The Effect Of Gene Intervention Of IL-10on Sinusoidal Capillarization Of CCL4-induced Hepatic Fibrosis In Rats

Objective:1.To establish the experimental rat hepatic fibrosis model inducedby CCl4，then observe the process of sinusoidal capillarization。2.To investigate the effect of IL-10on sinusoidal capillarizationof CCl4-induced hepatic fibrosis in rats.Methods:1.Fifty-six SD rats were divided randomly into control group(groupN),fibrosis model group(group M), Rats in group N were treated with2ml/kg normal sodium twice a week for eight weeks via intraperitonealinjection,and rats in group M were treated with2ml/kg a mixture of50%CCl4with castor oil twice a week for eight weeks.Rats were sacrificedat the end of3rdday、1stwk、2thwk、4thwk、6thwk、8thwk，and then collectedsample of liver tissue and serum. The grading and staging of hepaticfibrosis were measured by H-E staining and Masson staining.The change ofHA in serum was measured by radioimmunoassay. The structure of sinus wallin liver sinusoidal was observed by TEM.The expression of CD31、LN、Col-IVwere measured in liver tissue by S-P immunohistochemistry.2.Thirty SD rats were divided randomly into control group(groupN),fibrosis model group(group M),pcDNA3-rIL-10gene therapy group(groupI) and empty vector control group（group P）.Rats in group N were treatedwith2ml/kg normal sodium twice a week for eight weeks via intraperitonealinjection,and rats in other groups were treated with2ml/kg a mixture of50%CCl4with castor oil twice a week for eight weeks.At the beginning ofthe5thweek,rats in group N and M were injected weekly with Ringer’ssolution,rats in group I were injected weekly with the rIL-10recombinant plasmid pcDNA3-rIL-10and rats in group P were injected weekly with theempty vector pcDNA3.0via tail vein.At the end of the8thwk,all rats weresacrificed and then collected sample of liver tissue and serum. Thechange of ALT and AST in serum was measured by biochemistry analysis.The grading and staging of hepatic fibrosis were measured by H-E stainingand Masson staining.The changes of HA、LN、PCIII and Col-IV in serum weremeasured by radioimmunoassay. The structure of sinus wall in liversinusoidal was observed by TEM.The expression of CD31、LN、Col-IV weremeasured in liver tissue by S-P immunohistochemistry.Results:1.The liver pathohistology analysis confirmed that the experimentalrat hepatic fibrosis model induced by CCL4was establishedsuccessfully.In the initial stage of fibrosis,it was characterized by theinfiltration of inflammatory cells and necrosis of hepatocytes,and by thedisposition of collagen fibers and formation of fibrous septa during thelater stage. The necrosis of hepatocytes persisted,but the theinfiltration of inflammatory cells decreased.2.The results of TEM and immunohistochemistry showed that theformation of sinusoidal capillarization was occurred gradually. LSECslost fenestra significantly earlier than the formation of fibrous septa,but sinusoidal endothelial basement membrane was formed later than theformation of fibrous septa.3.Compared with the rats in group M and P,rats in group I showedsignificant therapeutic effects：decreased the degree of hepatocytenecrosis and degeneration and the number of inflammatory cells aroundcentral venous,reduced the disposition of collagen fibers and the levelsof ALT、AST、HA、LN、PCIII and Col-IV in serum(P<0.05).TEM showed there-opening of apertures and partial regression of BM. Masson stainingconfirmed the expression of collagen was significantly suppressed(P<0.05) and the immunohistochemistry confirmed positive levels of CD31、LN、Col-IV were decreased significantly in group I（P<0.05）.Conclusions： The gene intervention of rIL-10can alleviateefficiently the degree of inflammation and fibrosis in liver tissueinduce by CCL4,and reverse partially the the formation of sinusoidalcapillarization.The therapeutic effect of IL-10on hepatic fibrosis wasassociated with the reversal effect of IL-10on the sinusoidalcapillarization.