Bushen Jianpi Formula Combined with Entecavir for the Treatment of HBeAg-Negative Chronic Hepatitis B: A Multicenter, Randomized, Double-Blind, Placebo-Controlled Trial.

Background: Bushen Jianpi formula (BSJPF, also known as Lingmao formula) is a traditional Chinese medicine for chronic hepatitis B (CHB). The previous study has suggested that the treatment combination of BSJPF and entecavir (ETV) can achieve a significant loss of hepatitis B e antigen (HBeAg) and a significant decrease in serum level of hepatitis B virus (HBV) DNA in HBeAg-positive CHB patients with mildly elevated alanine aminotransferase. Objective: This study aimed to evaluate the efficacy and safety of BSJPF combined with ETV for treating HBeAg-negative CHB patients. Methods: A total of 640 patients were assigned randomly to the treatment group (receiving BSJPF combined with ETV for 96 weeks) or the control group (receiving a placebo combined with ETV for 96 weeks) in a 1 : 1 ratio. The primary endpoints are the rate of loss of hepatitis B surface antigen (HBsAg). The secondary outcomes included the rate of decrease in the HBsAg concentration to ≥1 lg·IU/mL, the HBV DNA suppression, the decline of the level of covalently closed circular DNA (cccDNA) in the liver, histological improvements, and the rate of ALT normalization. Results: The rate of HBsAg loss in the treatment group was significantly higher than that of the control group (5.5% versus 1.8%, P=0.031). There were 11.1% of patients in the treatment group who recorded a reduction in HBsAg ≥1 lg·IU/mL, which is better than 5.9% of patients in the control group (P=0.043). There was no significant difference between the two groups with regard to the rate of HBV DNA clearance, the reduction in intrahepatic cccDNA, and the rate of ALT normalization (P > 0.05). The rate of liver fibrosis improvement in the treatment group was better than that of the control group (35.5% versus 11.8%, P=0.031), but there was no difference in necroinflammatory improvement (P > 0.05). The adverse events (AEs) were similar between the two groups, except for the abnormal kidney function, with 2.2% in the control group and 0.0% in the treatment group (P=0.028). Conclusion: The combination of BSJPF and ETV can increase the rate of HBsAg loss and the rate of histological fibrosis improvement without serious adverse events in CHB patients. Trial Registration. This trial is registered with ChiCTR-IOR-16009880 on November 16, 2016-retrospectively registered, http://www.chictr.org.cn/showproj.aspx?proj=16836.

Beneficial Effects of Bushen Formula Combined with Enticavir on Chronic Hepatitis B Patients with Suboptimal Response to Enticavir by Regulating B-Cell Differentiation.

BACKGROUND/AIMS: To investigate the clinical effects of the combination therapy with Bushen Formula (BSF) plus enticavir (ETV) on chronic hepatitis B (CHB) patients with suboptimal response to ETV and explore the regulatory mechanisms of BSF on B cells-mediated humoral immunity. METHODS: Sixty-four HBeAg-positive CHB patients with suboptimal response to ETV were enrolled, and were randomly assigned into control group (C-Group, placebo combined with ETV for 12 months) or treatment group (T-Group, BSF combined with ETV for 12 months). Serum samples from 57 treatment-naïve CHB patients and 15 healthy controls were collected. Serum HBV DNA levels were evaluated by real-time PCR. Characteristics of peripheral blood B-cell subtypes were analyzed by flow cytometry. Serum HBV markers and B cell-activating factor (BAFF) levels were detected by ELISA. Chinese medicine symptom complex score was evaluated and recorded. RESULTS: After treatment, the rates of patients with a reduction of HBsAg > 0.5 log10 IU/ml or 1.0 log10 IU/ml and the rates of HBeAg clearance in T-Group were all higher than those in C-group, with no significant intergroup difference. Only in T-Group, Chinese medicine symptom complex score and the frequency of total B cells were significantly decreased, and the frequencies of Bm1, CD24+CD27-switched B cells and plasma cells were markedly increased after treatment compared with those before treatment. Compared with healthy controls, serum BAFF levels in treatment-naïve CHB patients were increased, and there was a significant positive correlation between serum BAFF and HBsAg levels. However, serum BAFF levels did not differ after treatment in T-Group and C-Group. CONCLUSIONS: The combination therapy with BSF plus ETV promotes the reduction of HBsAg level and the clearance of HBeAg in CHB patients with partial response to ETV through regulating the differentiation of B-cell subsets.

[Research on HBV DNA inhibition of plasmid acute infection mouse with betulinic acid].

Betulinic acid is a naturally occurring pentacyclic triterpenoid, which has antiretroviral, antimalarial, and anti-inflammatory properties. The purpose of this study is to investigate the HBV DNA replication inhibition in the mouse model with betulinic acid. Hydrodynamic injection method via the tail vein with the Paywl. 3 plasmid was used to establish the animal mode (n = 15), and the mice were randomly divided into the PBS control group (n = 5), Betulinic acid treatment group (n = 5) and lamivudine control group (n = 5). The day after successful modeling , the mice would have taken Betulinic acid (100 mg x kg(-1)), lamivudine (50 mg x kg(-1)), PBS drugs orally, once daily for 7 days, blood samples were acquired from the orbital venous blood at 3, 5, 7 days after the administering, HBsAg and HBeAg in serum concentration were measured by ELISA and the mice were sacrificed after 7 days, HBV DNA southern detections were used with part of mice livers. The results showed that betulinic acid significantly inhibited the expression of HbsAg in the mice model at the fifth day compared with the control group, and there was no significant differences between the effects of lamivudine and the PBS control group; both the betulinic acid and lamivudine groups had no significant inhibition for the HBeAg expression; the HBV DNA expressions of the liver tissue from the betulinic acid and lamivudine groups were inhibited compared with the control group. Taken together, these results reveal betulinic acid can inhibit the HBsAg expression and replication of the liver HBV DNA in the mouse model.

Beneficial therapeutic effect of Chinese herbal Bushen formula on CHB patients with mildly elevated alanine aminotransferase by down-regulating CD4+CD25+T cells.

ETHNOPHARMACOLOGICAL RELEVANCE: To investigate the clinical effects of the Bushen formula on chronic hepatitis B (CHB) patients with mildly elevated alanine aminotransferase (ALT) (1-2 times ULN) and its relevant immune mechanism. MATERIALS AND METHODS: Fifty CHB patients were enrolled, and they were categorized into two groups according to infection state, including CHB carrier group(24 patients) and mildly elevated ALT (1-2 times ULN) group (26 patients), and 12 healthy volunteers were collected as normal control group. In addition, patients with mildly elevated ALT were taking the Bushen formula for 6 months. Biochemical assessment, viral load quantitation and HBV markers were examined at the Department of Clinical Laboratory, Shanghai Shuguang Hospital. Peripheral blood mononuclear cells were separated from patients and healthy volunteers. T cells were separated from healthy blood, and Hepatitis B core antigen (HBcAg) was added to the culture media. The percentage of CD4+CD25+T cells and the expression levels of IFN-γ and IL-4 were examined by flow cytometry and the expression level of FoxP3 was examined by real-time PCR and flow cytometry. The Chinese medicine symptom complex score was calculated. RESULTS: The percentage of CD4+CD25+T cells and the expression level of Foxp3 in two CHB patient groups were higher than that in the normal control group, and the percentage of CD4+CD25+T cells and the expression level of Foxp3 in the CHB carrier group were highest. The experiment in vitro showed that both the percentage of CD4+CD25+T cells and the expression of Foxp3 were elevated by HBcAg in CD4+T cells separated from normal peripheral blood. In patients with mildly elevated ALT, the serum ALT level was significantly decreased from 81.77±18.24U/L to 64.92±20.60U/L and the serum HBV DNA level was significantly decreased from 6.54±0.95 (log) to 5.15±2.22 (log), and symptom scores were significantly decreased from 6.19±3.99 to 2.77±1.66 after administration of the Bushen formula. In addition, we found that the frequency of CD4+CD25+T cells correlated negatively with the expression level of IFN-γ in CD4+T cells from CHB patients with mildly elevated ALT, and the percentage of CD4+CD25+T cells was decreased from 11.07±4.30% to 8.70±3.49% and the expression level of IFN-γ in CD4+T cells was increased from 13.98±3.25% to 15.85±3.43% after administration of the Bushen formula. CONCLUSION: In CHB patients, the percentage of CD4+CD25+T cells and the expression of Foxp3 in peripheral blood were increased, which can be repeated in HBcAg-stimulated healthy CD4+T cells. The Bushen formula has positive effects on CHB patients with mildly elevated ALT (1-2 times ULN) by reducing serum ALT and HBV DNA levels, which is relevant with the decreasing of the percentage of CD4+CD25+T cells and the increasing of the expression level of IFN-γ in CD4+T cells.