Zhikang Capsule Ameliorates Inflammation, Drives Polarization to M2 Macrophages, and Inhibits Apoptosis in Lipopolysaccharide-induced RAW264.7 Cells.

OBJECTIVE: To explore the anti-inflammatory effect of the traditional Chinese medicine Zhikang capsule (ZKC) on lipopolysaccharide (LPS)-induced RAW264.7 cells. METHODS: Safe concentrations of ZKC (0.175, 0.35, and 0.7 mg/mL) were used after the half-maximal inhibitory concentration (IC50) of RAW264.7 cells was calculated through the CCK-8 assay. In addition, the optimal intervention duration of ZKC (0.7 mg/mL) on RAW264.7 cells was determined to be 6 h, since all proinflammatory mediators [tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), inteleukin-6 (IL-6), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and monocyte chemotactic protein-1 (MCP-1)] had a decreasing tendency and relatively down-regulated mRNA expression levels as compared with other durations (4, 8, and 12 h). RAW264.7 cells were pretreated with ZKC at various concentrations (0.175, 0.35 and 0.7 mg/mL) for 6 h and then stimulated with LPS (1 µg/mL) for an additional 12 h. RESULTS: In terms of inflammation, ZKC could reverse LPS-induced upregulation of TNF-α, IL-1ß, IL-6, COX-2, iNOS, and MCP-1 at both the mRNA and protein levels in RAW264.7 cells in a dose-dependent manner. In terms of the NF-κB signaling pathway, ZKC could reduce phosphorylated p65 and promote M2 polarization of RAW264.7 cells under LPS stimulation in a dose-dependent manner. Moreover, ZKC exhibited a protective effect on macrophages from apoptosis. CONCLUSION: ZKC exhibited obvious antiinflammatory and anti-apoptotic effects on LPS-induced RAW264.7 cells at the cellular level, and a weakened NF-κB signaling pathway may be a potential significant target.

Scoparone alleviates inflammation, apoptosis and fibrosis of non-alcoholic steatohepatitis by suppressing the TLR4/NF-κB signaling pathway in mice.

Scoparone, a naturally-occurring, bioactive compound isolated from the Chinese herb Artemisia capillaria, has been shown to ameliorate hepatotoxicity and cholestasis in liver diseases. However, the pharmacological effect of scoparone in non-alcoholic steatohepatitis (NASH) has not been elucidated. In this study, we investigated the protective effects and mechanisms of scoparone in NASH. In vivo, the NASH model was established in mice fed a methionine and choline-deficient (MCD) diet for 4weeks, with or without simultaneous scoparone treatment. In vitro, RAW264.7 cells induced by lipopolysaccharide (LPS) were pretreated with or without different concentrations of scoparone. Hepatic triglycerides and serum AST and ALT levels were examined by biochemical assays. Hepatic histology was assessed by H&E, oil red O and Masson's trichrome staining methods, which were applied to analyze the protective effects of scoparone in NASH. To further explore the underlying mechanism of scoparone, immunohistochemistry, TUNEL, qRT-PCR, and Western blotting assays were applied to liver tissue or LPS-induced RAW264.7 cells. We found that scoparone can effectively improve hepatic steatosis, apoptosis, inflammation, and fibrosis in an MCD diet-induced NASH murine model. Mechanistically, we demonstrated that scoparone treatment alleviates NASH- and lipopolysaccharide (LPS)-induced immune responses in macrophages partly by blocking TLR-4/NF-κB signaling in a dose-dependent manner. Taken together, our results present the potential protective effects and mechanism of scoparone in NASH, suggesting a potentially beneficial drug treatment for NASH.

Diallyl disulfide attenuates non­alcoholic steatohepatitis by suppressing key regulators of lipid metabolism, lipid peroxidation and inflammation in mice.

Non­alcoholic steatohepatitis (NASH) is a common clinicopathological condition. Currently, the pathogenesis of NASH remains unknown, and no optimal therapy option currently exists. It has previously been demonstrated that diallyl disulfide (DADS) was capable of attenuating liver dysfunction, as DADS supplementation had a positive impact on liver regeneration, proliferation and oxidative damage. Thus, DADS could serve as a potential therapeutic agent that can protect against the effects of NASH. The present study aimed to evaluate the effect of DADS on NASH and to understand the associated underlying molecular mechanisms. A methionine­ and choline­deficient diet (MCD) and high­fat diet (HFD) are the two common animal models that induce NASH. C57BL/6J mice were fed an MCD for 4 weeks, or an HFD for 20 weeks, in the present study. The mice were treated with or without DADS (20, 50 and 100 mg/kg) for 4 or 20 weeks. For the histopathological examination, hematoxylin and eosin staining, oil red O staining and immunohistochemical analyses were performed using the liver sections. Biochemical assays and ELISA were performed to measure the serum biochemical indicators of hepatic function and inflammatory indicators, respectively. Reverse transcription­quantitative PCR, immunofluorescence and western blotting were used to detect expression levels of the genes involved in the molecular mechanisms underlying DADS protection. MCD or HFD induced the histological features of NASH in mice, including significant vacuolated hepatocytes, marked inflammatory cell infiltration and severe micro­ and macro­vesicular steatosis. Serum alanine transferase and aspartate aminotransferase levels, as well as the contents of liver triglyceride and total cholesterol, were significantly increased in these two models. DADS attenuated these histological and biochemical changes. DADS ameliorated hepatic steatosis by regulating sterol regulatory element­binding transcription factor 1, apolipoprotein A1, cyclic AMP­responsive element­binding protein H and fibroblast growth factor 21. Furthermore, DADS was revealed to prevent lipotoxicity via peroxisome proliferator­activated receptor α elevation and stearoyl­coenzyme A desaturase 1 inhibition in HFD­fed mice. In addition, DADS markedly inhibited lipid peroxidation by modulating malondialdehyde and superoxide dismutase, and it also decreased tumor necrosis factor­α production, interleukin­6 production and macrophage influx, as well as suppressing nuclear factor­κB activation, indicating suppression of MCD­induced hepatic inflammation. Taken together, the results have shown that DADS exerts beneficial effects on MCD­ or HFD­induced NASH by suppressing key regulators of lipid metabolism, lipid peroxidation and inflammation.

Gadoxetic Acid-Enhanced Hepatobiliary-Phase Magnetic Resonance Imaging for Pyrrolizidine Alkaloid-Induced Hepatic Sinusoidal Obstruction Syndrome and Association with Liver Function.

Hepatic sinusoidal obstruction syndrome (HSOS) can be caused by pyrrolizidine alkaloids(PAs)-containing herbals. In this study, the aim of our study was to investigate the imaging features of PAs-induced HSOS on gadoxetic acid-enhanced magnetic resonance imaging (MRI), susceptibility-weighted imaging(SWI) and T2* weighted imaging (T2* WI). We analyzed medical records and MR images of 28 PAs-induced HSOS patients enrolled from Feb, 2013, to Apr, 2017. Abnormal liver function was observed in most of the PAs-induced HSOS patients. Heterogeneity of liver parenchyma in hepatobillary phase (HBP) of gadoxetic acid-enhanced MR scan was observed in 100% of the PAs-induced HSOS patients. Distributional patterns of heterogeneous hypointensity were multifocal distribution (mild) in 4 patients (14.29%), multifocal distribution (severe) in 15 cases (53.57%), and diffuse distribution in 9 patients (32.14%). Hypointense in SWI and T2*WI was observed in the patients of PAs-induced HSOS, and the distribution of hypointense in SWI and T2*WI was similar to that of portal-venous phase of MR scan. The severity of heterogeneous hypointensity scored by volume fraction in hepatobillary phase of gadoxetic acid-enhanced MRI was positively correlated with PT and INR, the severity of hypointensity in HBP was a risk factor of death events. In conclusion: Heterogenous hypointensity of liver parenchyma was an imaging sign of hepatobillary phase in gadoxetic acid-enhanced MRI; thus, it will provide evidences for the diagnosis of PA-induced HSOS.

Baicalin attenuates non-alcoholic steatohepatitis by suppressing key regulators of lipid metabolism, inflammation and fibrosis in mice.

AIMS: Baicalin (BA), an active flavonoid compound originating from the herb of Scutellaria baicalensis Georgi, has been previously shown to exert anti-inflammation and anti-oxidant effects in liver diseases. However, the potential role of BA in the regulation of non-alcoholic steatohepatitis (NASH) remains elusive. In this study, we newly explored the hepatoprotective effects of BA in MCD diet-induced NASH by ameliorating hepatic steatosis, inflammation, fibrosis and apoptosis. MAIN METHODS: NASH was induced in mice fed a methionine and choline-deficient (MCD) diet for 4weeks. The mice were simultaneously treated with or without BA for 4weeks. Serum liver functional markers and inflammatory indicators were assessed by biochemical and ELISA methods, respectively. The livers were histologically examined using H&E, Oil Red O and Masson's trichrome staining methods. The qRT-PCR, IHC and Western blotting assays were applied to analyze mechanisms underlying BA protection. KEY FINDINGS: BA treatment significantly attenuated MCD diet-induced hepatic lipid accumulation partly through regulating the expression of SREBP-1c, FASN, PPARα and CPT1a. BA treatment dramatically suppressed MCD diet-induced hepatic inflammation, which was associated with decrease in serum TNF-α, IL-1ß and MCP-1 production, macrophage influx and suppression of nuclear factor-κB activation. Additionally, BA was proved to prevent liver fibrosis, which appears to be mediated by inhibition of α-SMA, TGF-ß1 and Col1A1. Furthermore, BA markedly inhibited hepatocyte apoptosis and cleaved caspase-3 protein expression in MCD diet-induced mice. SIGNIFICANCE: These results provide a possible basis of the underlying mechanism for the application of BA in the treatment of NASH.

Diagnostic performance of Contrast-enhanced CT in Pyrrolizidine Alkaloids-induced Hepatic Sinusoidal Obstructive Syndrome.

Hepatic sinusoidal obstruction syndrome (HSOS) can be caused by pyrrolizidine alkaloids(PAs)-containing herbals. Since PAs exposure is obscure and clinical presentation of HSOS is unspecific, it is challenge to establish the diagnosis of PAs-induced HSOS. Gynura segetum is one of the most wide-use herbals containing PAs. The aim of our study is to describe the features of contrast-enhanced computed tomography (CT) in gynura segetum-induced HSOS, and then determine diagnostic performance of radiological signs. We retrospectively analyzed medical records and CT images of HSOS patients (71 cases) and the controls (222 cases) enrolled from January 1, 2008, to Oct 31, 2015. The common findings of contrast CT in PAs-induced HSOS included: ascites (100%), hepatomegaly (78.87%), gallbladder wall thickening (86.96%), pleural effusion (70.42%), hepatic vein narrowing (87.32%), patchy liver enhancement (92.96%), and heterogeneous hypoattenuation (100%); of these signs, patchy enhancement and heterogeneous hypoattenuation were valuable features. Then, the result of diagnostic performance demonstrated that contrast CT possessed better performance in diagnosing PAs-induced HSOS compared with various parameters of Seattle criteria. In conclusion, the patients with PAs-induced HSOS display distinct radiologic features at CT-scan, which reveals that contrast-enhanced CT provides an effective noninvasive method for diagnosing PAs-induced HSOS.

Zhikang Capsule ameliorates dextran sodium sulfate-induced colitis by inhibition of inflammation, apoptosis, oxidative stress and MyD88-dependent TLR4 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Zhikang Capsule (ZKC) is a traditional Chinese medicine (TCM) modified from classic formulas Qi-Li-San (an ancient formula dating to Qing Dynasty) and Fu-Jin-Sheng-Ji-San (written into The Golden Mirror of Medicine). ZKC contains 14 kinds of materials and has been widely used for the clinical therapy of inflammatory bowel diseases (IBD) for a long time. However, the therapeutic mechanisms of ZKC are still unclear. AIM OF THE STUDY: To determine the protective effect of ZKC on dextran sodium sulfate (DSS)-induced colitis and explore the underlying mechanisms. MATERIALS AND METHODS: C57BL/6 mice were fed with 3% DSS in drinking water for one week to induce experimental colitis. They were randomly assigned to six groups according to the treatment conditions. The histological changes of colon tissues were observed by hematoxylin and eosin (H&E) staining. The serum concentration of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1ß, and IL-12) and anti-inflammatory mediators (IL-4 and IL-10) was detected by enzyme-linked immune sorbent assays (ELISAs). The production of MPO, SOD, MDA, NO, and caspase-3 was assessed by biochemical assay kits. The expression of iNOS, ICAM-1, and NF-ΚB was evaluated by immunohistochemistry staining. The levels of TLR4, MyD88, and TRAF6 were determined by western blot. RESULTS: Histologic analysis exhibited that ZKC alleviated the inflammation, loss of goblet cells, and submucosal edema induced by DSS. ZKC significantly suppressed the pro-inflammatory cytokines and promoted the anti-inflammatory mediators. The antioxidation of ZKC was indicated by increased activity of SOD and reduced production of MDA, NO, and iNOS in ZKC-treated mice. Furthermore, ZKC repressed the colonic expression of caspase-3 and the activity of the MyD88-dependent TLR4 signaling pathway. CONCLUSIONS: This research demonstrated the protective effect of ZKC on DSS-induced colitis. For the first time, we identified four therapeutic mechanisms of ZKC, including effective inhibition of the inflammatory responses, significant alleviation of intestinal epithelium apoptosis, considerable prevention of oxidative stress, and selective down-regulation of the MyD88-dependent TLR4 signaling pathway. With high therapeutic effects and low toxic effects, ZKC exhibits great superiority over western medicines in IBD treatment.