Yin-chen Wu-ling powder alleviate cholestatic liver disease: Network pharmacological analysis and experimental validation.

BACKGROUND: Yin-chen Wu-ling Powder (YWP) has potential therapeutic effects on cholestatic liver disease (CLD), however, its active compounds and conceivable mechanism are as yet indistinct. METHODS: The network pharmacology and gene function annotation examined the multiple active ingredients, potential targets, and possible mechanisms of YWP in CLD treatment. Then the molecular docking reassured the reliability of the core compounds including the key genes and farnesoid X receptor (FXR). Finally, The Mdr2-/- mice were used to test the effect and mechanism of YWP against CLD. RESULTS: The network analysis identified nine main active ingredients, including quercetin, capillarisin, eupalitin, isorhamnetin, skrofulein, genkwanin, cerevisterol, gederagenin, and sitosterol. The PPI network predicted the ten hub genes involved were AKT1, MAPK1, MAPK14, IL6, RXRA, ESR1, IL10, NCOA1, CAV1, and EGFR. The KEGG and GO analysis showed that YWP might contribute to CLD treatment through the PI3K/Akt and MAKP signalings to manage pathological reactions, for instance, inflammatory responses. The molecular docking displayed a functional similarity among the core compounds with ursodeoxycholic acid (UDCA) and Obeticholic acid (OCA) on the effects on AKT1, MAPK1, MAPK14, RXRA, and ESR, and the affinity to FXR. In addition, the YWP could significantly attenuate hepatic injury and improve inflammatory response in Mdr2-/- mice. The mechanism exploration showed that YWP mainly decreased inflammatory response by inhibiting AKT/P38MAPK signaling. CONCLUSION: This study firstly revealed the multiple active ingredients, potential targets, and possible mechanism of YWP to treat CLD based on network pharmacology Analysis and molecular docking. YWP could alleviate cholestasis in Mdr2-/- mice by impairing inflammation via inhibiting AKT/P38MAPK Signaling.

Efficacy and safety of AnluoHuaxian pills on chronic hepatitis B with normal or minimally elevated alanine transaminase and early liver fibrosis: A randomized controlled trial.

ETHNOPHARMACOLOGICAL RELEVANCE: The AnluoHuaxian pill (AHP) is a widely used patented medicine for chronic hepatitis B (CHB) patients with advanced fibrosis or cirrhosis that has been used in China for more than 15 years. However, data are lacking on whether monotherapy with AHP can be effective in CHB patients with alanine aminotransferase (ALT) levels less than 2 times the upper limit of normal (ALT<2ULN) and early liver fibrosis (F ≤ 2). AIM OF THE STUDY: We aimed to investigate whether monotherapy with AHP improves liver histology in these patients. MATERIALS AND METHODS: In this double-blind, randomized, placebo-controlled trial, 270 CHB patients with ALT<2ULN and F ≤ 2 were treated in 12 hospitals in China. The patients were randomly assigned to an intervention (AHP) group and a placebo group at a ratio of 2:1. Of these 270 enrolled patients, 147 had paired liver biopsies. The primary end point was histological change after 48 weeks of treatment. RESULTS: Per-protocol analysis revealed that the rate of histologic improvement in liver fibrosis patients in the AHP group was significantly higher than that in the placebo group (37.7% vs. 19.5%, P = 0.035) after 48 weeks of treatment, which was consistent with results from intention-to-treat and sensitivity analyses. Moreover, after adjusting for baseline characteristics, AHP was superior to placebo with respect to improving liver fibrosis (odds ratio [OR] = 2.58, 95% confidence interval [CI]: (1.01, 6.63),P = 0.049) and liver histology (OR = 3.62, 95% CI: (1.42, 9.20),P = 0.007). In noninvasive measurement of liver fibrosis (FibroScan®), the level of liver stiffness measurement (LSM) had decreased significantly at 48 weeks (5.1 kPa) compared with that at baseline (5.7 kPa) (P = 0.008) in the AHP group, whereas it did not decrease significantly in the placebo group. Cirrhosis developed in one patient in the placebo group but in no patients in the AHP group. No serious side effects occurred in the AHP-treated patients. CONCLUSIONS: Treatment of CHB patients who had ALT<2ULN and F ≤ 2 with the traditional Chinese medicine AHP for 48 weeks improves liver fibrosis. However, due to the short duration of treatment and the limited sample size of liver pathology, the long-term benefits of AHP in reducing fibrosis and the risk of cirrhosis and hepatocellular carcinoma in these patients need to be further studied in the future.

Atractylenolide III ameliorates Non-Alcoholic Fatty Liver Disease by activating Hepatic Adiponectin Receptor 1-Mediated AMPK Pathway.

Background: Nonalcoholic fatty liver disease (NAFLD) is the most frequent cause of chronic liver diseases worldwide. At present, there are no effective pharmacological therapies for NAFLD except lifestyle intervention-mediated weight loss. Atractylenolide III (ATL III), the major bioactive component found in Atractylode smacrocephala Koidz, has been shown to exert anti-oxidant, anti-tumor, anti-allergic response, anti-bacterial effects and cognitive protection. Here we investigate the therapeutic potential and underlying mechanisms of ATL III for the treatment of NAFLD. Methods: Male C57BL/6J mice were fed a high-fat diet (HFD) and treated with ATL III. Lipid accumulation was analyzed by Oil Red O staining in liver tissues and free fatty acids (FFAs)-treated hepatocytes. AMP-activated protein (AMPK) and sirtuin 1(SIRT1) signaling pathways were inhibited by Compound C and EX527 in vitro, respectively. Small-interfering RNA (siRNA) was used to knockdown adiponectin receptor 1 (AdipoR1) expression in HepG2 cells. Results: ATL III treatment ameliorated liver injury and hepatic lipid accumulation in the HFD-induced NAFLD mouse model as demonstrated by that ATL III administration significantly reduced serum levels of alanine aminotransferase, glutamic oxaloacetic transaminase, triglycerides, total cholesterol and low-density lipoprotein. Furthermore, treatment with ATL III alleviated hepatic oxidative stress, inflammation and fibrosis in the HFD feeding model. To study the underlying mechanisms, we performed Computer Aided Design assay and found that open-formed AdipoR1 and adiponectin receptor 2 were the potential receptors targeted by ATL III. Interestingly, HFD feeding or FFAs treatment only reduced hepatic AdipoR1 expression, while such reduction was abolished by ATL III administration. In addition, in vitro treatment with ATL III activated the AdipoR1 downstream AMPK /SIRT1 signaling pathway and reduced lipid deposition in HepG2 cells, which was diminished by silencing AdipoR1. Finally, inhibition of AMPK or SIRT1, the AdipoR1 downstream signaling, abolished the protective effects of ATL III on lipid deposition and oxidative stress in FFAs-treated HepG2 cells. Conclusion: Our findings suggest that ATL III is a therapeutic drug for the treatment of NAFLD and such protective effect is mediated by activating hepatic AdipoR1-mediated AMPK/SIRT1 signaling pathway.

Chemical characters and protective effect of Baqi Lingmao formula on experimental liver injury.

OBJECTIVE: To investigate the chemical characters of water-extract of Baqi Lingmao formula (BQLM formula) and its effects on anti-liver injury in model mice and live cells. METHODS: BQLM formula was composed of ten herbal medicines. We determined the contents of alkaloids, saponins, phenolic acids and flavonoid in BQLM formula by UV spectrophotometry. The active components of alkaloids and phenolic acids in BQLM formula were identified by HPLC chromatography. The anti-hepatic injury effects of BQLM formula were investigated with concanavalin A (ConA)-induced hepatitis model of mice, human liver LO2 and HepG2.2.15 cells. RESULTS: BQLM formula (2 and 10 g/kg, orally) significantly improved the damages of liver tissues and functions caused by ConA in mice, reduced the infiltration of inflammatory cells into liver and inhibited the inflammatory cytokine secretion of interferon-γ, tumor necrosis factor-α and interleukin-6. BQLM formula simultaneously decreased the levels of alanine aminotransferase and aspartate aminotransferase of liver and serum, and recovered the superoxide dismutase and catalase activities of liver to normal levels in ConA-induced hepatic-injury mice. The serum of BQLM formula group stimulated the human liver LO2 cell proliferation in vitro. Further, BQLM formula obviously promoted the proliferation of normal hepatocytes (LO2 cells) and inhibited the hepatocytes death induced by ConA. It also significantly inhibited the proliferation of HepG2.2.15 cells and decreased the secretion of HBsAg and HBeAg in vitro. CONCLUSIONS: BQLM formula has anti-inflammation and anti-hepatitis virus Beffects, and is capable of improving liver injury in vivo and in vitro.

C-C chemokine hepatocellular carcinoma motif ligand 5-deficiency promotes hepatocellular carcinoma progression by affecting B cell recruitment.

OBJECTIVE: To evaluate the expression of C-C motif chemokine ligand 5 (CCL5) in hepatocellular carcinoma (HCC) and to explore its role in regulating the immune microenvironment and the related mechanism in tumor immunity. METHODS: The mRNA expression level of CCL5 in HCC and adjacent non-cancerous tissues was measured by quantitative polymerase chain reaction and the protein expression was examined by immunohistochemistry. Serum CCL5 expression was measured by an enzyme-linked immunosorbent assay (ELISA). C57BL/6 wild-type (WT) and Ccl5-knockout (Ccl5-/- ) mice were utilized to conduct the diethylnitrosamine-induced HCC model. The immune cell population was determined by flow cytometry, and peripheral serum immunoglobulin M (IgM) level was quantified by ELISA. RESULTS: CCL5 expression was low in HCC tissue and peripheral blood compared with adjacent non-cancerous tissues or controls, and its expression was correlated with the overall survival, cancer recurrence and distant metastasis. In the HCC mouse model, liver-to-body weight ratio was of the Ccl5-/- group were higher than that of the WT group. Moreover, compared with the WT mice, the number of B cells in the tumor tissue of the Ccl5-/- mice was lower, while there were no significant differences in the other immune cell populations. Furthermore, serum IgM level of the Ccl5-/- mice was significantly lower than that of the WT mice. CONCLUSION: CCL5 expression is decreased in HCC tissues. CCL5 deficiency reduces B cell recruitment and decreases IgM secretion in HCC, potentially leading to tumor progression.

Ganji Formulation for Patients with Hepatocellular Carcinoma Who Have Undergone Surgery: A Multicenter, Randomized, Double-Blind, Controlled Trial.

Objective. To ascertain the efficacy and safety of Ganji Formulation (GF) for patients with Hepatocellular carcinoma (HCC) who had undergone surgery. Materials and Methods. A total of 262 HCC patients who had undergone liver resection, local ablation, or transcatheter arterial chemoembolization (TACE) were divided randomly into the treatment group and control group. The former was treated with GF and the later with placebo, both for 6 months. The primary endpoint was overall survival (OS). Second endpoints were disease-free survival (DFS) or time to disease progression (TTP). Results. OS of the treatment group was significantly longer than that of the control group (P < 0.05). Subgroup analysis showed that, for patients who received TACE, the TTP was significantly longer in the treatment group than in the control group (P < 0.05). However, for patients who underwent liver resection or local ablation, there was no significant difference in DFS between the two groups (P > 0.05). Conclusion. GF could improve postoperative cumulative survival and prolong the TTP. This clinical trial number is registered with ChiCTR-IOR-15007349.

Utility and Safety of Tolvaptan in Cirrhotic Patients with Hyponatremia: a Prospective Cohort Study

Abstract: Introduction and aim. Hyponatremia is common in patients with decompensated cirrhosis and is associated with increased mortality. Tolvaptan, a vasopressor V2 receptor antagonist, can increase free wáter excretion, but its efficacy and safety in cirrhotic patients remain unclear. Material and methods. We studied the usage and safety of tolvaptan in cirrhotic patients in a real-life, non-randomized, multicenter prospective cohort study. Forty-nine cirrhotic patients with hyponatremia were treated with tolvaptan 15 mg daily, and 48 patients not treated with tolvaptan in the same period served as controls. Improvement in serum sodium level was defined as an increase in serum sodium from < 125 to ≥ 125 mmol/L or from 125-134 to ≥ 135 mmol/L on day 7. Results. Twenty-three (47%) patients in the tolvaptan group and 17 (35%) in the control group had normal serum sodium on day 7 (p = 0.25). Serum sodium improved in 30 (61%) patients in the tolvaptan group and 17 (35%) patients in the control group (p = 0.011). Adverse events occurred in 46-47% of patients in both groups, and tolvaptan was not associated with worsened liver function. No patient with normal serum sodium on day 7 died within 30 days of treatment, whereas 16% of those with persistent hyponatremia died (p = 0.0019). Conclusion. In conclusion, short-term tolvaptan treatment is safe and can improve serum sodium level in cirrhotic patients with hyponatremia. Normalization of serum sodium level is associated with better survival.

Hepatitis B core antigen upregulates B7-H1 on dendritic cells by activating the AKT/ERK/P38 pathway: a possible mechanism of hepatitis B virus persistence.

B7-H1 binding to programmed death-1 may deliver a coinhibitory signal to T cells that is involved in the regulation of T-cell activation and tolerance. B7-H1 plays a key role in dysfunction of dendritic cells (DCs) during chronic HBV infection, but the expression mechanism of B7-H1 remains unclear. One hundred and twenty-nine patients with chronic HBV infection were categorized into either the immune tolerance phase (HBV-IT), the immune clearance phase (HBV-IC), or the inactive carrier phase (HBV-IA). Twenty healthy volunteers were enrolled as controls. Another 16 patients with HBeAg-positive chronic Hepatitis B were enrolled, and entecavir was administrated at 0.5 mg per day for 6 months. The B7-H1 expression level on peripheral DCs was tested by flow cytometry. In vitro, expression levels of B7-H1 and signaling molecules on monocyte-derived DC (MO-DC) induced by recombinant hepatitis B virus C antigen (rhHBcAg) were examined by RT-PCR, flow cytometry, and western blotting, and the apoptosis rate was tested by flow cytometry. The percentages of peripheral DCs and myeloid DCs (mDCs) were decreased and B7-H1 levels were increased in patients compared with controls. Serum HBV-DNA levels were positively correlated with B7-H1 levels on mDCs in patients with HBV-IT. B7-H1 levels on peripheral DCs from patients with chronic hepatitis B decreased after antiviral therapy. In vitro studies demonstrated that the B7-H1 level on MO-DC was upregulated by rhHBcAg, which resulted from the activation of PI3K-AKT, ERK, and P38 signaling pathways, and the percentage of MO-DC was downregulated by rhHBcAg. In addition, rhHBcAg promoted the apoptosis of MO-DC. The data suggest that HBcAg induced B7-H1 upregulation by activating AKT, ERK, and P38 signaling pathways, which inhibited the clearance of HBV-DNA and the reduction of DCs contributed to immune tolerance, which may correlate with apoptosis.

Natural killer cells in hepatitis B virus infection

Natural killer cells are a unique type of lymphocytes with cytotoxic capacity, and play important roles against tumors and infections. Recently, natural killer cells have been increasingly valued in their effects in hepatitis B virus infection. Since hepatitis B virus is not cytopathic, the subsequent antiviral immune responses of the host are responsible for sustaining the liver injury, which may result in cirrhosis and even hepatocellular carcinoma. Many studies have confirmed that natural killer cells participate in anti-hepatitis B virus responses both in the early phase after infection and in the chronic phase viacytolysis, degranulation, and cytokine secretion. However, natural killer cells play dichotomic roles: they exert antiviral and immunoregulatory functions whilst contribute to the pathogenesis of liver injury. Here, we review the roles of natural killer cells in hepatitis B virus infection, introducing novel therapeutic strategies for controlling hepatitis B virus infection viathe modulation of natural killer cells.

Natural killer cells in hepatitis B virus infection.

Natural killer cells are a unique type of lymphocytes with cytotoxic capacity, and play important roles against tumors and infections. Recently, natural killer cells have been increasingly valued in their effects in hepatitis B virus infection. Since hepatitis B virus is not cytopathic, the subsequent antiviral immune responses of the host are responsible for sustaining the liver injury, which may result in cirrhosis and even hepatocellular carcinoma. Many studies have confirmed that natural killer cells participate in anti-hepatitis B virus responses both in the early phase after infection and in the chronic phase via cytolysis, degranulation, and cytokine secretion. However, natural killer cells play dichotomic roles: they exert antiviral and immunoregulatory functions whilst contribute to the pathogenesis of liver injury. Here, we review the roles of natural killer cells in hepatitis B virus infection, introducing novel therapeutic strategies for controlling hepatitis B virus infection via the modulation of natural killer cells.

Germacrone inhibits the proliferation of glioma cells by promoting apoptosis and inducing cell cycle arrest.

Germacrone is one of the major bioactive components of the traditional Chinese Medicinal plant Curcuma aromatica Salisb. and has been shown to possess anti­tumor properties. In the present study, the anti­proliferative effect of germacrone on human glioma cells and the molecular mechanism underlying its cytotoxicity were investigated. Treatment of the U87 and U251 human glioma cell lines with germacrone inhibited the cell proliferation in a dose­ and time­dependent manner as assessed by MTT assay, while significantly lower effects were observed on normal human astrocytes. Flow cytometric analysis and DNA fragmentation revealed that germacrone promoted apoptosis of glioma cells, associated with an increased expression of p53 and bax and decreased expression of bcl­2. Furthermore, flow cytometric cell cycle analysis revealed that germacrone induced G1 phase arrest, associated with an obvious decrease in the expression of cyclin D1 and CDK2 and an increased expression of p21. In conclusion, the present study suggested that germacrone may be a novel potent chemopreventive drug candidate for gliomas via regulating the expression of proteins associated with apoptosis and G1 cell cycle arrest.

Cell cycle-related kinase mediates viral-host signalling to promote hepatitis B virus-associated hepatocarcinogenesis.

BACKGROUND: Androgen receptor (AR) signalling contributes to male predominance in hepatocellular carcinoma (HCC), which is more pronounced in HBV-endemic areas. Cell cycle-related kinase (CCRK) is essential for AR-induced hepatocarcinogenesis but its molecular function in HBV-associated HCC remains obscure. OBJECTIVE: To determine the molecular function of CCRK in HBV-associated HCC. DESIGN: Transcriptional regulation was assessed by chromatin immunoprecipitation, promoter mutation and luciferase reporter assays. Hepatocellular proliferation and tumourigenesis were examined by colony formation, soft agar assays and using HBV X protein (HBx) transgenic mice with low-dose exposure to diethylnitrosamine. Protein expressions were examined in clinical samples and correlated with patient survival by log-rank Mantel-Cox test. RESULTS: Overexpression of CCRK, but not its kinase-defective mutant, activated ß-catenin/T cell factor signalling through phosphorylation of glycogen synthase kinase-3ß (GSK-3ß) at Ser9, led to upregulation of AR transcriptional activity and, subsequently, expression of HBx. The viral transactivator in turn induced CCRK expression through enhanced AR signalling, thus forming a positive regulatory loop. RNA interference silencing of CCRK, which suppressed the CCRK/GSK-3ß/ß-catenin/AR regulatory loop, significantly suppressed HBx-induced hepatocellular proliferation (p=0.001) and transformation (p<0.001) and remarkably reduced >80% diethylnitrosamine-mediated hepatocarcinogenesis in HBx transgenic mice. Finally, patients with HBV-associated HCC with concordant overexpression of CCRK, GSK-3ß phosphorylation at Ser9, active dephosphorylated ß-catenin and AR phosphorylation at Ser81 had poorer overall (HR=31.26, p<0.0001) and disease-free (HR=3.60, p<0.01) survival rates. CONCLUSIONS: Our findings highlight the critical role of CCRK in a self-reinforcing circuitry that regulates HBV-associated hepatocarcinogenesis. Further characterisation of this intricate viral-host signalling may provide new prognostic biomarkers and therapeutic targets for HCC treatment.

[Characteristic of liver pathology in HBeAg-positive and HBeAg-negative chronic hepatitis B patients with mildly elevated ALT].

UNLABELLED: To analyse the live pathology characteristics in mild ALT-elevated (1 x ULN less than ALT less than 2 x ULN ) HBeAg-positive and HBeAg-negative chronic hepatitis B (CHB) patients, and to explore the influence of the age and HBV DNA level to liver pathology in different HBeAg status patients. METHODS: All the patients who met the inclusion criteria form "eleventh five-year plan" National Science and Technology Major Project, the treatment program of integrative traditional and western medicine for CHB were enrolled in this study between October 2009 and March 2011 .B type ultrasound-guided liver biopsy was carried out in all patients and hepatitis B surface antigen (HBsAg) , HBeAg titer as well as HBV DNA level were detected at the same time. Hepatic tissue inflammation and fibrosis degree of patients according to HBeAg-positive and negative, age ( more than or equal to 40 years and less than 40 years), HBV DNA level (more than or equal to 10^5copy/ml and less than l0^5 copy/ml) were compared respectively. Chi-square test was used to compare the constitute percentage between the two samples. Multivariate logistic regression analysis was also performed to evaluate the correlation between different factors. RESULTS: There were no significant difference in the grade of liver inflammation and the stage of liver fibrosis between 389 HBeAg positive and 126 HBeAg-negative patients (X2=4.326 and X2=3.464, respectively, P values were all more than 0.05). In the group of patients with age less than 40 years, the distribution of different liver inflammation and fibrosis had no significant difference between HBeAg-positive and negative patients (X2=2.543 and X2=5.024, respectively, P values were all more than 0.05). In the group of patient with age more than or equal to 40 years, the percentage of moderate and severe inflammation (G3, G4) HBeAg-positive patients(32.9%) owned is much higher than that of HBeAg-negative patients(16.4%), X2=8.777, P less than 0.05.But the stage of liver fibrosis in HBeAg-positive patients was not significantly different than that of HBeAg-negative ones (X2=0.977, P more than 0.5). In the group of patients with HBV DNA more than or equal to 10^5copy/ml, the percentage of mild inflammation in HBeAg-positive patients (17.5%) was much high than that of HBeAg-negative patients(7.3%), X2=8.851, P less than 0.05. The stage of liver fibrosis between HBeAg-positive and negative patients was no significant difference (X2=8.227, P more than 0.05).In the patients with HBV DNA less than 10^5 copy/ml, The percentage of HBeAg-negative patients(29.6%) with mild inflammation(G1) was much higher than HBeAg-positive patients (6.9%), X2=6.357, P less than 0.05. There was no significant difference in the stage of liver fibrosis between HBeAg-positive and negative patients (X2=4.061, P more than 0.05). The results of multivariate logistic regression analysis showed that age was the independent risk factor for different degree of liver inflammation and fibrosis seriousness. CONCLUSION: The status of HBeAg has no association with the grade of liver inflammation and the stage of liver fibrosis in CHB patients with mildly elevated ALT. The percentage of moderate and severe inflammation in the HBeAg-positive patients with age more than or equal to 40 years was significantly elevated. The grade of liver inflammation has significant difference between HBeAg-positive and negative patients with different HBV DNA levels as well.

Interleukin-10 genotype correlated to deficiency syndrome in hepatitis B cirrhosis.

Traditional Chinese medicine (TCM) syndrome is an important basis for TCM diagnosis and treatment. As Child-Pugh classification as well as compensation and decompensation phase in liver cirrhosis, it is also an underlying clinical classification. In this paper, we investigated the correlation between single nucleotide polymorphisms (SNPs) of Interleukin-10 (IL-10) and TCM syndromes in patients with hepatitis B cirrhosis (HBC). Samples were obtained from 343 HBC patients in China. Three SNPs of IL-10 (-592A/C, -819C/T, and -1082A/G) were detected with polymerase chain-reaction-ligase detection reaction (PCR-LDR). The result showed the SNP-819C/T was significantly correlated with Deficiency syndrome (P = 0.031), but none of the 3 loci showed correlation either with Child-Pugh classification and phase in HBC patients. The logistic regression analysis showed that the Excess syndrome was associated with dizzy and spider nevus, and the Deficiency syndrome was associated with dry eyes, aversion to cold, IL-10-819C/T loci, and IL-10-1082A/G loci. The odds ratio (OR) value at IL-10-819C/T was 4.022. The research results suggested that IL-10-819C/T locus (TC plus CC genotype) is probably a risk factor in the occurrence of Deficiency syndrome in HBC patients.

HBcAg induces PD-1 upregulation on CD4+T cells through activation of JNK, ERK and PI3K/AKT pathways in chronic hepatitis-B-infected patients.

Hyper-expression of programmed death-1 (PD-1) is a hallmark of exhausted T cells. In chronic hepatitis-B virus (HBV)-infected patients, PD-1 upregulation on T cells was often observed. The mechanism of it has not been fully understood. In this study, we examined the dynamic changes of PD-1 expression on T cells during the natural history of chronic HBV infection and explored the signaling pathway of PD-1 upregulation by the hepatitis-B core antigen (HBcAg). Sixty-seven chronic HBV-infected patients were categorized into an immune tolerance group, an immune clearance group and an inactive virus carrier group, and 20 healthy volunteers were chosen as normal control group. Peripheral blood mononuclear cells from patients and healthy volunteers, and T lymphocytes from healthy volunteers were separated. Results showed that the PD-1 expression level on CD4(+)T cells in every phase of chronic HBV infection was significantly higher than that in healthy volunteers, whereas such effects were not observed on CD8(+)T cells. In the immune clearance phase, a positive correlation was found between serum HBV DNA level and the PD-1 expression level on CD4(+)T cells. In all phases, no correlation was shown between serum alanine amino transferase (ALT) level and PD-1 expression level. Phosphorylation of JNK, ERK and AKT was induced by HBcAg, and inhibitors of JNK, ERK and PI3K/AKT significantly decreased the HBcAg-induced PD-1 upregulation on CD4(+)T cells. In conclusion, the PD-1 expression level on CD4(+)T cells was upregulated in every phase of chronic HBV infection, which was induced by HBcAg through JNK, ERK and PI3K/AKT signaling pathways.

[Syndrome distribution among patients with chronic hepatitis C and interventions of integrated traditional Chinese and Western medicine: study protocol].

BACKGROUND: Chronic hepatitis C is one of the major causes of end-stage liver disease with a high incidence rate, amounting to a grave and serious problem of public health. Currently, interferon-based (with or without ribavirin) antiviral therapy has limited use due to its stringent indications, possible contraindications and side effects. Traditional Chinese medicine (TCM) may have advantages in the prevention and treatment of chronic hepatitis C and it is of significant value to discover the advantages. Through this research, a safe and effective treatment protocol of TCM or integrated TCM and Western medicine for chronic hepatitis C can be formed. To this end, during China's Eleventh Five-Year Plan, special research projects on acquired immune deficiency syndrome (AIDS), viral hepatitis and the other major infectious diseases were established. Our studies on chronic hepatitis C constitute one of the major special research topics. METHODS AND DESIGN: Clinical information of patients with chronic hepatitis C will be first collected in a large, multicenter epidemiological survey. Positive symptoms will be analyzed by rapid cluster analysis, principal constituent analysis and factor analysis, and syndrome types will be diagnosed based on expert advice. Concurrently, a large, multicenter, randomized, parallel-group prospective study will be launched based on evidence-based medical principles to evaluate the effects and safety of the treatment protocol for chronic hepatitis C. The evaluated indexes will include the normalization rate of liver function, virological improvement and quality of life improvement for the short-term efficacy and the incidence of liver cirrhosis and (or) primary liver cancer and mortality for the long-term efficacy. DISCUSSION: This study will investigate the TCM syndrome differentiation norms and the syndrome distribution rules of chronic hepatitis C and evaluate the efficacy and safety of a treatment protocol for chronic hepatitis C based on TCM theory or combined treatment of TCM and Western medicine. The study results will be helpful to developing a TCM treatment program for chronic hepatitis C. TRIAL REGISTRATION: The research program was registered in the Chinese Clinical Trial Registry in English and Chinese in January 2010. REGISTRATION NUMBER: ChiCTR-TRC-10000770.

[Effect of bushen recipe on the immune effector molecules of natural killer cells in patients with chronic hepatitis B].

OBJECTIVE: To investigate the immunoregulation mechanism of Bushen Recipe (BSR) in patients with chronic hepatitis B (CHB) of Gan-Shen yin-deficiency and lingering damp-heat syndrome (GSS). METHODS: Thirty-five patients with positive HBV DNA and abnormal alanine transaminase (ALT) level were assigned to the treatment group (22 patients) and the control group (13 patients), they were treated with BSR and alpha-2b interferon for 6 months respectively. Blood levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL) and HBV DNA were measured before and after treatment. And the expressions of immune effector molecules of nature killer (NK) cell, including perforin (PF), granzyme B (GrB), granulysin (GNLY), tumor necrosis factor-alpha (TNF-alpha) and gamma-interferon (gamma-IFN), were detected using flow cytometry. RESULTS: Levels of ALT and AST declined significantly in both groups after treatment (P < 0.05 or P < 0.01), showing insignificant difference between them. And the expressions (%) of PF and GNLY in the treatment group reduced significantly after treatment, from 69.62 +/- 27.58 to 34.86 +/- 31.60 for PF and from 64.54 +/- 25.96 to 25.72 +/- 24.98 for GNLY (both P < 0.05). In the treatment group and the control group, as compared with before treatment, the total scores of Chinese medicine symptoms were significantly declined after treatment (P < 0.01), and the total scores of Chinese medicine symtoms in the treatment group was significantly lower than that of the control group (P < 0.05). As compared with the total effective rate of Chinese medicine syndromes in the control group after treatment, that in the treatment group was significantly increased (P < 0.05). CONCLUSION: The mechanism of BSR in immuneregulating on patients with CHB of GSS is by way of declining the expressions of relative immune effector molecules to promote the recovery of the damaged liver.

[Expression characteristics of some immune effector molecules in CD8+ T lymphocytes from patients with chronic hepatitis B].

OBJECTIVE: To investigate the expressions of perforin (PF), granzyme B (GrB), granulysin (GNLY), TNF-alpha and IFN-gamma in peripheral CD8+ T lymphocytes and their correlation to infection status in patients with chronic hepatitis B (CHB). METHODS: ALT, AST, TB and HBV DNA copy were detected to evaluate the infection status in CHB patients, with healthy volunteers serving as the control group. According to the infection status, the CHB patients were divided into 4 groups, namely normal hepatic function and high HBV DNA level group, normal hepatic function and low HBV DNA level group, abnormal hepatic function and high HBV DNA level group and abnormal hepatic function and low HBV DNA level group. The expressions of some immune effector molecules in CD8+T cells were detected by flow cytometry, and the correlations between these immune effector molecules and the infection status were analyzed. RESULTS: The expressions of GrB, TNF-alpha and IFN-gamma in normal hepatic function and low HBV DNA level group were significantly higher than those in abnormal hepatic function and high HBV DNA level group (P<0.05). The expression of IFN-gamma in normal hepatic function and high HBV DNA level group was significantly higher than that in abnormal hepatic function and high HBV DNA level group (P<0.05). The expressions of PF and GNLY were similar among all the 4 groups. Positive correlations were noted between GrB, PF, GNLY, TNF-alpha and IFN-gamma. CONCLUSION: GrB, TNF-alpha and IFN-gamma in peripheral CD8+ T cells are inversely correlated to hepatic dysfunction and HBV DNA level in CHB patients.

[Expression profile of immune effector molecules in natural killer cells in patients with chronic hepatitis B].

OBJECTIVE: To investigate the expression profile of immune effector molecules in peripheral natural killer cells (NK) in patients with chronic hepatitis virus B. METHODS: According to the infection status, patients were divided into four experiment groups: normal hepatic function and high HBV DNA level group, normal hepatic function and low HBV DNA level group, abnormal hepatic function and high HBV DNA level group and abnormal hepatic function and low HBV DNA level group. The expression of perforin (PF), granzyme B (Gr B), granulysin (GNLY), tumor necrosis factor alpha (TNFa) and interferon gamma (IFNr) in NK cells were detected by flow cytometer. RESULTS: Compared with control group (31.50%+/-27.64%), the expression of GNLY was significantly increased in normal hepatic function and high HBV DNA level group (59.74%+/-30.82%) and normal hepatic function and low HBV DNA level group (61.89%+/-33.30%); the expression of IFNr in normal hepatic function and high HBV DNA level group (39.89%+/-21.30%) and abnormal hepatic function and high HBV DNA level group (37.54%+/-18.79%) was lower than that in normal control group (57.38%+/-23.69%); the expression of PF, GrB, GNLY in abnormal hepatic function and high HBV DNA level group (35.47%+/-29.64%, 66.55%+/-22.92%, 42.03%+/-33.17%) was lower than that in normal hepatic function and high HBV DNA level group (56.98%+/-38.34%, 81.53%+/-19.58%, 59.74%+/-30.82%) and normal hepatic function and low HBV DNA level groups (62.95%+/-31.98%, 84.51%+/-14.57%, 61.89%+/-33.3%); there were positive correlations between ef PF, Gr B, GNLY, TNFa, and IFNr. CONCLUSION: The expression of IFNr in NK cells from patients with high HBV DNA replication level is lower than that in normal control group; the expression of PF, Gr B and GNLY in NK cells from patients with normal hepatic function is higher than that in NK cells from patients with abnormal hepatic function.

[Quality control of non-alkaloids from Radix Sophorae tonkinensis].

OBJECTIVE: To establish a method for quality control of non-alkaloids from Radix Sophorae tonkinensis. METHODS: Sugar, organic-acid, polyphenolic and saponin were identified by TLC. The contents of Sugar, polyphenolic and saponin were determined by spectrophotometry and the content of organic-acids was determined by acid-based titration. RESULTS: The contents of sugar, polyphenolic and saponin were 54.37%, 4.74%, 4.40%, respectively. The average content of organic-acid was 9.40%. CONCLUSIONS: The established method is simple, accurate, reproducible and suitable for the quality control of non-alkaloids from Radix Sophorae tonkinensis.