Capsaicin receptor TRPV1 maintains quiescence of hepatic stellate cells in the liver via recruitment of SARM1.

BACKGROUND & AIMS: Capsaicin receptor, also known as transient receptor potential vanilloid 1 (TRPV1), is involved in pain physiology and neurogenic inflammation. Herein, we discovered the presence of TRPV1 in hepatic stellate cells (HSCs) and aimed to delineate its function in this cell type and liver fibrosis. METHODS: TRPV1 expression was examined in liver biopsies from patients with liver fibrosis using quantitative real-time PCR and immunostaining. Its contribution to liver fibrosis was examined in Trpv1-/- mice, upon lentiviral delivery of the TRPV1 gene, and in human and mouse primary HSCs, using patch clamp, intracellular Ca2+ mobilization determination, FACS analyses and gain/loss of function experiments. Binding of sterile alpha and Toll/interleukin-1 receptor motif-containing protein 1 (SARM1) to TRPV1 was determined using mass spectrometry, co-immunoprecipitation, surface plasmon resonance, bioluminescence resonance energy transfer, and NanoBiT. RESULTS: TRPV1 mRNA levels are significantly downregulated in patients with liver fibrosis and mouse models, showing a negative correlation with F stage and α-smooth muscle actin expression, a marker of HSC activation. TRPV1 expression and function decrease during HSC activation in fibrotic livers in vivo or during culture. Genetic and pharmacological inhibition of TRPV1 in quiescent HSCs leads to NF-κB activation and pro-inflammatory cytokine production. TRPV1 requires binding of its N-terminal ankyrin repeat domain to the TIR-His583 (Toll/interleukin-1 receptor) domain of SARM1 to prevent HSCs from pro-inflammatory activation. Trpv1-/- mice display increased HSC activation and more severe liver fibrosis, whereas TRPV1 overexpression is antifibrotic in various disease models. CONCLUSION: The antifibrotic properties of TRPV1 are attributed to the prevention of HSC activation via the recruitment of SARM1, which could be an attractive therapeutic strategy against liver fibrosis. IMPACT AND IMPLICATIONS: We identified the neuronal channel protein TRPV1 as a gatekeeper of quiescence in hepatic stellate cells, a key driver of liver fibrogenesis and chronic liver disease. Physiologically expressed in healthy liver and consistently downregulated during liver fibrosis development, its therapeutic re-expression is expected to have few side effects, making it an attractive target diagnostic tool and drug candidate for industry and clinicians.

Efficacy and Safety of Yanggan Jian in Hepatitis B Virus-related Decompensated Cirrhosis: A Randomized, Double-blind, Controlled Trial.

Background and Aims: The aim was to evaluate the efficacy and safety of Yanggan Jian (YGJ) in HBV-infected patients with decompensated cirrhosis. Methods: This randomized, double-blind controlled trial enrolled 160 patients with HBV-related decompensated cirrhosis who were already receiving or about to start antiviral therapy. Patients were randomly assigned to receive YGJ or placebo for 24 weeks, and were followed-up to 36 weeks. The primary outcome was the proportion of patients with a ≥2 point reduction in Child-Turcotte-Pugh (CTP) score from baseline at week 24. Secondary outcomes were CTP class and score, serum liver function indices, mortality, incidence of hepatocellular carcinoma and variceal bleeding. Results: The proportion of patients with a CTP score reduction ≥2 was significantly greater in the YGJ than in the placebo group (p=0.009); the percentage of patients with CTP class C was significantly less than that in the placebo group (p<0.05), and the YGJ group had a significantly greater mean change from baseline in CTP score at week 24 (p=0.034). The improvement in measured values and change from baseline of prothrombin time, serum albumin, platelets, cholinesterase, international normalized ratio, and activated partial thromboplastin time were significantly better with YGJ than with placebo. Between-group differences in cumulative rates of variceal bleeding, hepatocellular carcinoma, death, or the frequency of any adverse event (AE), AEs related to treatment, or discontinuation because of AEs were not significant. Conclusions: YGJ significantly improved CTP scores and hepatic synthetic and reserve function in patients with HBV-related decompensated cirrhosis, and was safe and well tolerated.

Serum Glial Cell Line-Derived Neurotrophic Factor (sGDNF) Is a Novel Biomarker in Predicting Cirrhosis in Patients with Chronic Hepatitis B.

Objectives: We assessed the potential of glial cell line-derived neurotrophic factor (GDNF) as a useful biomarker to predict cirrhosis in chronic hepatitis B (CHB) patients. Methods: A total of 735 patients from two medical centers (385 CHB patients and 350 healthy controls) were included to determine the association of serum and tissue GDNF levels with biopsy-proven cirrhosis. The diagnostic accuracy of serum GDNF (sGDNF) was estimated and compared with other indices of cirrhosis. Results: We showed significantly higher levels of sGDNF in CHB patients with fibrosis (28.4 pg/ml vs. 11.6 pg/ml in patients without) and patients with cirrhosis (33.8 pg/ml vs. 23.5 pg/ml in patients without). The areas under receiver operating curve (AUROCs) of sGDNF were 0.83 (95% confidence interval (CI): 0.80-0.87) for predicting liver fibrosis and 0.84 (95% CI: 0.79-0.89) for cirrhosis. Findings from the serum protein level and hepatic mRNA expression were consistent. Using the best cutoff to predict cirrhosis, we categorized the patients into sGDNF-high and sGDNF-low groups. The sGDNF-high group had significantly larger Masson's trichrome and reticulin staining-positive area, higher Scheuer score, and METAVIR fibrosis stage (all p < 0.001) but not steatosis. On multivariable regression, sGDNF was independently associated with cirrhosis with an odds ratio of 6.98 (95% CI: 1.10-17.94). Finally, we demonstrated that sGDNF outperformed AST to platelet ratio index, FIB-4, fibroscore, forn index, and fibrometer in differentiating F4 vs. F3. Conclusion: Using serum, tissue mRNA, and biopsy data, our study revealed a significant potential of sGDNF as a novel noninvasive biomarker for cirrhosis in CHB patients.

Efficacy and safety of bicyclol for treating patients with idiosyncratic acute drug-induced liver injury: A multicenter, randomized, phase II trial.

BACKGROUND AND AIMS: Evidence for using bicyclol in drug-induced liver injury (DILI) is limited. This study aimed to explore the efficacy and safety of bicyclol in acute DILI. METHODS: This was a multicenter, randomized, double-blinded, double-dummy, active-controlled, superiority and phase II trial. Patients with idiosyncratic acute DILI were randomized 1: 1:1 to low-dose bicyclol (25 mg times a day [TID]), high-dose bicyclol (50 mg TID) and polyene phosphatidylcholine (control) groups. The primary endpoint was the decrease from baseline in serum alanine aminotransferase (ALT) levels at post-treatment for 4 weeks. RESULTS: Overall, 241 patients were included in the full analysis set, with 81, 82 and 78 patients in the low-dose bicyclol, high-dose bicyclol, and control groups respectively. ALT levels decreased across groups (-249.2 ± 151.1, -273.6 ± 203.1, and -180.8 ± 218.2 U/L in the low-dose bicyclol, high-dose bicyclol and control groups, respectively; both p < .001, the bicyclol-dependent groups vs. control group). The ALT normalization rates at weeks 1, 2, 4, 6 and 8 were higher in the bicyclol-dependent groups than in the control group (p = .002 at week 1 and all p < .001 at weeks 2, 4, 6 and 8 respectively). The median times to ALT normalization in the low-dose bicyclol, high-dose bicyclol and control groups were 29, 16 and 43 days respectively. Adverse events, serious adverse events and adverse drug reactions were similar across groups. CONCLUSIONS: Bicyclol (25 and 50 mg TID) appeared efficacious and safe for treating idiosyncratic acute DILI, while bicyclol 50 mg TID showed higher efficacy. TRIAL REGISTRATION NUMBER: www. CLINICALTRIALS: gov (registration no. NCT02944552).

Glial cell line-derived neurotrophic factor contributes to alcoholic-induced liver injury by regulating the NF-κB pathway.

BACKGROUND: Alcoholic liver disease (ALD) is associated with high morbidity and mortality worldwide. The pathogenesis of ALD is not completely understood. Although accumulating evidence suggests an important role of glial cell line-derived neurotrophic factor (GDNF) in several diseases, there are no data concerning its role in ALD. This study compared patients with ALD with control subjects and used a mouse model and a cell culture model to investigate the function of GDNF in ALD and its mechanism of action in hepatocyte injury. METHODS: Serum levels of GDNF were measured in 25 patients with ALD and 25 healthy control subjects. A 4-week Lieber-DeCarli ethanol (EtOH) liquid diet combined with the Gao-Binge model was used in the mouse study. Mouse primary hepatocytes and Huh-7 cells were used for cell experiments. The parameters of liver injury, inflammatory cytokines, and lipid metabolism were measured. RESULTS: Patients with alcoholic hepatitis had higher serum GDNF than control subjects. Expression of GDNF mRNA and protein was markedly increased in mice in the chronic-plus-binge ALD mouse model. The level of GDNF mRNA was upregulated in primary hepatic stellate cells isolated from ethanol-fed mouse liver. Ethanol induced GDNF expression in LX2 cells. The levels of inflammatory cytokines (tumor necrosis factor α, interleukin 1ß, and monocyte chemotactic protein 1) were significantly increased after GDNF stimulation in primary hepatocytes and Huh-7 cells. After GDNF stimulation, levels of both p-AKT and p-NF-κB were significantly increased in primary hepatocytes and Huh-7 cells. The NF-κB activity induced by GDNF was significantly decreased by an NF-κB inhibitor, which limited hepatocyte injury and inflammation. CONCLUSIONS: The concentration of GDNF is increased in the circulation of ALD patients. GDNF promotes alcohol-induced liver injury and inflammation via the activation of NF-κB, which mediates hepatocyte injury and inflammatory cytokine expression. Based on these findings, GDNF is a potential therapeutic target for preventing or ameliorating liver injury in ALD.

Neutrophil-lymphocyte ratio and the risk of hepatocellular carcinoma in patients with hepatitis B-caused cirrhosis.

AIM: The neutrophil-to-lymphocyte ratio (NLR) has been reported as a prognostic marker of hepatocellular carcinoma (HCC); however, the relationship between NLR and risk of HCC occurrence has yet to be systematically elucidated. We aimed to investigate the association between NLR and HCC risk in patients with hepatitis B-caused cirrhosis (HBC) undergoing antiviral therapy. METHODS: A total of 1599 patients with HBC receiving entecavir or tenofovir at three tertiary hospitals between June 2014 and November 2017 were included. Cox proportional hazards regression was used to identify the association between NLR and risk of HCC occurrence by adjusting for potential risk factors. The cumulative incidence of HCC was evaluated using Kaplan-Meier analysis. RESULTS: At study enrollment, the median NLR was 2.0 (interquartile range, 1.4-3.0). The 3-year cumulative probabilities of HCC were 4.8, 8.4, 13.2, and 18.0% across the NLR quartiles, respectively (P < 0.001). Compared with the lowest quartile, higher NLR correlated with an increased HCC occurrence [NLR 1.4-2.0: adjusted hazard ratio (aHR), 1.18 (95% confidence interval (CI), 1.11-1.25); NLR 2.0-3.0: aHR, 2.09 (95% CI, 1.19-3.66); NLR > 3.0: aHR, 2.80 (95% CI, 1.59-4.95); P for trend = 0.001] in the fully adjusted models. In the subgroup analysis, elevated NLR was associated with increased HCC risk, regardless of stratification criteria. CONCLUSION: Elevated NLR is an independent risk factor for HCC occurrence in patients with HBC undergoing antiviral therapy.

Peroxisome proliferator-activated receptor γ inhibits hepatic stellate cell activation regulated by miR-942 in chronic hepatitis B liver fibrosis.

AIMS: Liver fibrosis is a chronic liver disease characterized by hepatic stellate cell (HSC) activation. Peroxisome proliferator-activated receptor gamma (PPARγ) play an important role in HSC activation. This study aimed to investigate the role of PPARγ in the progression of human hepatic fibrosis and the mechanism by which microRNA-942 regulates HSC activation. METHODS: 70 chronic hepatitis B (CHB) patients liver tissues were used to assess PPARγ, α-SMA and miR-942 levels by immunoblot and real-time PCR. Human primary HSCs or LX2 cells were used to perform multiple molecular experiments based on the transfection of small interfering RNA (siRNA) or co-transfection of microRNA inhibitor. Site-directed mutagenesis and luciferase reporter assays were used to identify miR-942 targets. miR-942 expression and localization in hepatic fibrosis and co-localization between α-SMA were determined by fluorescence in situ hybridization (FISH). KEY FINDINGS: The mRNA expression of PPARγ was decreased in activated HSCs and CHB patients with liver fibrosis, which was negatively correlated with F stage and α-SMA. miR-942 negatively regulates PPARγ expression via targeting the PPARγ 3'UTR. Inhibiting PPARγ promoted TGFß1 induced HSC activation, and this effect was blocked after inhibiting the miR-942. Moreover, miR-942 was mainly expressed in fibrous septa and negatively correlated with PPARγ in liver fibrosis. SIGNIFICANCE: PPARγ targeting by miR-942 and decreasing HSC activation in human hepatic fibrosis. Hence, regulating PPARγ may be a promising therapeutic strategy for hepatic fibrosis.

Nomogram for Individualized Prediction of Hepatocellular Carcinoma with Portal Vein Tumor Thrombosis on Conservative Treatment.

BACKGROUND: Portal vein tumor thrombosis (PVTT) is one of the major predictive factors for patients with hepatocellular carcinoma (HCC). The objective of this study was to establish a prognostic nomogram for identifying individual survival outcomes in patients with HCC and PVTT on conservative treatment based on specific factors. METHODS: Two hundred and ten patients with HCC and PVTT on conservative treatment in Beijing Ditan Hospital between June 2008 and May 2017 were studied retrospectively as a derivation cohort. We built a nomogram based on independent risk factors for survival prediction. The concordance index (c-index) and a calibration curve were used to evaluate the predictive accuracy. During the study, 102 patients were included at the Putuo Hospital and Third People's Hospital of Changzhou as a validation cohort. RESULTS: In the derivation cohort, the independent factors for overall survival were identified by multivariate analysis, namely, aspartate aminotransferase ≥119 IU/L, gamma-glutamyl transferase ≥115 IU/L, Child-Pugh class C liver function, creatinine ≥91 µmoI/L, α-fetoprotein ≥400 ng/ml, and largest tumor diameter ≥5 cm. The nomogram had a c-index of 0.737 (95% confidence interval, 0.692-0.782) and the calibration curves fitted well. The median survival time was 4.2 months in the derivation cohort, with an MST of 5 months for BCLC C stage and 1.8 months for BCLC D stage patients. Kaplan-Meier analysis showed significant statistical differences in the 6-month overall survival rates of the primary and validation cohorts after the total scores were divided into three quartiles (low risk: 0-85; intermediate risk: 86-210; high risk: ≥211; p < 0.0001 in both cohorts). CONCLUSIONS: The nomogram can be a more accurate and individualized prediction for 6-month overall survival of patients with HCC and PVTT on conservative treatment, and it is possible to consider further active interventions for patients in the low-risk group (0-85 scores) to achieve the aim of prolonging survival.

Comparison of macrovascular invasion-free survival in early-intermediate hepatocellular carcinoma after different interventions: A propensity score-based analysis.

Objectives: The purpose of this study was to compare macrovascular invasion (MVI)-free survival (MFS) at the three-year follow-up in patients with hepatocellular carcinoma (HCC) who underwent hepatic resection (HR), transcatheter arterial chemoembolization (TACE), or TACE combined with radiofrequency ablation (TACE-RFA). Materials and Methods: We retrospectively analyzed the medical records of 828 patients who were diagnosed with Barcelona Clinic Liver Cancer (BCLC) stage A or stage B HCC. Of these patients, 116 underwent HR, 395 underwent TACE-RFA, 239 underwent TACE, and 78 patients received conservative treatment (control group). A validation cohort of 158 patients was included. The MFS and overall survival (OS) before and after propensity score (PS) matching were evaluated using Kaplan-Meier analysis. Results: The baseline characteristics between the control and TACE groups were comparable. MFS was higher in the TACE group than in the control group at the three-year follow-up (p = 0.0091), and OS was similar in the two groups (p = 0.0549). PS matching was used to generate 68 pairs of patients in the control versus HR group and 74 pairs of patients in the control versus TACE-RFA group (1-to-1 matched). MFS was significantly higher in the HR or TACE-RFA groups than in the control group (p < 0.0001 (HR versus control) and p = 0.0001 (TACE-RFA versus control), respectively). Furthermore, for patients in the HR versus TACE-RFA versus TACE groups that were generated by PS matching, the Kaplan-Meier analysis showed that MFS and OS were higher with HR or TACE-RFA than with TACE at three years. In the study, similar results were obtained in the validation cohort. Conclusions: MFS and OS were higher with HR or TACE-RFA than with TACE for HCC patients without MVI.

Glial cell line-derived neurotrophic factor (GDNF) mediates hepatic stellate cell activation via ALK5/Smad signalling.

OBJECTIVE: Although glial cell line-derived neurotrophic factor (GDNF) is a member of the transforming growth factor-ß superfamily, its function in liver fibrosis has rarely been studied. Here, we investigated the role of GDNF in hepatic stellate cell (HSC) activation and liver fibrosis in humans and mice. DESIGN: GDNF expression was examined in liver biopsies and sera from patients with liver fibrosis. The functional role of GDNF in liver fibrosis was examined in mice with adenoviral delivery of the GDNF gene, GDNF sgRNA CRISPR/Cas9 and the administration of GDNF-blocking antibodies. GDNF was examined on HSC activation using human and mouse primary HSCs. The binding of activin receptor-like kinase 5 (ALK5) to GDNF was determined using surface plasmon resonance (SPR), molecular docking, mutagenesis and co-immunoprecipitation. RESULTS: GDNF mRNA and protein levels are significantly upregulated in patients with stage F4 fibrosis. Serum GDNF content correlates positively with α-smooth muscle actin (α-SMA) and Col1A1 mRNA in human fibrotic livers. Mice with overexpressed GDNF display aggravated liver fibrosis, while mice with silenced GDNF expression or signalling inhibition by GDNF-blocking antibodies have reduced fibrosis and HSC activation. GDNF is confined mainly to HSCs and contributes to HSC activation through ALK5 at His39 and Asp76 and through downstream signalling via Smad2/3, but not through GDNF family receptor alpha-1 (GFRα1). GDNF, ALK5 and α-SMA colocalise in human and mouse HSCs, as demonstrated by confocal microscopy. CONCLUSIONS: GDNF promotes HSC activation and liver fibrosis through ALK5/Smad signalling. Inhibition of GDNF could be a novel therapeutic strategy to combat liver fibrosis.

FAM83D is associated with gender, AJCC stage, overall survival and disease-free survival in hepatocellular carcinoma.

Prognostic significance of family with sequence similarity 83, member D (FAM83D) in hepatocellular carcinoma (HCC) patients has not been well-investigated using Gene Expression Omnibus (GEO) series and TCGA database, we compared FAM83D expression levels between tumor and adjacent tissues, and correlated FAM83D in tumors with outcomes and clinico-pathological features in HCC patients. Validated in GSE33006, GSE45436, GSE84402 and TCGA, FAM83D was significantly overexpressed in tumor tissues than that in adjacent tissues (all P<0.01). FAM83D up-regulation was significantly associated with worse overall survival (OS) and disease-free survival (DFS) in HCC patients (Log rank P=0.00583 and P=4.178E-04, respectively). Cox analysis revealed that FAM83D high expression was significantly associated with OS in HCC patients [hazard ratio (HR) = 1.44, 95% confidence interval (CI) = 1.005-2.063, P=0.047]. Additionally, patients deceased or recurred/progressed had significantly higher FAM83D mRNA levels than those living or disease-free (P=0.0011 and P=0.0238, respectively). FAM83D high expression group had significantly more male patients and advanced American Joint Committee on Cancer (AJCC) stage cases (P=0.048 and P=0.047, respectively). FAM83D mRNA were significantly overexpressed in male (P=0.0193). Compared with patients with AJCC stage I, those with AJCC stage II and stage III-IV had significantly higher FAM83D mRNA levels (P = 0.0346 and P=0.0045, respectively). In conclusion, overexpressed in tumors, FAM83D is associated with gender, AJCC stage, tumor recurrence and survival in HCC.

MicroRNA-942 mediates hepatic stellate cell activation by regulating BAMBI expression in human liver fibrosis.

MicroRNA (miRNA)-mediated gene regulation contributes to liver pathophysiology, including hepatic stellate cell (HSC) activation and fibrosis progression. Here, we investigated the role of miR-942 in human liver fibrosis. The expression of miR-942, HSC activation markers, transforming growth factor-beta pseudoreceptor BMP and activin membrane-bound inhibitor (BAMBI), as well as collagen deposition, were investigated in 100 liver specimens from patients with varying degree of hepatitis B virus (HBV)-related fibrosis. Human primary HSCs and the immortalized cell line (LX2 cells) were used for functional studies. We found that miR-942 expression was upregulated in activated HSCs and correlated inversely with BAMBI expression in liver fibrosis progression. Transforming growth factor beta (TGF-ß) and lipopolyssacharide (LPS), two major drivers of liver fibrosis and inflammation, induce miR-942 expression in HSCs via Smad2/3 respective NF-κB/p50 binding to the miR-942 promoter. Mechanistically, the induced miR-942 degrades BAMBI mRNA in HSCs, thereby sensitizing the cells for fibrogenic TGF-ß signaling and also partly mediates LPS-induced proinflammatory HSC fate. In conclusion, the TGF-ß and LPS-induced miR-942 mediates HSC activation through downregulation of BAMBI in human liver fibrosis. Our study provides new insights on the molecular mechanism of HSC activation and fibrosis.

Influence factors and a predictive scoring model for measuring the biochemical response of primary biliary cholangitis to ursodeoxycholic acid treatment.

AIMS: The biochemical response after ursodeoxycholic acid (UDCA) treatment contributes toward predicting the prognosis for primary biliary cholangitis (PBC) patients. This study aimed to establish a score model that can be used for predicting the biochemical response. PATIENTS AND METHODS: A total of 218 patients in the derivation group and 66 patients in the verification group were enrolled. Response endpoints were based on the Barcelona criteria combined with the Paris I criteria. We determined independent factors of the biochemical response by univariate and multivariate analyses. Then, we established a predictive score model on the basis of regression coefficients after adjusted multivariate analyses. RESULTS: The median follow-up duration in the derivation and the verification group was 12.9 and 12.2 months, respectively. Multivariate logistic regression analysis after adjusting for sex and age indicated that First-UDCA treatment [odds ratio (OR)=2.543, 95% confidence interval (CI): 1.234-5.240, P=0.011], baseline alanine aminotransferase level (OR=1.265, 95% CI: 1.089-1.471, P=0.002), and baseline total bilirubin level (OR=0.571, 95% CI: 0.420-0.776, P<0.001) were independent factors that influenced the biochemical response in PBC patients after 1 year of UDCA treatment. Therefore, the resulting biochemical response prediction score model represented the sum of the points corresponding to these three variables. The area under the receiver operating characteristic curve of the score model in the derivation group and the verification group was 0.763 (95% CI: 0.701-0.817, P<0.001) and 0.798 (95% CI: 0.681-0.887, P<0.001), respectively. CONCLUSION: We developed and verified an easy-to-use scoring model for the first time, which showed excellent predictive value for the biochemical response in PBC patients.

Xia-Yu-Xue Decoction Inhibits Intestinal Epithelial Cell Apoptosis in CCl4-Induced Liver Fibrosis.

BACKGROUND/AIMS: Intestine-derived endotoxin is thought to play a role in the development of liver fibrosis. However, the pathological change in the intestine during liver fibrosis is still poorly understood. Here, we investigated the effects of Xia-yu-xue decoction (XYXD) on intestinal inflammation, apoptosis, and tight junction integrity in the carbon tetrachloride (CCl4)-induced liver fibrosis. METHODS: Murine liver fibrosis was developed by CCI4 treatment three times per week over a 6-week period. The CCl4-treated mice were divided into two groups: the CCl4-water group (n=8, CCl4) and the CCl4-XYXD group (n=8, CCl4+XYXD). The CCl4+XYXD mice were treated with XYXD from the beginning of the first week. The expression of inflammatory cytokines and apoptotic molecules were examined using immunohistochemistry, real-time PCR, and western blot. The intestinal epithelial cell apoptosis was examined by TUNEL staining. The tight junction-related molecules, such as ZO-1, claudin, and occludin in the gut were measured by real-time PCR. RESULTS: In CCl4-treated mice damage of the intestinal epithelia and infiltration of inflammatory cells into the lamina propria and muscular layer were observed. Proinflammatory markers MCP-1, TNF-α, CXCL11, IL-6, and CD68 were significantly increased in the intestinal epithelia in CCI4-treated mice. The expression of pro-apoptotic molecules including Fas and Bax was increased in the intestinal epithelia in CCI4-treated mice compared with that in control. The number of TUNEL-positive intestinal epithelial cells was also markedly increased in CCl4-treated mice. The expression of the tight junction proteins including ZO-1, claudin, and occludin was significantly decreased in CCI4-treated mice compared with that in control mice. Notably, XYXD treatment ameliorated increased inflammatory markers and apoptosis-related molecules and decreased tight-junction proteins in CCl4-treated mice. CONCLUSION: CCl4-treatment increased expression of proinflammatory cytokines and pro-apoptotic molecules and disrupted tight junction integrity in the intestine. XYXD treatment ameliorated intestinal inflammation, cell death, and tight junction disintegrity induced by CCl4 treatment, suggesting that XYXD inhibits CCl4-mediated liver fibrosis at least in part by ameliorating the intestinal epithelial damage.

Sorafenib Inhibits Renal Fibrosis Induced by Unilateral Ureteral Obstruction via Inhibition of Macrophage Infiltration.

AIMS: Sorafenib, which has been used extensively for the treatment of renal cell cancer and advanced hepatocellular carcinoma (HCC), has also been shown to have antifibrotic effects in liver fibrosis. However, the effects of sorafenib on renal fibrosis are unknown. Therefore, we investigated whether sorafenib inhibited renal fibrosis in a mouse model of unilateral ureteral obstruction (UUO) and further explored the potential mechanism. METHODS: Mice underwent UUO followed by vehicle or sorafenib treatment. The expression of CD68, a macrophage marker, and the pro-inflammatory cytokines, MCP1 and CXCR3, were immunohistochemically analyzed. The involvement of macrophages in the formation of renal fibrosis was studied using confocal microscopy. RESULTS: Renal histopathology improved in the UUO-sorafenib mice. Sorafenib notably suppressed TGF-ß1-mediated renal fibrogenic effects. The mRNA and protein expressions of CD68, MCP1, and CXCR3 in the obstructed kidney were significantly decreased by sorafenib. Immunohistochemistry showed that CD68 and CXCR3 had a similar distribution, whereas MCP1 was observed predominantly in the tubular epithelial cells. Double immunofluorescence demonstrated that CD68-positive macrophages could co-localize with CXCR3. It also revealed that CXCR3 interacted with CXCL11 in the UUO mouse kidneys. Widespread adhesion of macrophages to myofibroblasts was markedly inhibited in UUO-sorafenib mouse kidneys. CONCLUSIONS: Taken together, the results indicated that sorafenib had protective effects against renal fibrosis; its mechanism of action was associated with inhibition of macrophage infiltration via the CXCR3/CXCL11 pathway. These data suggest the clinical potential of sorafenib for treatment of renal fibrosis and illustrate the immunological mechanisms underlying the protective effects of sorafenib.

Xia-yu-xue decoction (XYXD) reduces carbon tetrachloride (CCl4)-induced liver fibrosis through inhibition hepatic stellate cell activation by targeting NF-κB and TGF-ß1 signaling pathways.

BACKGROUND: Hepatic stellate cell (HSC) activation is activated mainly by endotoxin and transforming growth factor (TGF-ß1) in chronic liver injury, consequently, can be important therapeutic targets. Xia-yu-xue decoction (XYXD), a classical recipe used in China to treat liver fibrosis, and has been revealed to inhibit hepatic fibrosis in animal models, the mechanism of action of XYXD remains elusive. In the present study, we evaluated whether XYXD reduced endotoxin and pro-fibrogenic pathways induced by lipopolysaccharide (LPS) and TGF-ß1 in HSCs. METHODS: The in vivo effect of XYXD on fibrosis progression was assessed in mice model induced by carbon tetrachloride (CCl4), The in vitro effect of XYXD on mice GFP-Col-HSC cells was evaluated using LPS and TGF-ß1 stimulation. RESULTS: XYXD treatment reduced CCl4-induced liver fibrosis and decreased hepatic hydroxyproline (Hyp) content, the mRNA levels of smooth muscle actin (α-SMA) and Col 1(α1) in fibrotic liver. XYXD suppressed nuclear factor-κB (NF-κB) activation induced by LPS and TGF-ß1 assessed by using NF-κB-luciferase reporter. The expression of NF-κB target genes, chemokine (C-C motif) ligand 2 (CCL2) and chemokine (C-X-C motif) ligand 2 (CXCL2) induced by LPS was suppressed after XYXD treatment. The expression of TGF-ß1 targets genes, Col1(α1) and tissue inhibitor of metalloproteinases (TIMP1) induced by TGF-ß1 was inhibit after XYXD treatment. CONCLUSION: XYXD treatment attenuates liver fibrosis by inhibiting HSC activation via inhibition of NF-κB and TGF-ß1 signaling pathway, thereby blocking the synthesis of Col1 (α1) and TIMP-1. These findings from present study suggest that XYXD may be a therapeutic decoction for liver fibrosis in which NF-κB and TGF-ß1 are thought to take part.

Paeoniflorin exerts a nephroprotective effect on concanavalin A-induced damage through inhibition of macrophage infiltration.

BACKGROUND: It is well established that macrophage infiltration is involved in concanavalin A (conA)-induced liver injury. However, the role of macrophages in conA-induced renal injury remains unknown. The aims of this study were to investigate macrophage infiltration in conA-induced renal injury and determine whether paeoniflorin (PF) could inhibit macrophage infiltration into the kidney. METHODS: BALB/C mice were pre-treated with or without PF 2 h (h) before conA injection. At 8 h after con A injection, all the mice were sacrificed; The liver and kidney histology were studied. The renal CD68 expression was detected by immunohistochemical and real-time PCR analysis. The level of expression of C-X-C chemokine receptor type 3 (CXCR3) was analyzed by western blot, immunohistochemical and real-time PCR. The pathophysiological involvement of CXCR3 in macrophage infiltration were investigated using dual-colour immunofluorescence microscopy. RESULTS: PF administration significantly reduced the elevated serum levels of alanine transaminase (ALT), blood urea nitrogen (BUN), creatinine (Cr) and the severity of liver and renal damage compared with that in the conA-vehicle group. PF administration inhibited the increase in renal IL1ß mRNA expression and concentration. Furthermore, immunohistochemical analysis showed that macrophages secreted CXCR3 in the kidneys of the conA-vehicle mice. Immunofluorescence microscopy demonstrated CXCR3 bound tightly to C-X-C motif ligand 11 (CXCL11) in the kidneys of the conA-vehicle mice and showed that PF treatment could suppress CXCR3/CXCL11 over-activation. CONCLUSIONS: Macrophage infiltration was a notable pathological change in the kidneys of conA-treated mice. PF administration attenuated conA-induced renal damage, at least in part, by inhibiting the over-activated CXCR3/CXCL11 signal axis.

[A randomized placebo-controlled multicentre study of Fuzhenghuayu capsule for prevention of oesophageal variceal bleeding in patients with liver cirrhosis].

OBJECTIVE: To evaluate the efficacy of Fuzhenghuayu capsule for the prevention of oesophageal variceal bleeding in patients with liver cirrhosis. METHODS: A multicentre randomized placebo-controlled trial was conducted. A total of 181 liver cirrhosis patients were enrolled in the study and randomly assigned to different groups according to the level of oesophageal variceal bleeding. Patients with light oesophageal varices received Fuzhenghuayu capsule or a placebo. Patients with medium to heavy oesophageal varices received either Fuzhenghuayu capsule alone, Fuzhenghuayu capsule plus propranolol, or propranolol plus a placebo. Patients with a history of oesophageal variceal bleeding received either Fuzhenghuayu capsule plus propranolol, propranolol alone, or a placebo. For all patients, the treatment lasted 2 years. The primary end point of the study was oesophageal variceal bleeding. The secondary end points were liver cancer, death by any cause, and liver transplantation. Risk of bleeding and survival were statistically assessed. RESULTS: The median follow-up time was 50 months. The patients with small oesophageal varices who were treated with Fuzhenghuayu capsule showed a significantly higher cumulative probability of bleeding than their counterparts treated with the placebo (3.4% vs. 23.7%, x² = 4.829, P =0.028). The patients with medium to heavy oesophageal varices and no history of oesophageal variceal bleeding who were treated with Fuzhenghuayu capsule plus propranolol showed a remarkably higher cumulative probability of bleeding than their counterparts treated with propranolol alone (15.2% vs. 43.6%, x² =6.166, P =0.013). There were no significant differences between the patients treated with Fuzhenghuayu capsule alone and those treated with propranolol alone (P =0.147) or the patients treated with Fuzhenghuayu capsule plus propranolol and those treated with Fuzhenghuayu capsule alone (P =0.147). The patients with history of oesophageal variceal bleeding who were treated with Fuzhenghuayu capsule showed significantly higher cumulative probability of bleeding and median time of bleeding than their counterparts treated with propranolol alone (44.0% vs. 24.2% and 40.00 ± 17.92 months vs. 7.00 ± 2.35 months; x² = 4.433, P =0.035). There were no significant differences in the cumulative probability of liver cancer and survival among all of the groups. CONCLUSION: Fuzhenghuayu capsule can decrease the cumulative probability of bleeding in cirrhotic patients with light oesophageal varices. For cirrhosis patients with a history of oesophageal variceal bleeding, the combination of Fuzhenghuayu capsule plus propranolol can decrease the cumulative probability of bleeding with median or heavy varices.

Curative Effects of ZHENG-Based Fuzheng-Huayu Tablet on Hepatitis B Caused Cirrhosis Related to CYP1A2 Genetic Polymorphism.

Aim. To investigate the correlation of Fuzheng-Huayu tablet (FZHY) efficacy on chronic hepatitis B caused cirrhosis (HBC) and single nucleotide polymorphisms (SNPs) of CYP1A2. Methods. After 111 cases of HBC with 69 excess, 21 deficiency-excess, and 21 deficiency ZHENGs (ZHENG, also called traditional Chinese medicine syndrome) were treated by FZHY for 6 months, clinical symptoms, Child-Pugh score, and ZHENG score were observed. Three of the SNPs in CYP1A2 gene were detected and analyzed using SNaPshot assay. Results. In ZHENG efficacy between effective and invalid groups, there was significant difference (P < 0.001). The ZHENG deficiency was significantly correlated with FZHY efficacy (P < 0.05). AA genotype of CYP1A2-G2964A was significantly different with GG genotype (P < 0.05) between CYP1A2 Genotypes and FZHY efficacy on ZHENG. More importantly, GA plus AA genotype of CYP1A2-G2964A was significantly different with deficiency ZHENG (P < 0.05) between CYP1A2 genotypes and FZHY efficacy on ZHENG. Conclusion. FZHY improved ZHENG score of HBC, and these efficacies may relate to CYP1A2-G2964A sites. It was suggested that CYP1A2-G2964A locus is probably a risk factor for ZHENG-based FZHY efficacy in HBC.