| Background and Objective:Liver fibrosis is one of the most common disease with reversible of intrahepatic connective tissue dysplasia caused by a lot of pathogenic factors, at the same time,it is also a dynamic development,and a pathological stage which make various liver diseases eventually develop into cirrhosis.In the liver and gallbladder surgery clinical, biliary obstruction and cholestasis are common pathological states.Longer obstruction can cause permanent liver damage,and even develop into liver fibrosis and biliary cirrhosis,which seriously harm to the patient’s quality of life and survival rate. In recent years,although we have a better understanding of cholestatic liver fibrosis pathogenesis,the clinical treatment is still ineffective. Despite some patients accepted a technically successful biliary drainage surgery, jaundice subsided slowly and poor liver function recovery,and the high incidence of postoperative complications,making the mean prolonged hospital stay,increased cost of treatment and the psychological burden,therefore early prevention and occlusion of liver fibrosis is the key to change the prognosis of the disease.Previous studies have shown that the liver progenitor cells,oval cells,has been found in the chronic liver disease.This hepatic progenitor cells mainly located in periportal areas,fiber spacing,periportal zone adjacent hepatic parenchyma,and inflammatory pseudo-lobular borders with the dual potential of proliferation and differentiation into mature hepatocytes and biliary epithelial cells.With the specific markers of the oval cells found,research about oval cells has been hot,especially in the research of liver damage repairing and liver fibrosis.These provide the latest way for the study of liver injury protection,treatment and the formation mechanism of liver fibrosis.Cholestatic hepatic fibrosis belong to "jaundice" category. YCHT derives from Han· Zhang Zhong Jing "Treatise on",and it consists of Capillaris,Gardenia and Rhubarb with the power of heat and dampness jaundice.Modern research shows that YCHT has gallbladder,liver,antipyretic, anti-inflammatory,analgesic,anti-bacterial,anti-virus. In recent years,the study of YCHT on antifibrotic mechanism is not uncommon.Therefore,in this study,we explore the roles of YCHT on hepatoprotection and antifibrosis by observation YCHT on impact of hepatic oval cells and hepatic stellate cell in experimental rat liver fibrosis model, using histological and PCR techniques,by ligating Wistar rats’ common bile duct to replicate the pathological states of biliary obstruction cholestatic liver fibrosis.Methods:Male Wistar rat40is randomly divided into four groups,namely the sham group (10),the common bile duct ligation group (10),the recanalization group (10) and the recanalization+Chinese intervention group (10).The sham group is only for laparotomy but not ligation,and the rest of the group is carried out bile duct ligation to prepare cholestatic liver fibrosis model.Bile duct recanalization occur on the common bile duct ligation for7days and10days, respectively, and recanalization for1day is given traditional Chinese medicine orally or equal amount of saline for three days. Postoperative raised under the same conditions.The7days and10day after the operation in the sham group and the common bile duct ligation group,and the3day after the recanalization operation in the recanalization group and the recanalization+Chinese intervention group,five in each group were sacrificed. Take right lobe of the liver10%formalin-fixed histological analysis;Take liver left lobe-80℃frozen for RT-PCR detection reserved.1、Through the naked eye and HE staining,observe of bile duct obstruction in liver morphological changes at different time points.2、Through immunohistochemistry,observe dynamically the number and distribution of oval cells at different time points,by OV-6marker.3、Through the PCR detection to each purpose gene expression:alpha-smooth muscle action protein (alpha-SMA) and type I collagen (COL I),observe dynamically each gene expression at different time points.Results:1、General results:(1)The sham group:liver specimens,each lobe rosy and smooth surface.(2)The common bile duct ligation group:After Wistar rat biliary obstruction,the liver is gradually yellowing and swelling,partly hard texture, liver edema and congestion. We can find formalin-fixed part of rat liver with the rough surface samples and grey color and the performance of early liver fibrosis.With the increase of obstruction of the amount of days liver fibrosis significantly increased.(3)The recanalization group:After biliary recanalization,liver jaundice is gradually fading. (4)The recanalization+Chinese intervention group:Compared to the recanalizatio-n group,liver disease is no change in the naked eye.2、HE dyeing results:(1)The sham group:No inflammatory cell infiltration,and no liver cell degeneration and necrosis.(2)The common bile duct ligation group:After Wistar rat biliary obstruction7days,the hepatic lobule structure integrity,inflammatory cell infiltration,the liver cell swelling,partly degeneration and necrosis,moreover haemorrhage and necrosis,liver tissue collect abbacy visible bile duct proliferation and fibrous tissue cell hyperplasia;After biliary obstruction10days, the lobular structure damage, liver cell degeneration and necrosis, fibrosis degree progress gradually deteriorated.(3)The recanalization group:After bile duct recanalization, the liver tissue still inflammatory changes,liver cell degeneration and necrosis,the degree of swelling reduced,partly still visible fibrosis.(4)The recanalization+Chinese intervention group:compared to the recanalization group,liver lesions slightly improved.3、Immunohistochemical results:OV-6positive cells express parts mainly located in collect abbacy bile ducts and fiber spacing and liver interstitial new born bile duct and no small bile duct cells lumen of cytoplasm. OV-6immunohistochemical staining cell number and positive distribution with obstruction the extension of time and increase. In the sham group there is very little positive expression.And in the common bile duct ligation group,OV-6positive cells area after modeling7days and10days increased compared with the sham group, the difference was statistically significant. Compared with the common bile duct ligation group, after modeling for7days, the recanalization group OV-6positive cells area has been slightly increased, and after modeling10days, the recanalization group OV-6positive cells area has also been slightly increased, but the difference was no statistically significant. In the recanalization+Chinese intervention group,OV-6expression area in the corresponding time points increased compared with the recanalization group,the difference was statistically significant.4、RT-PCR resultsWe analyze relative gene expression levels using2-ΔΔCt. Results are as follows:in the common bile duct ligation group for7days, alpha-SMA mRNA relative expression levels increased by6.72times compared with the sham group(P<0.05),in the recanalization group alpha-SMA mRNA relative expression is2.59times higher than the sham group,and in the recanalization+Chinese intervention group,alpha-SMA mRNA relative expression levels is only1.38times higher than the sham group(P>0.05); compared with the sham group, alpha-SMA mRNA relative expression was significantly increased to13.38times in the common bile duct ligation group for10days(P<0.05), while in the recanalization group alpha-SMA mRNA expression was increased5.94times, and in the recanalization+Chinese intervention group alpha-SMA mRNA relative expression levels only increased by1.56times compared with the sham group(P>0.05). In the common bile duct ligation group for7days, COL1mRNA expression increased by9.94times compared with the sham group(P<0.05), and further increased to10.71times in the common bile duct ligation group for10days(P<0.05); COL1mRNA expression is3.95times higher than the sham group in the recanalization group for7days, and increased to5.41times in the recanalization group for10days(P>0.05); in the recanalization+Chinese intervention group COL1mRNA expression did not change significantly compared with the sham group at7days and10days(P>0.05). Thus alpha-SMA and COL1gene expression gradually increased with the increase of obstruction of the amount of days; early recanalization alpha-SMA and COL1gene expression reduced compared with late recanalization; alpha-SMA and COL1gene expression trends in each group are consistent; YCHT can make alpha-SMA mRNA and COL1mRNA expression markedly decreased in cholestatic hepatic fibrosis rats model.Conclusion:1、This study observed liver pathological changes at different time points,by ligating Wistar rats’ common bile duct to successfully replicate cholestatic hepatic fibrosis rats model, and confirmed that the liver of the BDL rats present cholestasis, progressive aggravating fibrosis performance with the increase of obstruction of the amount of days, YCHT on reducing liver pathological damage in liver fibrosis rat is superior to simple recanalization,which can promote liver pathology and functional improvement after recanalization.2、The study found through immunohistochemistry that YCHT can promote the proliferation of hepatic oval cells improve the process of liver fibrosis by up-regulating OV-6expression in cholestatic hepatic fibrosis rats model.3、The study found through RT-PCR that YCHT can inhibit alpha-SMA and COL1gene expression in the liver fibrosis rats to improve the degree of liver fibrosis to some extent.4、The study found that liver fibrosis is a complex process involving multi-factor,and YCHT with up-regulation hepatic oval cells OV-6expression, degradation of collagen deposition and apoptosis of hepatic stellate cells maybe one of the mechanism on YCHT improving the progression of liver fibrosis. |
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