Effect of Additional Clostridium butyricum on the Intestinal Flora of Chronic Hepatitis B Patients Treated with Entecavir.

INTRODUCTION: To explore the influence of intestinal flora on the occurrence, development and antiviral therapy of chronic hepatitis B (CHB), 16S rDNA amplification sequencing was performed to investigate the intestinal flora in CHB patients treated with entecavir (ETV) and Clostridium butyricum (CB). METHODS: CHB patients were divided into the ETV group (treatment with ETV alone) and ETV + CB group (treatment with ETV and CB). After 8-week treatment, feces samples were collected and processed for 16S rDNA amplicon sequencing; blood samples were collected for the biochemical, immunologic and virologic evaluations, which were compared between groups. RESULTS: ETV treatment for 8 weeks significantly decreased the serum levels of alanine aminotransferase (ALT), interleukin-6 (IL-6), IL-8, tumor necrosis factor-α (TNF-α) and HBV DNA compared to those before treatment, but there were no marked differences between the ETV group and ETV + CB group. The intestinal flora changed significantly in the CHB patients after ETV + CB treatment: there were marked differences in 13 unique species before treatment and 4 unique species after ETV + CB treatment; at the phylum level, the top five bacteria with significant difference between patients before treatment and ETV + CB patients were Firmicutes, Actinobacteria, Cyanobacteria, Euryarchaeota and Synergistetes. There were significant differences in 25 unique species in the ETV group and 4 unique species in the ETV + CB group; at the phylum level, the top five bacteria with significant difference between ETV patients and ETV + CB patients were Actinobacteria, Fusobacteria, Proteobacteria, Saccharibacteria and Synergistetes. CONCLUSION: ETV treatment improves the serum biochemical, immunologic and virologic variables, but additional CB fails to further improve these variables. Of note, additional CB affects the intestinal flora in the CHB patients treated with ETV.

[Effects of bone morphogenic proteins-7 on production of collagen from hepatocytes and hepatic stellate cells during liver fibrosis].

OBJECTIVE: To study the effects of bone morphogenic proteins-7 (BMP-7) on the production of collagen from hepatocytes and hepatic stellate cells (HSC) during liver fibrosis, and the possible mechanism thereof. METHODS: Ten SD rats underwent repeated peritoneal injection of pig serum twice a week for 8 weeks so as to establish liver fibrosis models. Another 5 rats were injected intraperitoneally with normal saline as control group. Then the rats were sacrificed with their livers taken out. Primary hepatocytes and HSC were isolated from the fibrotic livers and cultured. BMP-7 at the concentrations of 10, 50, or 100 mg/L was added into the culture media for 24 and 96 hours respectively. ELISA was used to detect the levels of collagen of type I, III, and IV. BMP-7 at the concentration of 50 mg/L was added into the culture media for 96 h, then immunofluorescence staining was conducted on the cells to examine the levels of TbetaR-I and TbetaR-II, TGF-beta1 receptors on the cell surface, and the Smad2/3 level. Real-time RT-PCR was used to detect the TGF-beta1 mRNA expression in the cells. RESULTS: The levels of collagen of type I and III in the HSC treated by BMP-7 were all significantly lower than those in the control group especially 50 mg/L BMP-7 decreased the expression of collagen I by 40% +/- 6% (P < 0.01) and decreased the collagen III expression by 60% +/- 8% (P < 0.01), however, the levels of type IV collagen in the hepatocytes treated by BMP-7 were not significantly different from those of the control group. The levels of collagen of type I, III, and IV in the hepatocytes treated by BMP-7 were not significantly different from those of the control group. The impact of 50 mg/L BNP-7 on the HSC was stronger than that of the 10 mg/L BMP-7, however, not significantly different from that of the 100 mg/L BMP-7. There were not significant differences in the TbetaR-I and TbetaR-II levels in the HSC culture fluid treated by 50 mg/L BMP-7. 96 h after the treatment of 50 mg/L BMP-7 the Smad2/3 immunofluorescent strength in the HSC and hepatocytes decreased remarkably, and the TGF-beta1 level in the HSC decreased to the level 88% as high as that before treatment. CONCLUSION: BMP-7 inhibits the production of type I, and III collagen in activated HSC with the possible mechanism of inhibition of phosphorylated Smad2/3 in HSCs and down-regulation of the expression of TGF-beta1.

Migration of hepatic stellate cells in fibrotic microenvironment of diseased liver model.

BACKGROUND: In liver fibrosis, alterations within the space of Disse microenvironment facilitate the progression of chronic liver disease. The normal basement membrane-like matrix in the space of Disse converts to a matrix rich in fibril-forming collagens during the fibrosis. This study aimed to investigate the impact of alterations in the space of Disse microenvironment on the migration of hepatic stellate cells (HSCs) in the process of liver fibrosis, and to explore the novel mechanism of liver fibrosis from the viewpoint of cell migration. METHODS: A modified in vitro Boyden chamber system was employed to partially mimic the in vitro microenvironment of the Disse space in normal liver and in fibrosis. The effects of fibrogenetic growth factors on the migration of HSCs in simulated liver fibrosis were assessed by cell migration and cell proliferation experiments. RESULTS: Enhanced platelet-derived growth factor (PDGF)-BB, transforming growth factor-beta1 (TGF-beta1) and/or epithelial growth factor (EGF) in liver fibrosis resulted in an increase in migratory capacity of activated HSCs. The enhanced migration of HSCs induced by PDGF-BB was proliferation-independent. The elevation of basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) during liver fibrosis had no effect on the migration of HSCs. CONCLUSIONS: The study provides valuable insights into the role of the space of Disse microenvironment in regulating the migratory behavior of HSCs. TGF-beta1, PDGF-BB and EGF, which increase in liver fibrosis, induce the migration of activated HSCs. However, bFGF and VEGF have no effect although they also increase during liver fibrosis.

[Mechanism of hepatic stellate cell migration during liver fibrosis].

OBJECTIVE: To study the mechanism of migration of hepatic stellate cells (HSCs) within the space of Disse microenvironment during liver fibrosis, and to explore the novel pathogenesis of liver fibrosis from the view of cell migration. METHODS: Human HSCs of the line LX2-HSC were cultured. A modified in vitro Boyden chamber system was used to partially mimic the microenvironment of Disse space of normal basement membrane like matrix or that in fibrosis. HSCs were put in the upper chamber, and transforming growth factor (TGF)-beta1, plate-derived growth factor (PDGF)-BB, epidermal growth factor (EGF), vascular epithelial growth factor (VEGF), bfibroblast growth factor (bFGF), and collagen type I or type IV were put into the lower chamber. Four hours later cell migration assay was conducted. HSCs were put into 6-well plate and then added with TGF-beta1, PDGF-BB, EGF, VEGF, bFGF, and collagen type I or type IV, zymography was used to examine the activity of matrix metalloproteinases 2 and 9. SDS-gel immunoblotting was used too. RESULTS: Stimulation of HSCs with PDGF-BB, TGF-beta1, and/or EGF resulted in an increase in their migratory capacity and up-regulated MMP-2 activity. And the increase of MMP-2 could enhance Migration of HSC by 4.9-fold. The migration of HSCs (3.2-fold) was induced by type I collagen and inhibited by type IV collagen (1.2-fold). Migration induced by PDGF-BB, TGF-beta1, and collagen I could be inhibited by alpha1- and/or alpha2-integrin blocking antibodies. CONCLUSION: In liver fibrosis, alterations within the space of Disse microenvironment facilitate the migration of HSCs; the mechanism is associated with up-regulation of MMP-2 and with mediation of alpha1beta1 and alpha2beta1 integrins. Extracellular matrix by it self shows feedback actions to migration of HSCs.

Inhibitory effect of bone morphogenetic protein-7 on hepatic fibrosis in rats.

AIM: Hepatic cirrhosis is a serious clinical problem caused by the accumulation of extracellular matrix, which can ultimately progress into hepatic failure. Transforming growth factor-beta1 (TGF-ß1) plays a pivotal role in extracellular matrix production. Bone morphogenetic protein-7 (BMP-7), as a member of the TGF-ß1 superfamily, has been well proved to be capable of reversing renal fibrosis in mice. In this study, we aim to investigate the potential effect of BMP-7 on hepatic fibrosis in rats. METHODS: Sprague-Dawley rats were randomly divided into five groups. In the hepatic fibrosis model group (n=8), rats was treated with porcine serum at 0.5 ml each time, twice a week. In the negative control group (n=10), rats were intraperitoneally injected with equal amount and frequency saline. Rats were injected with BMP-7 (100 µg/kg weight) before porcine serum intraperitoneal injection in the preventive group (n=9). For the early (n=10) and late (n=8) treatment group, rats were received with BMP-7 (100 µg/kg weight) every other day since the second and fourth week respectively after porcine serum injection. After eight weeks, the degree of liver fibrosis in rats was evaluated and the expression of TGF-ß1 in liver tissues was detected by Western blot and immunohistochemistry. RESULTS: The grade of hepatic fibrosis was significant attenuated by BMP-7 prevention and treatment compared with the rats in negative control group (P<0.05). In addition, the expression of TGF-ß1 greatly decreased in the BMP-7 preventive and treatment groups detected by both Western blot and immunohistochemistry. CONCLUSIONS: BMP-7 can attenuate and even prevent the level of hepatic fibrosis in rats through inhibiting the expression of TGF-ß1 in the liver fibrotic tissues. Therefore, it may be a potential clinical drug for the prevention and treatment of hepatic fibrosis.