Abstract

BackgroundWhat was the relationship of Fuzheng Huayu recipe (FZHY) inhibiting hepatocyte apoptosis and HSC activation at different stage of liver fibrosis? In order to answer this question, the study was carried out to dynamically observe FZHY’s effect on hepatocyte apoptosis and HSC activation and further explored underling mechanism of FZHY against hepatocyte apoptosis.MethodsMice were randomly divided into four groups: normal, model, FZHY, and N-acetylcystein (NAC) groups. Acute hepatic injury and liver fibrosis in mice were induced by CCl4. Three days before the first CCl4 injection, treatment with FZHY powder or NAC respectively was started. In vitro, primary hepatocytes were pretreated with FZHY medicated serum or Z-VAD-FMK and then incubated with ActD and TNF-α. Primary HSCs were treated with DNA from apoptotic hepatocytes incubated by Act D/TNF-α or FZHY medicated. Liver sections were analyzed for HE staining and immunohistochemical evaluation of apoptosis. Serum ALT and AST, Alb content and TNF-α expression in liver tissue were detected. Hyp content was assayed and collagen deposition was visualized. Expressions of α-SMA and type I collagen were analyzed by immunofluorescence and immunoblotting. Flow cytometry, immunofluorescence, and DNA ladder for hepatocyte apoptosis and immunoblotting for TNF-R1, Bcl-2 and Bax were also analyzed.ResultsMice showed characteristic features of massive hepatocytes apoptosis in early stage of liver injury and developed severe hepatic fibrosis in later phase. FZHY treatment significantly alleviated acute liver injury and hepatocyte apoptosis, and inhibited liver fibrosis by decreasing α-SMA expression and hepatic Hyp content. In vitro, primary hepatocytes were induced by TNF-α and Act D. The anti-apoptotic effect of FZHY was generated by reducing TNFR1 expression and balancing the expressions of Bcl-2 and Bax. Meanwhile, the nuclear DNA from apoptotic hepatocytes stimulated HSC activation in a dose dependent manner, and the DNA from apoptotic hepatocytes treated with FZHY or Z-VAD-FMK reduced HSC activation and type I collagen expression.ConclusionThese findings suggested that FZHY suppressed hepatocyte apoptosis through regulating mediators in death receptor and mitochondrial pathways, and the effect of FZHY on hepatocyte apoptosis might play an important role in inhibiting liver fibrosis.

Highlights

  • What was the relationship of Fuzheng Huayu recipe (FZHY) inhibiting hepatocyte apoptosis and hepatic stellate cells (HSCs) activation at different stage of liver fibrosis? In order to answer this question, the study was carried out to dynamically observe FZHY’s effect on hepatocyte apoptosis and HSC activation and further explored underling mechanism of FZHY against hepatocyte apoptosis

  • These findings suggested that FZHY suppressed hepatocyte apoptosis through regulating mediators in death receptor and mitochondrial pathways, and the effect of FZHY on hepatocyte apoptosis might play an important role in inhibiting liver fibrosis

  • FZHY exerted both anti-fibrotic and anti-apoptotic functions, but whether the effect of FZHY on liver fibrosis was primarily due to its inhibitory effect on hepatocyte apoptosis

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Summary

Introduction

What was the relationship of Fuzheng Huayu recipe (FZHY) inhibiting hepatocyte apoptosis and HSC activation at different stage of liver fibrosis? In order to answer this question, the study was carried out to dynamically observe FZHY’s effect on hepatocyte apoptosis and HSC activation and further explored underling mechanism of FZHY against hepatocyte apoptosis. What was the relationship of Fuzheng Huayu recipe (FZHY) inhibiting hepatocyte apoptosis and HSC activation at different stage of liver fibrosis? Liver fibrosis is a wound healing process elicited by chronic liver injury [1] Liver injuries, such as hepatocytic inflammatory necrosis and apoptosis, are the precursors of liver fibrosis [2]. FZHY consists of six Chinese medicinal herbs [10], namely Radix Salvia Miltiorrhizae (Danshen), Cordyceps (Chongcao), Semen Persicae (Taoren), Gynostemma Pentaphyllammak (Jiaogulan), Pollen Pini (Songhuafen), Fructus Schisandrae Chinensis (Wuweizi). It has been approved by SFDA as a drug and widely used to treat hepatic fibrosis in China since 2002. Its efficacy against liver fibrosis was confirmed in phase II clinical trial carried out in US (www.clinicaltrials.gov)

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