Studies On The Prophylaxis And Treatment Hepatic Fibrosis Of Hydroxycamptothecin In A Rat And Part Of The Active Mechanism

Objective:To observe the preventive and therapeutic effect of hydroxycamptothecin (HCPT) in CC14-induced liver fibrosis in rats by detect the Liver function and the degree of fibrosis; Preliminary study the mechanism of the HCPT by detect the Bax,Bcl-2mRNA level,the a-SMA expression and the TUNEL stain in liver. To explore new ways for the treatment of liver fibrosis and to provide theoretic evidence for expanding the clinical apply of the drug.Methods:64 SD rats were randomly divided into 5 groups:Normal group, Model group, Low dose treatment group, Intermediate-dose group and high-dose group.Hepatic fibrosis was induced in rat by abdominal cavity injection of CC14,each rat give 40%CC14 (0.2 ml/kg bodyweight).twice a week,8 weeks total. The normal group inject saline only,the low-dose group inject HCPT(0.25mg/kg bodyweight) 3 times a week,the intermediate-dose group and the high-dose group each apply 0.5mg/kgN 1 mg/kg instead,8 weeks total. Each group remained 10 rats in the eighth week.We check the following sample:①etected the ALT,AST,ALB,TP and A/G levels on serum by using automatic biochemical analyzer;②Detected the WBC and PLT levels on serum by using automatic blood cell analyzer;③Take the same position for the paraffin sections of livers then make the HE stain and masson stain to observe the degree of hepatic fibrosis;④ake the paraffin sections of liver tissue for a-SMA immunohistochemistry and TUNEL stain;⑤ake the same position for the RT-PCR test of livers to observe hepatic Bax/Bcl-2mRNA levels and the Bax/Bcl-2mRNA ratioResults:1. Liver function in each group:The ALT and AST levels on serum of the model group were much higher than the normal group (P<0.05).However,the 3 therapy groups were much lower than the model group (P<0.05).The ALB and A/G levels on serum of the model group were much lower than the normal group (P<0.05) However,the 3 therapy groups were much higher than the model group (P<0.05)2. Test of WBC, PLT in each group:The WBC of the normal group were much higher than the others (P<0.05),there was no significant difference between the model group and the 3 treatment groups (P>0.05).The normal group was the only one which has significant difference to the model group (P<0.05). there was no significant difference between the others (P>0.05)3. Gross morphology of livers in each group:The appearance of normal group's livers are red and smooth, they have sharp edges and soft texture.The livers of the model group were reduced Significantly, Severe adhesion with the adjacency tissues,have uneven surface, hard texture and rough edges.The livers of the 3 therapy groups were bigger than the model group. The livers have slightly dark surface,still soft texture but have not see hyperplastic nodules significantly.4. HE and Masson staining pathology and hepatic fibrosis index:①Histopathology: Has integrity Lobular structural, no fibrosis and no inflammatory cell infiltration of the normal group.The model group have a lot of pseudolobuli, inflammatory cell infiltration,hepatocyte ballooning and steatosis of bullous.However, the 3 therapy groups'lobular structure are clear, Proliferation of fibrous tissue in varying degrees of portal area,fewer Pseudolobules,but a lot fatty degeneration of liver cells Near the portal area,and few inflammatory cells infiltration can be seen,②The histopathological instalment:The statistics of liver fibrosis show that The histopathological instalment of the model group were much higher than the normal group (P<0.05),but the the 3 therapy groups are lower than the model group (P<0.05). The intermediate-dose group and the high-dose group which are better than the low dose group (P<0.05).5. Hepatic Bax,Bcl-2 mRNA and Bax/Bcl-2mRNA levels in rats by RT-PCR: Bax,Bcl-2mRNA levels on liver of the model group were much lower than the normal group (P<0.05).However,the 3 therapy groups were much lower than the model group (P<0.05). The Bax/Bcl-2mRNA ratio of the model group were much lower than the 3 therapy groups(P<0.05) and the intermediate-dose group is the highest one(P<0.05) 6. Hepatic a-SMA levels in rats by immunohistochemistry.-The a-SMA levels of the model group were much higher than the therapy groups (besides low-dose group) (P<0.05).The high-dose and the intermediate-dose group have no significant difference (P>0.05)7. TUNEL staining of liver tissue:TUNEL staining showed positive staining liver cells mainly in the liver portal area, especially in interval in the fiber like the immunohistochemical staining region for the a-SMA protein. No obvious staining of liver cellsConclusion:HCPT could significantly inhibit hepatic fibrosis in CC14-induced in rats. The mechanism is that HCPT may inhibit the expression of a-SMA protein and the proliferation of HSCs.To induce apoptosis of activated HSCs by changing the ratio between the Bax/Bcl-2mRNA, improving the process of liver fibrosis, reaching the therapeutic effect of liver fibrosis.At the same time, we found the dosage, route of administration, dosing interval of HCPT in this study are safe and feasible.This results also are sense to prophylaxis and treatment other tissues and organs of fibrosis (eg pulmonary fibrosis, etc.) by the HCPT.