Nd701 Experimental Hepatic Fibrosis

Hepatic fibrosis is the result of wound-healing response to chronic liver injury due to various etiologies such as parasitic disease,chronic viral infection,immunologic attack,toxic damage etc. Because of the worldwide prevalence of these etiologies,hepatic fibrosis is a kind of severe disease with high morbidity and mortality.It is predominantly characterized by excessive accumulation of extracellular matrix(ECM) caused by both an increased synthesis and decreased or unbalanced degradation of ECM.The activation of hepatic stellate cell(HSC) is critical step in the development of hepatic fibrosis and the proliferation and fibrosis of activated HSC are attributed to over-aggradation of ECM.Although hepatic fibrosis is thought to be a reversible pathological state through proper cure,no established effective therapy of hepatic fibrosis is available for clinical use yet.It is hoped that understanding the molecular pathophysiology of hepatic fibrosis will lead to novel therapeutic strategies and antifibrogenic drugs.ND701 is a phenolic compound extracted from Tibetan Salvia miltorrhiza Bunge which is used as herbal plant and has many intresting biological activities,such as adstringent,antioxidative,antiinflammatory,antimutagen,antibacterial and antiviral,antitumor,antihepatitis and protecting the liver,inhibiting blood clots and enhancing fibrolysis.Now it has been developed by Shandong natural drug technology research center as a novel drug in chronic persisting hepatitis and liver fibrosis with proprietary intellectual property rights.The aim is to study the therapeutic effects and mechanisms of ND701 on hepatic fibrosis.1.The model of immunological hepatic fibrosis induced by bovine albumin was prepared,the animals were randomly divided into six groups(normal groups,control groups,ND701 groups and positive progoups) according to the body-weight and the level of albumin(Alb),fibronectin(FN). The therapeutic groups were givern different ND701 dilutions,fufangbiejiaranganpian orally,at the same time,saline was given to normal and control groups for 60 days.And at 15th and 30th days, serum alanine aminotransferase(ALT),Alkaline Phosphatase(ALP),aspartate aminotransferase (AST),total bilirubin(Tbil),albumin(Alb) and total protein(TP) were assayed by spectrophotometry. At 60th day all rats were killed,hyaluronic acid(HA),laminin(LN) and procollagenⅢ(PcⅢ) in serum were assessed by ELISA.Hepatic tissue sections were fixed in 4%formaldehyde and embedded in paraffin.Paraffin sections were stained with hematoxylin-eosin(HE),Massion used for hepatic fibrosis.The results showed that hepatocyte was destroyed and fibrosis was induced when rats were given repeat immune activation.The parameters of hepatic function and the degree of hepatic fibrosis deteriorated in control group compared with normal group(serum transaminase activities,ALP,Tbi,ALb/TP,HA,PcⅢ,collagenⅣand hydroxyproline Hyp).Based on histopathological sections,immune activation elicited extensive necrosis of hepatocyte,plasmoedama,karyopyknosis,disappearance of Nigeria's body,enhance of plasmoeosinophilic and even the rupture of nuclear membrane.So only the outline of cell was seen and fibrous tissue proliferated excessively in liver.Intragastric administration of ND701(30 mg/kg,15 mg/kg) significantly for 15 and 30 days decreased the activity of serum transaminase and ALR At the 30th day,ND701(30mg/kg,15mg/kg) could significantly improve hepatic function and at the 60th day, ND701(30mg/kg,15mg/kg) could significantly inhibit the progression of hepatic fibrosis with the lower HA,LN,PcⅢand Hyp contents(P＜0.05 or 0.01 vs model group).Histopathological section showed that ND701 could alleviate the change of liver.2.In the vitro experiments,hepatic stellate cell(HSC) were incubated with various concentrations of ND701.The effect of ND701 on the activation of HSC was assayed by the alteration of cell adherence and cell morphology.Use auto-activation model,cell proliferation was investigated by SRB assay.The levels of transforming growth factor-β1(TGF-β1) and connective tissue growth factor(CTGF-) in cell lysis solution and fibronectin(FN) in culture supernatants were determined by ELISA.The results showed that ND701(1μg/ml,2μg/ml,4μg/ml,8μg/ml,16μg/ml)could inhibit the activation of HSC in dose dependent manner and ND701(2μg/ml,4μg/ml,8μg/ml,16μg/ml) could inhibit the proliferation of activated HSC and weaker its ability to synthesizeTGF-β1(no significance),CTGF-and FN(P＜0.05 or 0.01).The results showed that ND701 could improve hepatic function,inhibit the activation and proliferation of HSC,reduce the contents of connective tissue.So ND701 had obviously therapeutical effect on hepatic fibrosis which due to its ability to inhibit the generation of TGF-β1 and CTGF.