Ischemia/reperfusion (I/R) is a pathophysiologic process that occurs during hemorrhagic shock, liver resection and liver transplantation. Baicalein, the main active ingredient of the Scutellaria root, exerts anti-inflammatory and anti-apoptotic properties in the setting of I/R injury in the heart and brain. However, the role of baicalein in liver I/R injury and its regulatory mechanisms remain poorly understood. This study was designed to evaluate the effects of baicalein in a model of liver I/R in mice and to explore the possible mechanisms. Baicalein (100 mg/kg) was intraperitoneally injected 1 h before warm ischemia. Pretreatment with baicalein protected against liver I/R injury, as indicated by the decreased serum aminotransferase levels and the reduced histopathologic abnormalities. Baicalein also significantly reduced cellular hepatic apoptosis in response to I/R injury. Moreover, pretreatment with baicalein significantly inhibited nuclear factor-kappa B (NF-κB) activation and the subsequent proinflammatory cytokine production, and decreased leukocyte infiltration. In vitro studies, baicalein treatment inhibited the proinflammatory cytokine production via the modulation of NF-κB signaling pathway in lipopolysaccharide-stimulated macrophages. Taken together, these results suggest that baicalein could protect against liver I/R injury via inhibition of inflammation by down-regulating NF-κB activity, and suppression of cellular hepatic apoptosis. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Ischemia/reperfusion (I/R) injury is triggered when the liver is transiently deprived of oxygen and reoxygenated. It is a leading cause of liver injury after liver transplantation, liver resection and trauma [1,2]. I/R injury is a complex phenomenon leading to an injurious inflammatory response, which is characterized by the activation of resident Kupffer cells and the infiltration of leukocytes, as well as the production of inflammatory cytokines [3]. Activated immune cells (e.g., Kupffer cells) release a large amount of inflammatory mediators, including reactive oxygen species (ROS), tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-1. These inflammatory mediators result in a direct cellular damage, and indirectly lead to the migration and accumulation of neutrophils in the liver via inducing the expression of chemokines and ⁎ Corresponding author at: Department of Geriatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, No.1095 Jiefang Avenue, Wuhan 430030, China. Tel./fax: +86 27 8366 3306/3659. E-mail address: [email protected] (Q. Hu).
http://dx.doi.org/10.1016/j.intimp.2014.11.014 1567-5769/© 2014 Elsevier B.V. All rights reserved.
adhesion molecules [4,5]. Activated neutrophils result in additional, prolonged injury via the release of oxidants and proteases [6]. Baicalein (5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one) is a main active ingredient derived from the dried root of Scutellaria, which is a popular herb in traditional Chinese medicine. Baicalein shows a variety of biological activities, including anti-inflammatory [7] , anti-apoptotic [8], anti-oxidant [9], anti-thrombotic [10], and anticancer [11] properties. Previous investigations have shown that baicalein acts as an anti-inflammation agent, inhibits the lipopolysaccharide (LPS)-induced inflammation, improves the vasoreactivity and the survival rate, as well as reverses the organ injury in septic animals [12,13]. Baicalein also exerts a cytoprotective role in H2O2-induced apoptosis by inhibiting the mitochondria-dependent caspase activation [8]. Recent studies have demonstrated that baicalein is able to decrease I/R injury in the brain and heart [14,15]. Moreover, baicalein protects animals from D-galactosamine(GaIN)/LPS induced acute liver failure [16] and carbon tetrachloride (CCl4)-induced liver damage [17] via inhibition of inflammation and apoptosis in murine models. However, its impact on liver I/R injury and its molecular mechanisms remain unclear.
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Prompted by the close associations between the pathological features of liver I/R and the protective properties of baicalein, we hypothesized that baicalein could protect against hepatic I/R injury. To test this hypothesis, we investigated the beneficial effects of baicalein, as well as its plausible signaling mechanisms in a mouse model of liver I/R.
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2.4. Liver and kidney damage assessment Serum alanine transaminase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine levels were measured using an automated chemical analyzer (Hitachi Co, Tokyo, Japan). 2.5. Histopathology
2. Materials and methods 2.1. Experimental design Experimental design was described in Fig. 1. To investigate whether baicalein could protect against liver I/R injury, mice were pretreated with either baicalein (100 mg/kg, i.p. Sigma-Aldrich, St. Louis, MO) or vehicle (dimethyl sulfoxide, DMSO, Sigma-Aldrich) 1 h prior to warm ischemia. Mice were killed at 1, 6 and 24 h of reperfusion. Liver injury, inflammatory cytokine, neutrophil infiltration, apoptosis and nuclear factor-kappa B (NF-κB) activation were analyzed. To investigate the possible mechanism of the anti-inflammatory effects of baicalein, murine macrophage cell line RAW264.7 and primary murine peritoneal macrophages were used in vitro cell culture studies. RAW264.7 cells and peritoneal macrophages were pretreated with various doses of baicalein (1–10 μm) or vehicle (DMSO) for 1 h and then exposed to LPS (Escherichia coli serotype O55:B05 type, Sigma-Aldrich, 10 ng/mL) for different periods. The production of inflammatory cytokine and the activation of NF-κB were analyzed.
Liver tissue was fixed in 4.5% buffered formalin for at least 24 h. Paraffin embedding was performed using standard techniques. Sections (4 μm) were stained with Hematoxylin–Eosin and assessed for tissue damage. 2.6. Myeloperoxidase (MPO) immunohistochemistry The presence of neutrophils in the liver was assessed by myeloperoxidase (MPO) staining. Briefly, after de-paraffinization, rehydration and antigen retrieval, the sections were incubated with MPO antibody (1:50; Abcam, Cambridge, UK) for 1 h at room temperature. After washing, the sections were incubated with goat anti-rabbit secondary antibody and visualized with diaminobenzidine. MPO-positive neutrophils were counted in 5 high-power fields (HPFs) per section at a magnification of 400 ×. The results are expressed as the mean number of MPO-positive neutrophils per HPF. 2.7. Caspase-3 activity assay
2.2. Animals Male inbred C57BL/6 mice (8–10 weeks old, weighing within 20– 22 g) were purchased from Wuhan university Center for Animal Experiment (Wuhan, China). All animals were housed under standard animal care conditions and had free access to water and food. All procedures were carried out according to the ethical guidelines of the Animal Care and Use Committee of Huazhong University of Science and Technology. 2.3. Partial hepatic warm I/R The partial hepatic warm I/R model was generated as described previously [18]. In brief, mice were completely anesthetized with pentobarbital (60 mg/kg, i.p.). After opening the abdomen and dissecting the interlobular ligaments, a microvascular clamp was used to interrupt the arterial and portal venous blood supply to the left lateral and median liver lobes for 60 min.
Relative caspase-3 activity in the liver tissues was detected with a caspase-3 colorimetric assay kit (Abcam) according to the manufacturer's instructions. 2.8. RAW264.7 and peritoneal macrophage cell culture The murine macrophage cell line RAW264.7 was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Peritoneal macrophages were isolated as described previously [19]. Cells were plated in 24-well plates at a density of 5 × 105 cells/ well for cytokine assay or in 6-well plates at a density of 2 × 106 cells/ well for Western blotting. 2.9. Assessment of cell viability RAW264.7 cells were cultured in a 96-well plate at a density of 5 × 103 cells/well. Following cultured with baicalein at different doses
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Fig. 2. In vivo toxicity profile of baicalein. Mice were treated with either baicalein at doses of 10, 50 or 100 mg/kg or vehicle (DMSO), liver and renal functions were assessed after the injections. (A) The serum ALT and AST levels were analyzed as a measure of hepatocellular injury. (B) The serum BUN and creatinine levels were analyzed as a measure of renal damage. The data are shown as mean ± SD. n = 4 per group.
for 3 h, cells were treated with 20 mg/mL) and incubated for 4 h. The medium were then removed and the remaining MTT-formazan crystals were dissolved in 200 nm, using 200 μL of DMSO as blank.
2.13. Enzyme-linked immunosorbent assay (ELISA) The levels of TNF-α and IL-6 in serum and cultured medium were analyzed using commercially available ELISA kits (R&D Systems, Minneapolis, MN).
2.10. Isolation of nuclear and cytoplasmic protein Nuclear and cytoplasmic extraction kit was bought from Pierce (Thermo Pierce, Rockford, IL). Cytoplasmic and nuclear protein from liver tissues or RAW264.7 cells was isolated according to the instructions of the manufacturer.
2.11. NF-κB activity assay NF-κB activity was measured using nuclear extracts from liver tissues or RAW264.7 cells. Activation of NF-κB was quantified using the TransAMNF-κB assay kits (Active Motif, Carlsbad, CA) according to the producer's instructions.
2.12. Gel electrophoresis and Western blotting Western blotting was performed as described previously [19]. Membranes were incubated overnight with primary rabbit anti-cleaved caspase-3 antibody (1:1000, Cell Signaling Technology, Beverly, MA), rabbit anti-cleaved caspase-7 antibody (1:1000, Cell Signaling Technology), rabbit anti-NF-κB (p65) antibody (1:1000, Cell Signaling Technology), rabbit anti-IκBα antibody (1:1000, Cell Signaling Technology), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:20,000; Sigma-Aldrich), and rabbit lamin B1 antibody (1:1000, Abcam). The membranes were developed with ECL Western blotting substrate (GE Healthcare, Buckinghamshire, UK) and Western blots were visualized on the Kodak Image Station (Carestream Health Inc, Rochester, NY).
2.14. Quantitative polymerase chain reaction (PCR) Real time PCR for TNF-α and IL-6 was performed as described previously [20]. The sequences of primers are as follows: TNF-α forward: 5′TGCTGGGAAGCCTAAAAGG-3′; reverse: 5′-CGAATTTTGAGAAGATGATC CTG-3′, IL-6 forward: 5′-TCAATTCCAGAAACCGCTATGA-3′ reverse: 5′CACCAGCATCAGTCCCAAGA-3, and ß-actin forward: 5′-AGAGGGAAAT CGTGCGTGAC-3′; reverse: 5′-CAATAGTGATGACCTGGCCGT-3′. 2.15. Statistical analysis The data are expressed as means ± SD. Differences between groups were evaluated for significance using a one-way ANOVA combined with Bonferroni's post hoc test. All tests were performed using SigmaStat v3.5 (Systat-Software, Erkrath, Germany). A p-value below 0.05 was considered to indicate statistical significance. 3. Results 3.1. Baicalein pretreatment protects against liver I/R injury The serum ALT, AST, BUN and creatinine levels were measured at 24 h and 7 day after baicalein injection to evaluate liver and kidney function, respectively. As shown in Fig. 2, baicalein doses of up to 100 mg/kg did not alter liver and kidney function compared to vehicle. The histologic examination of liver and kidney sections did not show histopathologic abnormalities after baicalein injection (data not shown).
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Cleaved Caspase3 Cleaved Caspase7 GAPDH Fig. 3. Baicalein attenuates liver injury and hepatic apoptosis. Mice were treated with either baicalein (100 mg/kg) or vehicle (DMSO) 1 h prior to ischemia. (A) The ALT and AST levels were analyzed as a measure of hepatocellular injury. (B) Routine histopathology was performed on formalin-fixed liver sections obtained from mice that had undergone 60 min of ischemia followed by 6 h of reperfusion (Original magnification ×400). (C) The caspase-3 activity was measured after 60 min of ischemia and 6 h of reperfusion. (D) The cleaved caspase-3 and cleaved caspase-7 cleavage were measured by Western blotting analysis. The data are shown as mean ± SD. n = 6 per group. *p b 0.05 compared to the vehicle-treated I/R group.
As shown in Fig. 3A, liver warm I/R resulted in significant increases in serum levels of ALT and AST. However, baicalein treatment significantly decreased the serum ALT and AST levels when compared to the vehicle control group. The liver histologic examination showed tissue injuries after I/R injury, including severe hepatocellular necrosis and cytoplasmic vacuolization of hepatocytes. In contrast, minimal damage was noted in baicalein-treated mice (Fig. 3B).
proinflammatory cytokines. Similarly, the serum levels of TNF-α and IL-6 were significantly lower in the baicalein-treated animals compared to the vehicle controls (Fig. 4A). The presence of neutrophils in the livers was assessed by MPO staining. After 60 min of warm ischemia and 6 h of reperfusion, the numbers of MPO-positive stained neutrophils were increased following I/R injury. The neutrophil infiltration was significantly less in the baicalein-treated animals than in the vehicle-injected animals (Fig. 4B).
3.2. Baicalein pretreatment decreases hepatic apoptosis following I/R injury
3.4. Baicalein modulates the NF-κB signaling pathway following I/R injury
As shown in Fig. 3C, after 60 min of warm ischemia and 6 h of reperfusion, the caspase-3 activity was significantly increased in the vehicletreated animals. However the caspase-3 activity in the baicalein-treated mice was markedly decreased compared to the vehicle control group. Furthermore, baicalein treatment significantly decreased the expression of cleaved caspase-3 and cleaved caspase-7 following I/R injury compared to the vehicle control group (Fig. 3D).
We then investigated whether baicalein pretreatment could affect the activation of NF-κB in the livers following I/R injury. As shown in Fig. 5A, compared to the sham operated animals, liver I/R in the vehicle-treated animals resulted in an increase of NF-κB in the nucleus and a decrease of IκBα in the cytoplasm. However, baicalein pretreatment significantly inhibited the increased levels of nuclear NK-κB and the decreased levels of cytoplasmic IκBα. The NF-κB activity was significantly inhibited in the baicalein-treated animals in response to I/R injury compared to the vehicle-injected animals (Fig. 5B).
3.3. Baicalein pretreatment decreases the production of inflammatory mediators and the infiltration of neutrophils following I/R injury Liver I/R in the vehicle-treated animals resulted in significant increases in the expression levels of TNF-α and IL-6 after reperfusion when compared to the sham operated animals. However, the baicalein-treated animals inhibited the increased expression of these
3.5. Baicalein pretreatment suppresses the LPS-induced proinflammatory cytokine production in macrophages As shown in Fig. 6A, the production of TNF-α and IL-6 was significantly increased in cultured medium of RAW264.7 cells after
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Fig. 4. Baicalein attenuates I/R induced-proinflammatory cytokine production and neutrophil infiltration. (A) The hepatic TNF-α and IL-6 mRNA expression levels were measured by quantitative PCR after 60 min of ischemia and 6 h of reperfusion. (B) The serum TNF-α and IL-6 levels were assessed by ELISA. (C) MPO staining was performed on the liver sections from mice at 6 h after reperfusion (Original magnification ×400). The numbers of MPO-positive neutrophils that infiltrated the livers were determined. Representative images from 6 mice/group were selected. The data are shown as mean ± SD. n = 6 per group. *p b 0.01 compared to the vehicle-treated I/R group.
stimulated with LPS. However, baicalein pretreatment showed a dose-dependent reduction of LPS-stimulated TNF-α and IL-6 production. To further investigate the anti-inflammatory effects of baicalein, peritoneal macrophages were isolated and pretreated with baicalein prior to LPS stimulation. The levels of TNF-α and IL-6 in the cultured medium were increased dramatically after treatment with LPS for 3 h. However, baicalein pretreatment significantly blunted the production of TNF-α and IL-6 (Fig. 6B). To exclude any possible interference on cell viability, the RAW264.7 cells were treated with the
escalating concentration of baicalein. As shown in Fig. 6C, baicalein concentrations of up to 10 μM did not affect the cell viability. 3.6. Baicalein pretreatment suppresses the activation of NK-κB by LPS in macrophages To investigate whether the anti-inflammatory effects of baicalein was mediated via inhibition of the NF-κB signaling pathway, we examined the effects of baicalein on the expression levels of IκBα and NF-κB.
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Fig. 5. Baicalein inhibits the activation of NF-κB following I/R injury. Mice were subjected to 60 min of ischemia after pretreatment with either baicalein (100 mg/kg) or vehicle (DMSO). Liver samples were harvested at 1 h of reperfusion. (A) The cytoplasmic IκBα and the nuclear NF-κB (p65) were measured by Western blotting analysis. Representative blots are presented and GAPDH and lamin B1 are served as a loading control for the cytoplasm and nuclear fractions, respectively. (B) The NF-κB activation during I/R injury was assessed by ELISA. The data are shown as mean ± SD. n = 6 per group. *p b 0.01 compared to the vehicle-treated I/R group.
As shown in Fig. 6D, Western blotting analysis showed that the levels of cytoplasmic IκBα were decreased, and the levels of nuclear NF-κB were increased in cells stimulated with LPS. However, baicalein pretreatment significantly prevented the degradation of cytoplasmic IκBα and the increase of nuclear NF-κB. Furthermore, baicalein pretreatment significantly blocked the NF-κB activity induced by LPS (Fig. 6E). 4. Discussion In the current study, we documented that baicalein treatment attenuated liver I/R injury via inhibition of the NF-κB activation and the subsequent inflammatory response, and suppression of the cellular apoptosis. Additionally, we confirmed that baicalein inhibited the expression of inflammatory cytokines and the activation of NF-κB in LPSstimulated macrophages. To our knowledge, this study is the first to provide in vivo evidence of the potential therapeutic value of baicalein in liver I/R injury. Several pharmacologic chemicals exhibit protective effects on liver I/R injury in animal models. However, none of them have shown any benefits in clinical trials of liver transplantation and liver resection in part due to the lack of safety and effectivity [21]. We demonstrated that baicalein doses of up to 100 mg/kg did not alter liver and kidney function in vivo. In addition, the anti-inflammatory effects of baicalein were not resulted from the increased cell death of macrophages for reducing the proinflammatory cytokine production. Our results were supported by Zong et al. [22], who demonstrated that mice received feed with 0.05% baicalein (dose: 100 mg/kg/day baicalein) for 12 weeks didn't affect their body weight. Hsieh et al. showed that baicalein concentrations of up to 30 μM did not affect the viability of HMC-1 cell in vitro [23]. In fact, baicalein is one of the major flavonoids from the root of Scutellaria, which has been used extensively in traditional Asian medicine to treat a variety of medical conditions for hundreds of years. The inflammatory response following reperfusion plays an important role in the pathophysiology of hepatic I/R injury [4,24,25]. The higher levels of circulation proinflammatory cytokines, such as TNFα and IL-6, have been shown to correlate with the early postoperative rejections and the infections during human liver transplantation [26]. Moreover, the neutralization of proinflammatory cytokines, such as TNF-α and IL-1, reduces the liver injury and mortality in a rat model of hepatic I/R [25,27]. Thus, an anti-inflammatory strategy via modulating the exaggerated inflammatory response to protect against liver I/R has been demonstrated in numerous studies [3,28]. It has demonstrated that baicalein reduces the production of TNF-α, inducible nitric oxide synthase (iNOS) nitric oxide in LPS-stimulated macrophages, and effectively protects animals from endotoxin shock
[12,13]. Furthermore, pretreatment with baicalein prevents D-GalN/ LPS-induced liver damage [16] and CCl₄-induced acute liver injury [17] in part by reducing inflammatory cytokine production. Additionally, baicalein preconditioning attenuates the myocardial I/R injury via inhibition of inflammation [15], as demonstrated recently. In agreement with these studies, we demonstrated that baicalein treatment significantly inhibited the production of inflammatory cytokines and the infiltration of neutrophils, and led to the reduction of the liver injury after I/R. These data indicate that the protective effects conferred by baicalein may be partially via the inhibition of inflammation. The mechanism of the anti-inflammatory effects of baicalein involves its ability to inhibit NF-κB signaling pathway, which plays an important role in liver I/R injury [28]. NF-κB is activated upon I/R injury and regulates the production of proinflammatory cytokines/ chemokines [28]. The inhibition of NF-κB activation leads to a decrease of proinflammatory cytokine production and an amelioration of I/R-induced tissue injuries [29]. It has been reported that the inhibitory effects of baicalein on LPS-induced pro-inflammatory cytokine expression both in vitro and in vivo are mediated by blocking NF-κB activation [12,13,16]. In this study, we identified that baicalein could effectively disrupt the activation of the NF-κB singling pathway by showing its activity on inhibiting the degradation of IκBα and the nuclear translocation of NF-κB in the livers following I/R. To investigate the possible mechanism of the anti-inflammatory effects of baicalein, the murine macrophage cell line RAW264.7 and the primary murine peritoneal macrophages were used in vitro cell culture studies. Kupffer cells, the hepatic resident macrophages, are important for triggering the inflammatory response in liver I/R injury [3,30]. Here, we clearly demonstrated that baicalein pretreatment inhibited the production of proinflammatory cytokines in LPS-stimulated macrophages. Consistent with the finding in vivo, the LPS-induced IκBα degradation and the NF-κB nuclear translocation were inhibited by baicalein administration in LPS-stimulated RAW264.7 cells. These findings suggest that the modulation of the IκBα/NF-κB signaling pathway in macrophages accounts, at least in part, for the antiinflammatory and the protective effects of baicalein. Since recent studies suggest that baicalein also exhibits protective effects via the suppression of apoptosis [8,16,17], we observed apoptotic cell death following liver I/R injury. Our results demonstrated that baicalein treatment inhibited apoptotic cell death as indicated by the decreases of I/R-induced caspase protein cleavage and caspase-3 activity. This finding was partly supported by baicalein reducing plasma cytokines levels, such as TNF-α, ROS and nitric oxide, which can initiate the apoptotic cascade via the death receptor/caspase pathway [31]. Baicalein pretreatment exhibits significant anti-
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IκBα GAPDH NF-κB Lamin B1
Fig. 6. Baicalein inhibits the activation of NF-κB in RAW264.7 cells activated by LPS. (A) RAW264.7 cells and (B) peritoneal macrophages were pretreated with various doses of baicalein (1–10 μM) or vehicle (DMSO) for 1 h and then exposed to LPS (10 ng/mL) for 3 h. The TNF-α and IL-6 concentrations in the cultured medium were measured by ELISA. (C) The cell viability was determined by MTT assay in RAW264.7 cells. (D) The cytoplasmic IκB and the nuclear NF-κB (p65) were measured in RAW264.7 cells stimulated with LPS (10 ng/mL) for 30 min after 1 h of pretreatment with baicalein (10 μM) or DMSO. Representative blots are presented and GAPDH and lamin B1 are served as a loading control for the cytoplasm and nuclear fractions, respectively. (E) The NF-κB activation was assessed by ELISA. The data are shown as mean ± SD. n = 3 per group. *p b 0.01 compared to the vehicle-treated group.
apoptotic protection against cardiomyocyte I/R injury via mitochondrial oxidant signaling [32]. Additionally, the molecular mechanisms underlying baicalein inhibition of D-GalN/LPS-induced apoptosis are associated with the protection of mitochondria, increasing the Bcl-2/ Bax ratio and blocking the release of cytochrome c [16]. In conclusion, we found that baicalein possessed strong beneficial effects against liver I/R injury in a mouse model. Mechanistically, the action of baicalein involves the attenuation of NF-κB activation and the
subsequent expression of inflammatory cytokine, and suppression of apoptosis.
Acknowledgments This project was supported by grants from the National Natural Science Fund of China (NSFC) (81102689, 81300343) and the Specialized
A. Liu et al. / International Immunopharmacology 24 (2015) 72–79
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