International Immunopharmacology 19 (2014) 365–372
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Protective effect of tormentic acid from Potentilla chinensis against lipopolysaccharide/D-galactosamine induced fulminant hepatic failure in mice Xing Lin a, Shijun Zhang a, Renbin Huang a, Shimei Tan a, Shuang Liang b, Xiaoyan Wu b, Lang Zhuo a, Quanfang Huang b,⁎ a b
Guangxi Medical University, Nanning 530021, China The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530023, China
a r t i c l e
i n f o
Article history: Received 15 December 2013 Received in revised form 27 January 2014 Accepted 9 February 2014 Available online 21 February 2014 Keywords: Tormentic acid Potentilla chinensis Lipopolysaccharide/D-galactosamine Fulminant hepatic failure
a b s t r a c t A compound was isolated from Potentilla chinensis, and it was identified as tormentic acid (TA) based on its physicochemical properties and spectral data. The hepatoprotective effect of TA was evaluated using an acute liver failure model induced by lipopolysaccharide (LPS)/D-galactosamine (D-GalN). The results revealed that TA significantly prevented LPS/D-GalN-induced fulminant hepatic failure, as evidenced by the decrease in serum aminotransferase and total bilirubin activities and the attenuation of histopathological changes. TA alleviated the pro-inflammatory cytokines including TNF-α and NO/iNOS by inhibiting nuclear factor-κB (NF-κB) activity. Moreover, TA strongly inhibited lipid peroxidation, recruited the anti-oxidative defense system, and increased HO-1 activity. In addition, TA significantly attenuated increases in TUNEL-positive hepatocytes through decreasing the levels of cytochrome c, as well as caspases-3, 8 and 9, while augmenting the expression of Bcl-2. In conclusion, TA protects hepatocytes against LPS/D-GalN-induced injury by blocking NF-κB signaling pathway for anti-inflammatory response and attenuating hepatocellular apoptosis. Consequently, TA is a potential agent for preventing acute liver injury and may be a major bioactive ingredient of Potentilla chinensis. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Fulminant hepatic failure is a dramatic clinical syndrome that results from severely impaired liver function. Lipopolysaccharide (LPS), when combined with D-galactosamine (D-GalN), induces liver injury, and this is a well-known experimental model for liver failure caused by massive hepatocyte death [1]. This model mimics clinical liver dysfunction and is useful for evaluating treatment efficiency [2]. LPS is a major component of the outer membrane of Gram-negative bacteria and is an endotoxin that might contribute to hepatic failure [3]. D-GalN is a specific hepatotoxic agent that depletes the uridine triphosphate pool, thereby inhibiting macromolecule synthesis [4]. D-GalN potentiates the toxic effects of LPS in the liver [5]. Due to thousands of years of experience, herbal medicines are a rich source of new therapeutic agents. Many compounds with new structural features and novel mechanisms of action have been isolated from herbal medicines. Natural products are excellent sources of novel anti-hepatitis drugs that may be applicable to liver disease treatments [6]. Potentilla chinensis (Rosaceae P. chinensis Ser.) is a traditional herb commonly used in popular folk medicine in China for treating immune disorders ⁎ Corresponding author. Tel.: +86 771 5645433. E-mail address: [email protected] (Q. Huang).
http://dx.doi.org/10.1016/j.intimp.2014.02.009 1567-5769/© 2014 Elsevier B.V. All rights reserved.
and liver diseases [7]. However, the active ingredients in P. chinensis and the mechanism of its anti-hepatic failure remain unknown. Therefore, this study was conducted to isolate and identify an anti-hepatic failure compound from this well-known Chinese herb. In this study, a pure compound was isolated from P. chinensis. The chemical structures were established based on the physicochemical properties and spectral data of the compound. Furthermore, the LPS/D-GalN model was used to evaluate the protective mechanisms of the compound during fulminant hepatic failure, particularly regarding the extent of oxidative damage and apoptosis. Silymarin is a polyphenolic flavonoid derived from milk thistle (Silybum marianum) that has anti-inflammatory, cytoprotective, and anticarcinogenic effects [8]. It has been reported that silymarin inhibited nitric oxide production and iNOS gene expression by inhibiting nuclear factor-kappa B (NF-κB)/Rel activation [9]. Similarly, some researches reported that silymarin blocked TNF-induced activation of NF-κB in a dose- and time-dependent manner, and this effect was mediated through inhibition of phosphorylation and degradation of IκBα, an inhibitor of NF-κB. Silymarin blocked the translocation of p65 to the nucleus without affecting its ability to bind to the DNA [10]. In addition, silymarin also blocked NF-κB activation induced by phorbol ester, LPS, okadaic acid, and ceramide [10]. In this paper, silymarin was used as a positive control.
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2. Materials and methods 2.1. Materials P. chinensis was purchased from Nanning Qianjinzi Chinese Pharmaceutical Co. Ltd. (Nanning, China). A voucher specimen (CALX12071306) was identified by Q.F. Huang at the First Affiliated Hospital of Guangxi Traditional Chinese Medicine University. Silymarin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione reductase (GSH-Rd) and catalase kits were obtained from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). The interleukin-6 and 10 (IL-6 and 10) and tumor necrosis factor-α (TNF-α) kits were purchased from Wuhan Boster Bio-engineering Co. Ltd. (Wuhan, China). 2.2. Herb extraction and chemical analysis The dried and powdered whole plant of P. chinensis (5 kg) was extracted with 95% ethanol at room temperature. After the solvent was evaporated under reduced pressure, the residue from the ethanol extract was successively extracted with petroleum ether, chloroform and acetic ether. The acetic ether fraction (285.2 g) was then subjected to silica gel chromatographic column (200–300 mesh, Yantai, PR China; 10 × 300 cm), eluted with CHCl3-MeOH-H2O (60:35:5, each 500 mL) to produce seven fractions (Frs. 1–7). Fr. 5 was further separated using a silica gel column (3 × 80 cm, 200–300 mesh) via successive elutions with a CHCl3/CH3OH gradient (100:0–0:100, each 200 mL), to yield five sub-fractions (Frs. I–V). Fr. IV was successively separated by Sephadex LH-20 column chromatography (eluted with MeOH) and HPLC (eluted with 35% aqueous MeOH), giving a compound (74.8 mg). The structure of the compound was elucidated based on its physicochemical properties and spectral data. 2.3. Animals and treatments Male C57BL/6 mice (20–22 g) were purchased from Vital River Laboratory Animal Technology Co. (Beijing, China). The research was conducted according to protocols approved by the Institutional Ethical Committee of Guangxi Medical University. After a period of one week, the mice were divided into seven groups with 15 mice per group as follows: Group I, normal control, the mice received the same volume of saline. Group II, TA control, the mice received tormentic acid (3 mg/kg). Group III, model control, the mice received LPS (50 μg/kg) and D-GalN (800 mg/kg). Group IV–VI, TA treatment, the mice received tormentic acid (0.75, 1.5 or 3 mg/kg) plus LPS (50 μg/kg) and D-GalN (800 mg/kg). Group VII, silymarin treatment, the mice received silymarin (50 mg/kg) plus LPS (50 μg/kg) and D-GalN (800 mg/kg). The animals were administered various drugs (TA or silymarin) intraperitoneally once daily for 3 days prior to challenge experimentation. Mice in groups III–VII were then challenged intraperitoneally with LPS/ D-GalN. Blood was sampled from the mouse eyes 1.5 h after LPS/D-GalN administration. The mice were then euthanized by intraperitoneally injecting a high dose of pentobarbitone sodium (150 mg/kg) 6 h after LPS/D-GalN injection. The liver samples were dissected and immediately washed with ice-cold saline. 2.4. Histological analysis and detection of apoptotic cells Liver tissues were fixed in 4% phosphate buffered formalin, dehydrated in graded alcohols and embedded in paraffin blocks. Five-
micrometer-thick paraffin sections were then rehydrated and stained with hematoxylin and eosin (H&E). Apoptotic cells were detected using TUNEL staining with an in situ Apoptosis Detection Kit (TaKaRa) according to the manufacturer's instructions. 2.5. Estimating AST, ALT and total bilirubin activities Serum levels of AST, ALT and total bilirubin were measured using commercially available kits (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China) according to the manufacturer's instructions. 2.6. Determination of serum TNF-α, IL-6 and IL-10 levels The serum TNF-α, IL-6 and IL-10 levels were determined using an enzyme-linked immunosorbent assay (ELISA) (Amersham Pharmacia Biotec, NJ, USA) according to the manufacturer's protocol. 2.7. Serum nitric oxide (nitrite/nitrate) analysis On a serum nitric oxide (NO) (nitrite/nitrate) analysis, a serum at 1.5 h post-injection of LPS/D-GalN was used. The sum of the serum nitrite − and nitrate (NO− 2 and NO3 , stable metabolites of nitric oxide) was measured using a commercial kit (BIOMOL, Plymouth Meeting, PA, USA). 2.8. HO-1 activity assay The HO-1 enzymatic activity in the liver was measured via bilirubin generation, as previously described [11]. The extracted bilirubin was measured using the difference in absorbance at 464 and 530 nm (ε = 40 mM− 1 cm− 1). 2.9. Hepatic lipid peroxidation assays Lipid peroxidation in the liver was determined by measuring the level of malondialdehyde (MDA), an end product of lipid peroxidation, using a thiobarbituric acid method. The level of hepatic MDA was measured according to the protocol established in our previous study [12]. 2.10. Estimation of hepatic antioxidant enzyme activities and glutathione content Liver tissue was homogenized on ice with Tris–HCl (5 mmol/L containing 2 mmol/L EDTA, pH 7.4). The homogenates were centrifuged at 1000 × g for 15 min at 4 °C. The supernatants were immediately used for the SOD, GSH-Px, GSH-Rd and catalase assays. All enzymes were evaluated using commercially available kits (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China) according to the manufacturer's instructions. In addition, the hepatic GSH content was measured using a Glutathione Assay Kit (BioVision, CA, USA). 2.11. RT-PCR for TNF-α and iNOS To examine the mRNA expression levels of TNF-α and inducible nitric oxide synthase (iNOS) in the liver, the total RNA from snap-frozen tissues was extracted using an RNeasy Mini Kit (QIAGEN Inc.). First-strand cDNA was prepared by following the instructions for the SuperScript™ FirstStrand Synthesis System for RT-PCR Kit (Life Technologies). Reverse transcription and amplification by PCR were performed as previously described [13]. The following primer sequences were used: TNF-α: 5′-ATG AGC ACA GAA AGC ATG ATC-3′ (sense) and 5′-TAC AGG CTT GTC ACT CGA ATT-3′ (antisense); iNOS: 5′-GTG AGG ATC AAA AAC TGG GG-3′ (sense) and 5′-ACC TGC AGG TTG GAC CAC-3′ (antisense); and β-actin: 5′-GAT GGT GGG TAT GGG TCA GAA GGA-3′ (sense) and 5′-GCT CAT TGC CGA TAG TGA TGA CCT-3′ (antisense). The PCR products were
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subjected to electrophoresis and ethidium bromide staining. The gels were analyzed by densitometry. β-Actin was used as the internal control. 2.12. Measurement of nuclear NF-κB p65 content Nuclear was extracted from the liver with a Nuclear Extraction Kit (Active Motif, Carlsbad, USA). The nuclear NF-κB p65 content was measured using a NF-κB/p65 ActivELISA kit (Imgenex, San Diego, USA). 2.13. Western blot immunoassay for cytochrome c and Bcl-2 The cytosolic proteins in liver samples were prepared using cytoplasmic extraction reagents according to the manufacturer's instructions (Pierce Biotechnology, Rockford, IL, USA). Briefly, equivalent aliquots of protein extract were separated on 12% SDS-PAGE gel. After electrophoretic separation, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes, incubated with primary antibodies against cytochrome c (BD Biosciences) and Bcl-2 (Santa Cruz Biotechnology Inc., Santa Cruz, USA) and treated with secondary antibodies. The intensity of the immunoreactive bands was determined using a densitometer equipped with Image QuaNT software (Molecular Dynamics, Sunnyvale, CA). The signals were normalized relative to β-actin (Sigma chemical Co.) for cytochrome c and Bcl-2 proteins. 2.14. Evaluation of caspase-3, caspase-8 and caspase-9 activities The liver samples were homogenized in lysis buffer. After centrifugation for 15 min at 25000 ×g, the supernatant was incubated with a substrate peptide (DEVD-AFC for caspase-3, IETD-AFC for caspase-8 and LEHD-AFC for caspase-9). The change in fluorescence (excitation at 400 nm and emission at 490 nm) was monitored after a 120-minute incubation according to the procedure reported by the previous studies [14]. 2.15. Statistical analysis Statistical analysis was performed using SPSS 11.5 for Windows. Differences between the groups were assessed using a one-way analysis of variance (ANOVA) with a Tukey's test for post-hoc multiple comparisons. The data are presented as the means ± SE. A P-value b0.05 was considered to be statistically significant. 3. Results and discussion
Fig. 1. Chemical structure of tormentic acid.
3.2. Effects of TA on histopathological changes The liver in both the normal control and TA control groups revealed normal lobular architecture and cellular structure. However, livers exposed to LPS/D-GalN exhibited multiple extensive areas with portal inflammation and cellular necrosis, as well as a moderate increase in inflammatory cell infiltration. These pathological alterations were ameliorated by 3 mg/kg TA or 50 mg/kg silymarin treatment (Fig. 2). The result suggests that TA has a hepatoprotective effect on this model of hepatic damage.
3.3. Effects of TA on serum ALT, AST and total bilirubin levels Significant elevations of AST, ALT and total bilirubin levels in the serum which are released from the cytosolic part of the liver cells often suggest the pathological condition of severe liver injury [15]. To evaluate the extent of liver injury in mice, we conducted an analysis of serum AST and ALT activities and total bilirubin level. As shown in Fig. 3A and B, significant increases in the serum ALT, AST and total bilirubin levels were observed in LPS/D-GalN animals compared to mice with saline treatment (normal control). Conversely, mice treated with TA or silymarin exhibited a significant decrease in the levels of these parameters. In addition, TA had no effect on the basal serum AST, ALT or total bilirubin level. The data further confirm that TA exerts a hepatoprotective effect during the LPS/D-GalN-induced model of hepatic damage.
3.1. Chemistry The structure of the compound isolated from P. chinensis was elucidated based on its physicochemical properties and spectral data as follows: mp: 265–267 °C, ESI-MS m/z 487 [M–H]−, IR (KBr) cm−1: 3392, 2964, 2918, 2834, 1601, 1452, 1394, 1233, 1157, 1052. 1H-NMR (C5D5 N, 400 MHz) δ: 5.58 (1H, brs, 12-H), 4.12 (1H, m, 2-H), 3.38 (1H, d, J = 9.2 Hz, 3-H), 3.15 (1H, m, 11-H), 3.06 (1H, s, 18-H), 1.73 (3H, s), 1.43 (3H, s), 1.31 (3H, s), 1.09 (3H, d, J = 10.5 Hz), 0.99 (3H, s), 0.98 (3H, s), 0.96 (3H, s). 13 C-NMR (Py-d 5, 125 MHz) δ: 47.2 (C-1), 68.5 (C-2), 83.3 (C-3), 38.9 (C-4), 55.6 (C-5), 18.4 (C-6), 33.3 (C-7), 40.4 (C-8), 47.6 (C-9), 38.1 (C-10), 23.5 (C-11), 126.9 (C-12), 138.9 (C-13), 41.7 (C-14), 28.7 (C-15), 25.9 (C-16), 48.2 (C-17), 54.2 (C-18), 72.3 (C-19), 42.2 (C-20), 26.7 (C-21), 38.2 (C-22), 26.4 (C-23), 23.1 (C-24), 16.6 (C-25), 17.5 (C-26), 24.6 (C-27), 179.8 (C-28), 26.5 (C-29), 16.7 (C-30). These data indicate that the compound is tormentic acid (TA), which has a molecular formula and molecular weight of C30H48O5 and 488.71, respectively. Its chemical structure is shown in Fig. 1.
3.4. Effects of TA on serum IL-6 and IL-10 levels Previous studies demonstrated that various cytokines can protect cells from inflammation byproducts [16]. IL-6 is a typical inflammatory cytokine that regulates the acute phase response in the liver. Furthermore, IL-6 is essential for liver regeneration after a partial hepatectomy and confers resistance to liver injury by hepatic toxins and ischemic injury [17]. The IL-6 blockade aggravates liver injury and induces lethality in LPS/D-GalN hepatitis model [18]. IL-10 is an anti-inflammatory cytokine that reduces the lethality and hepatic injury induced by LPS/D-GalN [19]. Previous study has revealed that IL-10 inhibits the synthesis and secretion of numerous pro-inflammatory cytokines and protects the liver by decreasing liver apoptosis during the LPS-induced hepatitis model [20]. In our study, the serum IL-6 content was lower, while IL-10 level was significantly higher in mice dosed with TA (Fig. 3C and D). In addition, there were no significant differences in the levels of IL-6 and IL-10 between the TA control group and normal control group.
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Fig. 2. TA-mediated histological changes (H&E staining, 100 ×). A1: normal control group, A2: TA control group, A3: model control group, A4: 1.5 mg/kg TA-treated group, A5: 3 mg/kg TA-treated group, and A6: 50 mg/kg silymarin treated group.
3.5. Effects of TA on serum TNF-α content and hepatic TNF-α mRNA level
3.7. Effects of TA on lipid peroxidation and antioxidant enzyme activities
During LPS/D-GalN-induced acute liver injury, TNF-α played an important role in hepatocyte apoptosis or necrosis [21]. LPS directly activates Kupffer cells to produce TNF-α, inducing hepatocyte apoptosis during the early stages of the liver injury in D-GalN-sensitized mice. Furthermore, TNF-α-induced neutrophil transmigration occurs during the later stages of the liver injury, causing massive hepatocyte necrosis and organ failure [22]. In the present study, administration of LPS/D-GalN markedly increased serum TNF-α content and hepatic TNF-α mRNA expression, while treatment with TA or silymarin attenuated its levels (Fig. 3E and F). As mentioned above, LPS/D-GalN has been shown to induce the inflammatory cells to release various pro-inflammatory cytokines, including TNF-α and IL-6, followed by the production of antiinflammatory cytokines, such as IL-10 and IL-4. Exposure to large doses of LPS causes extreme “endotoxemia” or “septic shock” whereby the exposed host exhibits hypotension, disseminated intravascular coagulation, and widespread tissue injury that can result in death due to multiple organ failure [23]. However, the problem is not the overexpression of pro-inflammatory cytokines, but rather that they are not adequately balanced by endogenous anti-inflammatory mediators [24]. We found that by increasing the dose of TA, levels of TNF-a and IL-6 decreased, while the content of IL-10 increased. We think that TA protects LPS/D-GalN-induced acute liver injury mainly by modulating the balance of pro-inflammatory and anti-inflammatory cytokines.
Free radicals generate the lipid peroxidation process in an organism. Malondialdehyde (MDA) is one of the final products of polyunsaturated fatty acid peroxidation in the cells. An increase in free radicals causes overproduction of MDA. Its level is commonly known as a marker of oxidative stress [27]. As shown in Fig. 4C, the hepatic MDA level, which is the end-product of lipid peroxidation, increased significantly in the model group. Treatment with TA or silymarin effectively decreased the MDA level. In addition, the effects of TA on the hepatic SOD, GSH-Px, GSH-Rd and catalase activities, as well as on the GSH content, are shown in Fig. 4D. The four hepatic antioxidant enzyme activities and the GSH content significantly decreased 6 h after LPS/D-GalN administration. In contrast, treatment with 3 mg/kg TA or 50 mg/kg silymarin significantly increased the activities of these enzymes, as well as the GSH content. TA alone had negligible effects on the basal lipid peroxidation and antioxidant enzyme activities. These results indicate that TA inhibits lipid peroxidation and effectively recruits the anti-oxidative defense system in liver injury induced by LPS/D-GalN.
3.6. Effects of TA on nitric oxide (NO) content and iNOS mRNA level Nitric oxide (NO) is a highly reactive oxidant, produced by parenchymal and nonparenchymal liver cells from L-arginine via the action of inducible nitric oxide synthase (iNOS) [25]. It is well-known that NO participates in diverse physiological and pathological processes, and may exert toxicological effects such as inhibition of mitochondrial respiration and DNA synthesis as well as formation of peroxynitrite, a more long-lived cytotoxic oxidant when over-generated under stress conditions [26]. In the present study, significant increases in the serum NO content and the liver iNOS mRNA level were observed in the model control group. Treatment with 1.5 or 3 mg/kg TA or with 50 mg/kg silymarin significantly inhibited these increases (Fig. 4A and B). Therefore, inhibiting NO also contributes to the hepatoprotective effect of TA.
3.8. Effect of TA on HO-1 activity Heme oxygenase (HO) is a rate-limiting enzyme that catalyzes the breakdown of heme into antioxidant and anti-inflammatory agents such as biliverdin, carbon monoxide, and iron. Previous studies have demonstrated that HO-1 expression is rapidly upregulated in the liver after administration of toxins such as carbon tetrachloride, endotoxin and LPS/D-GalN [11]. Chemically inducing HO-1 expression with a compound such as hemin is cytoprotective against LPS/D-GalN-induced acute hepatic injury in rats [28]. This study confirmed a significant increase in the HO-1activity after LPS/D-GalN injection. These alterations were augmented by TA treatment (Fig. 4E), suggesting that TA induces HO-1 activity and/or suppresses the degradation of this enzyme. TA alone had no significant effect on the basal HO-1 activity. 3.9. Effect of TA on nuclear NF-κB p65 content Nuclear factor-kappa B (NF-κB) is a master regulator of the hepatic inflammatory response. Under basal conditions, NF-κB is present in the cytoplasm of hepatocytes in a latent form, bound to the NF-κB inhibitory protein, inhibitor kappa B (IκB). Upon exposure to pro-inflammatory stimuli, the IκB kinase (IKK) complex is activated and catalyzes the phosphorylation of IκB. Phosphorylated IκB is then targeted for degradation by
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Fig. 3. Effects of TA on the serum ALT and AST (A) and total bilirubin levels (B), serum IL-6 (C) and IL-10 contents (D), serum TNF-α (E), and hepatic TNF-α mRNA level (F). The results are presented as the means ± SE. ⁎P b 0.05 compared to the LPS/D-GalN model control group.
the 26S proteosome complex, thereby liberating NF-κB to migrate to the cell nucleus and direct transcription of target genes [29]. It is clear that NF-κB directly regulates inflammatory cytokines, such as TNF-α, IL-6 and iNOS [30,31]. Because these inflammatory mediators are implicated in the pathophysiology of organ failure after shock and sepsis, inhibition of NF-κB activation could be a very effective strategy to reduce an excessive inflammatory response. In our study, treating mice with 3 mg/kg TA or 50 mg/kg silymarin significantly decreased the nuclear NF-κB p65 content compared to the model group (Fig. 4F). Thus, we speculate that TA inhibited the excessive release of inflammatory cytokines caused by LPS/D-GalN which may result from reduced NF-κB activation.
3.10. Effects of TA on apoptotic cell, apoptosis-related proteins and caspase Apoptosis has been observed in animal models for LPS/D-GalN injury. There are two major apoptotic pathways: the extrinsic pathway triggered by the activation of caspase-8 and the intrinsic pathway triggered by the activation of caspase-9 [32]. Caspase-3 is a predominant downstream effector activated by caspases 8 and 9 [33]. Massive activation of caspases is a crucial process during the induction of apoptosis associated with the pathogenesis of acute hepatic failure [34]. In addition, the caspase cascade is primarily associated with the release of cytochrome c from mitochondria, as well as caspases 3 and 9, during apoptosis [32]. The Bcl-2 family, including pro-apoptotic and anti-apoptotic proteins,
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Fig. 4. Effects of TA on serum NO content (A), liver iNOS mRNA level, lipid peroxidation (C), antioxidant enzyme activities (D), hepatic HO-1 activity (E), and nuclear NF-κB p65 content (F). The results are presented as the means ± SE. ⁎P b 0.05 compared to the LPS/D-GalN model control group.
helps control apoptosis, and the overexpression of Bcl-2 is antiapoptotic [35]. In this study, apoptotic hepatocytes were detected using TUNEL staining. Numerous TUNEL positive hepatocytes were observed in liver tissues obtained 6 h after LPS/D-GalN injection. However, few positive hepatocytes were observed in livers treated with 3 mg/kg TA or 50 mg/kg silymarin (Fig. 5A). Furthermore, the apoptosis-related proteins, including cytochrome c and Bcl-2, were detected using a Western blot immunoassay. As shown in Fig. 5B, after injecting LPS/D-GalN, the level of cytochrome c expression increased significantly compared to the normal control group. TA administration reduced cytochrome c expression relative to the LPS/D-GalN group. While Bcl-2 showed little expression in the LPS/D-GalN group, treatment with TA augmented it. Furthermore, the activities of caspases 3, 8 and 9 in the cytosol fraction 6 h after LPS/D-GalN administration increased rapidly relative to the normal
control group. The elevated caspase 3, 8 and 9 activities were attenuated by treatment with 3 mg/kg TA or 50 mg/kg silymarin. In addition, TA alone had a negligible effect on the basal levels of apoptotic cell, apoptosis-related proteins and caspase (Fig. 5C). Thus, the protective effect of TA against LPS/D-GalN-induced hepatic injury might be mediated by its anti-apoptotic effects. 4. Conclusion In conclusion, TA protects hepatocytes against LPS/D-GalN-induced acute liver injury in a dose-dependent manner, possibly by modulating the balance of pro-inflammatory and anti-inflammatory cytokines via inhibition of NF-κB, and attenuating hepatocellular apoptosis. Therefore, TA is a potential agent for preventing acute liver injury, and TA may be a major bioactive ingredient of P. chinensis.
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Fig. 5. Effects of TA on apoptotic cells (A), apoptosis-related proteins (B), and caspase (C). A: TUNEL staining (200×), A1: normal control group, A2: TA control group, A3: model control group, A4: 1.5 mg/kg TA-treated group, A5: 3 mg/kg TA-treated group, and A6: 50 mg/kg silymarin treated group. The results are presented as the means ± SE. ⁎P b 0.05 compared to the LPS/D-GalN model control group.
Acknowledgments The authors gratefully acknowledge the financial support provided by the National Natural Science Foundation of China (Nos. 81260674; 81260505), the Guangxi Natural Science Foundation (Nos. 2013GXNSFAA019146; 2013GXNSFAA019150), and the Foundation for the Guangxi Key Laboratory for Prevention & Treatment of Regional High-Incidence Diseases (KFJJ2010-71; KFJJ2011-37). References [1] Rahman TM, Hodgson HJ. Animal models of acute hepatic failure. Int J Exp Pathol 2000;81:145–57. [2] Wang F, Wen T, Chen X-Y, Wu H. Protective effects of pirfenidone on D-galactosamine and lipopolysaccharide-induced acute hepatotoxicity in rats. Inflamm Res 2008;57:183–8. [3] Bohlinger I, Leist M, Gantner F, Angermüller S, Tiegs G, Wendel A. DNA fragmentation in mouse organs during endotoxic shock. Am J Pathol 1996;149:1381. [4] Wu J, Danielsson Å, Zern MA. Toxicity of hepatotoxins: new insights into mechanisms and therapy. Expert Opin Investig Drugs 1999;8:585–607.
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Protective effect of tormentic acid from Potentilla chinensis against lipopolysaccharide/D-galactosamine induced fulminant hepatic failure in mice Xing Lin a, Shijun Zhang a, Renbin Huang a, Shimei Tan a, Shuang Liang b, Xiaoyan Wu b, Lang Zhuo a, Quanfang Huang b,⁎ a b
Guangxi Medical University, Nanning 530021, China The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530023, China
a r t i c l e
i n f o
Article history: Received 15 December 2013 Received in revised form 27 January 2014 Accepted 9 February 2014 Available online 21 February 2014 Keywords: Tormentic acid Potentilla chinensis Lipopolysaccharide/D-galactosamine Fulminant hepatic failure
a b s t r a c t A compound was isolated from Potentilla chinensis, and it was identified as tormentic acid (TA) based on its physicochemical properties and spectral data. The hepatoprotective effect of TA was evaluated using an acute liver failure model induced by lipopolysaccharide (LPS)/D-galactosamine (D-GalN). The results revealed that TA significantly prevented LPS/D-GalN-induced fulminant hepatic failure, as evidenced by the decrease in serum aminotransferase and total bilirubin activities and the attenuation of histopathological changes. TA alleviated the pro-inflammatory cytokines including TNF-α and NO/iNOS by inhibiting nuclear factor-κB (NF-κB) activity. Moreover, TA strongly inhibited lipid peroxidation, recruited the anti-oxidative defense system, and increased HO-1 activity. In addition, TA significantly attenuated increases in TUNEL-positive hepatocytes through decreasing the levels of cytochrome c, as well as caspases-3, 8 and 9, while augmenting the expression of Bcl-2. In conclusion, TA protects hepatocytes against LPS/D-GalN-induced injury by blocking NF-κB signaling pathway for anti-inflammatory response and attenuating hepatocellular apoptosis. Consequently, TA is a potential agent for preventing acute liver injury and may be a major bioactive ingredient of Potentilla chinensis. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Fulminant hepatic failure is a dramatic clinical syndrome that results from severely impaired liver function. Lipopolysaccharide (LPS), when combined with D-galactosamine (D-GalN), induces liver injury, and this is a well-known experimental model for liver failure caused by massive hepatocyte death [1]. This model mimics clinical liver dysfunction and is useful for evaluating treatment efficiency [2]. LPS is a major component of the outer membrane of Gram-negative bacteria and is an endotoxin that might contribute to hepatic failure [3]. D-GalN is a specific hepatotoxic agent that depletes the uridine triphosphate pool, thereby inhibiting macromolecule synthesis [4]. D-GalN potentiates the toxic effects of LPS in the liver [5]. Due to thousands of years of experience, herbal medicines are a rich source of new therapeutic agents. Many compounds with new structural features and novel mechanisms of action have been isolated from herbal medicines. Natural products are excellent sources of novel anti-hepatitis drugs that may be applicable to liver disease treatments [6]. Potentilla chinensis (Rosaceae P. chinensis Ser.) is a traditional herb commonly used in popular folk medicine in China for treating immune disorders ⁎ Corresponding author. Tel.: +86 771 5645433. E-mail address: [email protected] (Q. Huang).
http://dx.doi.org/10.1016/j.intimp.2014.02.009 1567-5769/© 2014 Elsevier B.V. All rights reserved.
and liver diseases [7]. However, the active ingredients in P. chinensis and the mechanism of its anti-hepatic failure remain unknown. Therefore, this study was conducted to isolate and identify an anti-hepatic failure compound from this well-known Chinese herb. In this study, a pure compound was isolated from P. chinensis. The chemical structures were established based on the physicochemical properties and spectral data of the compound. Furthermore, the LPS/D-GalN model was used to evaluate the protective mechanisms of the compound during fulminant hepatic failure, particularly regarding the extent of oxidative damage and apoptosis. Silymarin is a polyphenolic flavonoid derived from milk thistle (Silybum marianum) that has anti-inflammatory, cytoprotective, and anticarcinogenic effects [8]. It has been reported that silymarin inhibited nitric oxide production and iNOS gene expression by inhibiting nuclear factor-kappa B (NF-κB)/Rel activation [9]. Similarly, some researches reported that silymarin blocked TNF-induced activation of NF-κB in a dose- and time-dependent manner, and this effect was mediated through inhibition of phosphorylation and degradation of IκBα, an inhibitor of NF-κB. Silymarin blocked the translocation of p65 to the nucleus without affecting its ability to bind to the DNA [10]. In addition, silymarin also blocked NF-κB activation induced by phorbol ester, LPS, okadaic acid, and ceramide [10]. In this paper, silymarin was used as a positive control.
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2. Materials and methods 2.1. Materials P. chinensis was purchased from Nanning Qianjinzi Chinese Pharmaceutical Co. Ltd. (Nanning, China). A voucher specimen (CALX12071306) was identified by Q.F. Huang at the First Affiliated Hospital of Guangxi Traditional Chinese Medicine University. Silymarin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione reductase (GSH-Rd) and catalase kits were obtained from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). The interleukin-6 and 10 (IL-6 and 10) and tumor necrosis factor-α (TNF-α) kits were purchased from Wuhan Boster Bio-engineering Co. Ltd. (Wuhan, China). 2.2. Herb extraction and chemical analysis The dried and powdered whole plant of P. chinensis (5 kg) was extracted with 95% ethanol at room temperature. After the solvent was evaporated under reduced pressure, the residue from the ethanol extract was successively extracted with petroleum ether, chloroform and acetic ether. The acetic ether fraction (285.2 g) was then subjected to silica gel chromatographic column (200–300 mesh, Yantai, PR China; 10 × 300 cm), eluted with CHCl3-MeOH-H2O (60:35:5, each 500 mL) to produce seven fractions (Frs. 1–7). Fr. 5 was further separated using a silica gel column (3 × 80 cm, 200–300 mesh) via successive elutions with a CHCl3/CH3OH gradient (100:0–0:100, each 200 mL), to yield five sub-fractions (Frs. I–V). Fr. IV was successively separated by Sephadex LH-20 column chromatography (eluted with MeOH) and HPLC (eluted with 35% aqueous MeOH), giving a compound (74.8 mg). The structure of the compound was elucidated based on its physicochemical properties and spectral data. 2.3. Animals and treatments Male C57BL/6 mice (20–22 g) were purchased from Vital River Laboratory Animal Technology Co. (Beijing, China). The research was conducted according to protocols approved by the Institutional Ethical Committee of Guangxi Medical University. After a period of one week, the mice were divided into seven groups with 15 mice per group as follows: Group I, normal control, the mice received the same volume of saline. Group II, TA control, the mice received tormentic acid (3 mg/kg). Group III, model control, the mice received LPS (50 μg/kg) and D-GalN (800 mg/kg). Group IV–VI, TA treatment, the mice received tormentic acid (0.75, 1.5 or 3 mg/kg) plus LPS (50 μg/kg) and D-GalN (800 mg/kg). Group VII, silymarin treatment, the mice received silymarin (50 mg/kg) plus LPS (50 μg/kg) and D-GalN (800 mg/kg). The animals were administered various drugs (TA or silymarin) intraperitoneally once daily for 3 days prior to challenge experimentation. Mice in groups III–VII were then challenged intraperitoneally with LPS/ D-GalN. Blood was sampled from the mouse eyes 1.5 h after LPS/D-GalN administration. The mice were then euthanized by intraperitoneally injecting a high dose of pentobarbitone sodium (150 mg/kg) 6 h after LPS/D-GalN injection. The liver samples were dissected and immediately washed with ice-cold saline. 2.4. Histological analysis and detection of apoptotic cells Liver tissues were fixed in 4% phosphate buffered formalin, dehydrated in graded alcohols and embedded in paraffin blocks. Five-
micrometer-thick paraffin sections were then rehydrated and stained with hematoxylin and eosin (H&E). Apoptotic cells were detected using TUNEL staining with an in situ Apoptosis Detection Kit (TaKaRa) according to the manufacturer's instructions. 2.5. Estimating AST, ALT and total bilirubin activities Serum levels of AST, ALT and total bilirubin were measured using commercially available kits (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China) according to the manufacturer's instructions. 2.6. Determination of serum TNF-α, IL-6 and IL-10 levels The serum TNF-α, IL-6 and IL-10 levels were determined using an enzyme-linked immunosorbent assay (ELISA) (Amersham Pharmacia Biotec, NJ, USA) according to the manufacturer's protocol. 2.7. Serum nitric oxide (nitrite/nitrate) analysis On a serum nitric oxide (NO) (nitrite/nitrate) analysis, a serum at 1.5 h post-injection of LPS/D-GalN was used. The sum of the serum nitrite − and nitrate (NO− 2 and NO3 , stable metabolites of nitric oxide) was measured using a commercial kit (BIOMOL, Plymouth Meeting, PA, USA). 2.8. HO-1 activity assay The HO-1 enzymatic activity in the liver was measured via bilirubin generation, as previously described [11]. The extracted bilirubin was measured using the difference in absorbance at 464 and 530 nm (ε = 40 mM− 1 cm− 1). 2.9. Hepatic lipid peroxidation assays Lipid peroxidation in the liver was determined by measuring the level of malondialdehyde (MDA), an end product of lipid peroxidation, using a thiobarbituric acid method. The level of hepatic MDA was measured according to the protocol established in our previous study [12]. 2.10. Estimation of hepatic antioxidant enzyme activities and glutathione content Liver tissue was homogenized on ice with Tris–HCl (5 mmol/L containing 2 mmol/L EDTA, pH 7.4). The homogenates were centrifuged at 1000 × g for 15 min at 4 °C. The supernatants were immediately used for the SOD, GSH-Px, GSH-Rd and catalase assays. All enzymes were evaluated using commercially available kits (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China) according to the manufacturer's instructions. In addition, the hepatic GSH content was measured using a Glutathione Assay Kit (BioVision, CA, USA). 2.11. RT-PCR for TNF-α and iNOS To examine the mRNA expression levels of TNF-α and inducible nitric oxide synthase (iNOS) in the liver, the total RNA from snap-frozen tissues was extracted using an RNeasy Mini Kit (QIAGEN Inc.). First-strand cDNA was prepared by following the instructions for the SuperScript™ FirstStrand Synthesis System for RT-PCR Kit (Life Technologies). Reverse transcription and amplification by PCR were performed as previously described [13]. The following primer sequences were used: TNF-α: 5′-ATG AGC ACA GAA AGC ATG ATC-3′ (sense) and 5′-TAC AGG CTT GTC ACT CGA ATT-3′ (antisense); iNOS: 5′-GTG AGG ATC AAA AAC TGG GG-3′ (sense) and 5′-ACC TGC AGG TTG GAC CAC-3′ (antisense); and β-actin: 5′-GAT GGT GGG TAT GGG TCA GAA GGA-3′ (sense) and 5′-GCT CAT TGC CGA TAG TGA TGA CCT-3′ (antisense). The PCR products were
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subjected to electrophoresis and ethidium bromide staining. The gels were analyzed by densitometry. β-Actin was used as the internal control. 2.12. Measurement of nuclear NF-κB p65 content Nuclear was extracted from the liver with a Nuclear Extraction Kit (Active Motif, Carlsbad, USA). The nuclear NF-κB p65 content was measured using a NF-κB/p65 ActivELISA kit (Imgenex, San Diego, USA). 2.13. Western blot immunoassay for cytochrome c and Bcl-2 The cytosolic proteins in liver samples were prepared using cytoplasmic extraction reagents according to the manufacturer's instructions (Pierce Biotechnology, Rockford, IL, USA). Briefly, equivalent aliquots of protein extract were separated on 12% SDS-PAGE gel. After electrophoretic separation, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes, incubated with primary antibodies against cytochrome c (BD Biosciences) and Bcl-2 (Santa Cruz Biotechnology Inc., Santa Cruz, USA) and treated with secondary antibodies. The intensity of the immunoreactive bands was determined using a densitometer equipped with Image QuaNT software (Molecular Dynamics, Sunnyvale, CA). The signals were normalized relative to β-actin (Sigma chemical Co.) for cytochrome c and Bcl-2 proteins. 2.14. Evaluation of caspase-3, caspase-8 and caspase-9 activities The liver samples were homogenized in lysis buffer. After centrifugation for 15 min at 25000 ×g, the supernatant was incubated with a substrate peptide (DEVD-AFC for caspase-3, IETD-AFC for caspase-8 and LEHD-AFC for caspase-9). The change in fluorescence (excitation at 400 nm and emission at 490 nm) was monitored after a 120-minute incubation according to the procedure reported by the previous studies [14]. 2.15. Statistical analysis Statistical analysis was performed using SPSS 11.5 for Windows. Differences between the groups were assessed using a one-way analysis of variance (ANOVA) with a Tukey's test for post-hoc multiple comparisons. The data are presented as the means ± SE. A P-value b0.05 was considered to be statistically significant. 3. Results and discussion
Fig. 1. Chemical structure of tormentic acid.
3.2. Effects of TA on histopathological changes The liver in both the normal control and TA control groups revealed normal lobular architecture and cellular structure. However, livers exposed to LPS/D-GalN exhibited multiple extensive areas with portal inflammation and cellular necrosis, as well as a moderate increase in inflammatory cell infiltration. These pathological alterations were ameliorated by 3 mg/kg TA or 50 mg/kg silymarin treatment (Fig. 2). The result suggests that TA has a hepatoprotective effect on this model of hepatic damage.
3.3. Effects of TA on serum ALT, AST and total bilirubin levels Significant elevations of AST, ALT and total bilirubin levels in the serum which are released from the cytosolic part of the liver cells often suggest the pathological condition of severe liver injury [15]. To evaluate the extent of liver injury in mice, we conducted an analysis of serum AST and ALT activities and total bilirubin level. As shown in Fig. 3A and B, significant increases in the serum ALT, AST and total bilirubin levels were observed in LPS/D-GalN animals compared to mice with saline treatment (normal control). Conversely, mice treated with TA or silymarin exhibited a significant decrease in the levels of these parameters. In addition, TA had no effect on the basal serum AST, ALT or total bilirubin level. The data further confirm that TA exerts a hepatoprotective effect during the LPS/D-GalN-induced model of hepatic damage.
3.1. Chemistry The structure of the compound isolated from P. chinensis was elucidated based on its physicochemical properties and spectral data as follows: mp: 265–267 °C, ESI-MS m/z 487 [M–H]−, IR (KBr) cm−1: 3392, 2964, 2918, 2834, 1601, 1452, 1394, 1233, 1157, 1052. 1H-NMR (C5D5 N, 400 MHz) δ: 5.58 (1H, brs, 12-H), 4.12 (1H, m, 2-H), 3.38 (1H, d, J = 9.2 Hz, 3-H), 3.15 (1H, m, 11-H), 3.06 (1H, s, 18-H), 1.73 (3H, s), 1.43 (3H, s), 1.31 (3H, s), 1.09 (3H, d, J = 10.5 Hz), 0.99 (3H, s), 0.98 (3H, s), 0.96 (3H, s). 13 C-NMR (Py-d 5, 125 MHz) δ: 47.2 (C-1), 68.5 (C-2), 83.3 (C-3), 38.9 (C-4), 55.6 (C-5), 18.4 (C-6), 33.3 (C-7), 40.4 (C-8), 47.6 (C-9), 38.1 (C-10), 23.5 (C-11), 126.9 (C-12), 138.9 (C-13), 41.7 (C-14), 28.7 (C-15), 25.9 (C-16), 48.2 (C-17), 54.2 (C-18), 72.3 (C-19), 42.2 (C-20), 26.7 (C-21), 38.2 (C-22), 26.4 (C-23), 23.1 (C-24), 16.6 (C-25), 17.5 (C-26), 24.6 (C-27), 179.8 (C-28), 26.5 (C-29), 16.7 (C-30). These data indicate that the compound is tormentic acid (TA), which has a molecular formula and molecular weight of C30H48O5 and 488.71, respectively. Its chemical structure is shown in Fig. 1.
3.4. Effects of TA on serum IL-6 and IL-10 levels Previous studies demonstrated that various cytokines can protect cells from inflammation byproducts [16]. IL-6 is a typical inflammatory cytokine that regulates the acute phase response in the liver. Furthermore, IL-6 is essential for liver regeneration after a partial hepatectomy and confers resistance to liver injury by hepatic toxins and ischemic injury [17]. The IL-6 blockade aggravates liver injury and induces lethality in LPS/D-GalN hepatitis model [18]. IL-10 is an anti-inflammatory cytokine that reduces the lethality and hepatic injury induced by LPS/D-GalN [19]. Previous study has revealed that IL-10 inhibits the synthesis and secretion of numerous pro-inflammatory cytokines and protects the liver by decreasing liver apoptosis during the LPS-induced hepatitis model [20]. In our study, the serum IL-6 content was lower, while IL-10 level was significantly higher in mice dosed with TA (Fig. 3C and D). In addition, there were no significant differences in the levels of IL-6 and IL-10 between the TA control group and normal control group.
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Fig. 2. TA-mediated histological changes (H&E staining, 100 ×). A1: normal control group, A2: TA control group, A3: model control group, A4: 1.5 mg/kg TA-treated group, A5: 3 mg/kg TA-treated group, and A6: 50 mg/kg silymarin treated group.
3.5. Effects of TA on serum TNF-α content and hepatic TNF-α mRNA level
3.7. Effects of TA on lipid peroxidation and antioxidant enzyme activities
During LPS/D-GalN-induced acute liver injury, TNF-α played an important role in hepatocyte apoptosis or necrosis [21]. LPS directly activates Kupffer cells to produce TNF-α, inducing hepatocyte apoptosis during the early stages of the liver injury in D-GalN-sensitized mice. Furthermore, TNF-α-induced neutrophil transmigration occurs during the later stages of the liver injury, causing massive hepatocyte necrosis and organ failure [22]. In the present study, administration of LPS/D-GalN markedly increased serum TNF-α content and hepatic TNF-α mRNA expression, while treatment with TA or silymarin attenuated its levels (Fig. 3E and F). As mentioned above, LPS/D-GalN has been shown to induce the inflammatory cells to release various pro-inflammatory cytokines, including TNF-α and IL-6, followed by the production of antiinflammatory cytokines, such as IL-10 and IL-4. Exposure to large doses of LPS causes extreme “endotoxemia” or “septic shock” whereby the exposed host exhibits hypotension, disseminated intravascular coagulation, and widespread tissue injury that can result in death due to multiple organ failure [23]. However, the problem is not the overexpression of pro-inflammatory cytokines, but rather that they are not adequately balanced by endogenous anti-inflammatory mediators [24]. We found that by increasing the dose of TA, levels of TNF-a and IL-6 decreased, while the content of IL-10 increased. We think that TA protects LPS/D-GalN-induced acute liver injury mainly by modulating the balance of pro-inflammatory and anti-inflammatory cytokines.
Free radicals generate the lipid peroxidation process in an organism. Malondialdehyde (MDA) is one of the final products of polyunsaturated fatty acid peroxidation in the cells. An increase in free radicals causes overproduction of MDA. Its level is commonly known as a marker of oxidative stress [27]. As shown in Fig. 4C, the hepatic MDA level, which is the end-product of lipid peroxidation, increased significantly in the model group. Treatment with TA or silymarin effectively decreased the MDA level. In addition, the effects of TA on the hepatic SOD, GSH-Px, GSH-Rd and catalase activities, as well as on the GSH content, are shown in Fig. 4D. The four hepatic antioxidant enzyme activities and the GSH content significantly decreased 6 h after LPS/D-GalN administration. In contrast, treatment with 3 mg/kg TA or 50 mg/kg silymarin significantly increased the activities of these enzymes, as well as the GSH content. TA alone had negligible effects on the basal lipid peroxidation and antioxidant enzyme activities. These results indicate that TA inhibits lipid peroxidation and effectively recruits the anti-oxidative defense system in liver injury induced by LPS/D-GalN.
3.6. Effects of TA on nitric oxide (NO) content and iNOS mRNA level Nitric oxide (NO) is a highly reactive oxidant, produced by parenchymal and nonparenchymal liver cells from L-arginine via the action of inducible nitric oxide synthase (iNOS) [25]. It is well-known that NO participates in diverse physiological and pathological processes, and may exert toxicological effects such as inhibition of mitochondrial respiration and DNA synthesis as well as formation of peroxynitrite, a more long-lived cytotoxic oxidant when over-generated under stress conditions [26]. In the present study, significant increases in the serum NO content and the liver iNOS mRNA level were observed in the model control group. Treatment with 1.5 or 3 mg/kg TA or with 50 mg/kg silymarin significantly inhibited these increases (Fig. 4A and B). Therefore, inhibiting NO also contributes to the hepatoprotective effect of TA.
3.8. Effect of TA on HO-1 activity Heme oxygenase (HO) is a rate-limiting enzyme that catalyzes the breakdown of heme into antioxidant and anti-inflammatory agents such as biliverdin, carbon monoxide, and iron. Previous studies have demonstrated that HO-1 expression is rapidly upregulated in the liver after administration of toxins such as carbon tetrachloride, endotoxin and LPS/D-GalN [11]. Chemically inducing HO-1 expression with a compound such as hemin is cytoprotective against LPS/D-GalN-induced acute hepatic injury in rats [28]. This study confirmed a significant increase in the HO-1activity after LPS/D-GalN injection. These alterations were augmented by TA treatment (Fig. 4E), suggesting that TA induces HO-1 activity and/or suppresses the degradation of this enzyme. TA alone had no significant effect on the basal HO-1 activity. 3.9. Effect of TA on nuclear NF-κB p65 content Nuclear factor-kappa B (NF-κB) is a master regulator of the hepatic inflammatory response. Under basal conditions, NF-κB is present in the cytoplasm of hepatocytes in a latent form, bound to the NF-κB inhibitory protein, inhibitor kappa B (IκB). Upon exposure to pro-inflammatory stimuli, the IκB kinase (IKK) complex is activated and catalyzes the phosphorylation of IκB. Phosphorylated IκB is then targeted for degradation by
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Fig. 3. Effects of TA on the serum ALT and AST (A) and total bilirubin levels (B), serum IL-6 (C) and IL-10 contents (D), serum TNF-α (E), and hepatic TNF-α mRNA level (F). The results are presented as the means ± SE. ⁎P b 0.05 compared to the LPS/D-GalN model control group.
the 26S proteosome complex, thereby liberating NF-κB to migrate to the cell nucleus and direct transcription of target genes [29]. It is clear that NF-κB directly regulates inflammatory cytokines, such as TNF-α, IL-6 and iNOS [30,31]. Because these inflammatory mediators are implicated in the pathophysiology of organ failure after shock and sepsis, inhibition of NF-κB activation could be a very effective strategy to reduce an excessive inflammatory response. In our study, treating mice with 3 mg/kg TA or 50 mg/kg silymarin significantly decreased the nuclear NF-κB p65 content compared to the model group (Fig. 4F). Thus, we speculate that TA inhibited the excessive release of inflammatory cytokines caused by LPS/D-GalN which may result from reduced NF-κB activation.
3.10. Effects of TA on apoptotic cell, apoptosis-related proteins and caspase Apoptosis has been observed in animal models for LPS/D-GalN injury. There are two major apoptotic pathways: the extrinsic pathway triggered by the activation of caspase-8 and the intrinsic pathway triggered by the activation of caspase-9 [32]. Caspase-3 is a predominant downstream effector activated by caspases 8 and 9 [33]. Massive activation of caspases is a crucial process during the induction of apoptosis associated with the pathogenesis of acute hepatic failure [34]. In addition, the caspase cascade is primarily associated with the release of cytochrome c from mitochondria, as well as caspases 3 and 9, during apoptosis [32]. The Bcl-2 family, including pro-apoptotic and anti-apoptotic proteins,
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Fig. 4. Effects of TA on serum NO content (A), liver iNOS mRNA level, lipid peroxidation (C), antioxidant enzyme activities (D), hepatic HO-1 activity (E), and nuclear NF-κB p65 content (F). The results are presented as the means ± SE. ⁎P b 0.05 compared to the LPS/D-GalN model control group.
helps control apoptosis, and the overexpression of Bcl-2 is antiapoptotic [35]. In this study, apoptotic hepatocytes were detected using TUNEL staining. Numerous TUNEL positive hepatocytes were observed in liver tissues obtained 6 h after LPS/D-GalN injection. However, few positive hepatocytes were observed in livers treated with 3 mg/kg TA or 50 mg/kg silymarin (Fig. 5A). Furthermore, the apoptosis-related proteins, including cytochrome c and Bcl-2, were detected using a Western blot immunoassay. As shown in Fig. 5B, after injecting LPS/D-GalN, the level of cytochrome c expression increased significantly compared to the normal control group. TA administration reduced cytochrome c expression relative to the LPS/D-GalN group. While Bcl-2 showed little expression in the LPS/D-GalN group, treatment with TA augmented it. Furthermore, the activities of caspases 3, 8 and 9 in the cytosol fraction 6 h after LPS/D-GalN administration increased rapidly relative to the normal
control group. The elevated caspase 3, 8 and 9 activities were attenuated by treatment with 3 mg/kg TA or 50 mg/kg silymarin. In addition, TA alone had a negligible effect on the basal levels of apoptotic cell, apoptosis-related proteins and caspase (Fig. 5C). Thus, the protective effect of TA against LPS/D-GalN-induced hepatic injury might be mediated by its anti-apoptotic effects. 4. Conclusion In conclusion, TA protects hepatocytes against LPS/D-GalN-induced acute liver injury in a dose-dependent manner, possibly by modulating the balance of pro-inflammatory and anti-inflammatory cytokines via inhibition of NF-κB, and attenuating hepatocellular apoptosis. Therefore, TA is a potential agent for preventing acute liver injury, and TA may be a major bioactive ingredient of P. chinensis.
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Fig. 5. Effects of TA on apoptotic cells (A), apoptosis-related proteins (B), and caspase (C). A: TUNEL staining (200×), A1: normal control group, A2: TA control group, A3: model control group, A4: 1.5 mg/kg TA-treated group, A5: 3 mg/kg TA-treated group, and A6: 50 mg/kg silymarin treated group. The results are presented as the means ± SE. ⁎P b 0.05 compared to the LPS/D-GalN model control group.
Acknowledgments The authors gratefully acknowledge the financial support provided by the National Natural Science Foundation of China (Nos. 81260674; 81260505), the Guangxi Natural Science Foundation (Nos. 2013GXNSFAA019146; 2013GXNSFAA019150), and the Foundation for the Guangxi Key Laboratory for Prevention & Treatment of Regional High-Incidence Diseases (KFJJ2010-71; KFJJ2011-37). References [1] Rahman TM, Hodgson HJ. Animal models of acute hepatic failure. Int J Exp Pathol 2000;81:145–57. [2] Wang F, Wen T, Chen X-Y, Wu H. Protective effects of pirfenidone on D-galactosamine and lipopolysaccharide-induced acute hepatotoxicity in rats. Inflamm Res 2008;57:183–8. [3] Bohlinger I, Leist M, Gantner F, Angermüller S, Tiegs G, Wendel A. DNA fragmentation in mouse organs during endotoxic shock. Am J Pathol 1996;149:1381. [4] Wu J, Danielsson Å, Zern MA. Toxicity of hepatotoxins: new insights into mechanisms and therapy. Expert Opin Investig Drugs 1999;8:585–607.
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