Effect Of CTGF ShRNA And TIMP-1shRNA On Rats With Hepatic Fibrosis

Abstract: Objectives: To investigate the effect of CTGF short hairpin RNA(shRNA) and TIMP-1shRNA plasmids on the transcriptions of TIMP-1, CTGF,Col-Ⅰ and Col-Ⅲ mRNAs and the expression of CTGF, TIMP-1, Col-I andCol-Ⅲ proteins in the rats with hepatic fibrosis, and further to explore theprophylaxis mechanism of shRNA, and to find a new method of gene therapyof hepatic fibrosis. Methods:50Clean-level Wistar male rats were randomlydivided into5groups. Group A：double RNA interference group；Group B:CTGF interference group；Group C: TIMP-1interference group；Group D:empty vector treatment control group；Group E: fibrosis model control group.The rats were injected with1%of the DMN solution by intraperitonealinjection and each injection volume was80μl/100g, three times a week for threeweeks. The interference was started from the3rd week by the tail vein ofhigh-pressure injection of plasmid. Group A, the injection of psiRNA-GFP-Com (targeting CTGF and TIMP-1); Group B, the injection ofpsiRNA-GFP-CTGF (targeting CTGF only); Group C, the injection ofpsiRNA-GFP-TIMP-1(targeting TIMP-1only); Group D, the injection ofempty plasmid vector (psiRNA-DUO-GFPzeo); Group E: no injection.Injections were administrated once every two weeks and totally for four times.All rats were sacrificed and their liver tissues were collected at the12thweek.The liver tissues were fixed with neutral formaldehyde, and then pathologicalchanges of liver tissue were observed by HE staining and the expression of CTGF, TIMP-1, Col-Ⅰ and Col-Ⅲ protein in liver tissue were detected byimmunohistochemistry. After being extracted from the liver tissues, the RNAwhich were reverse-transcribed to be cDNA, and the transcriptions of CTGFmRNA, TIMP-1mRNA, Col-Ⅰ mRNA and Col-Ⅲ mRNA in liver tissue wereassayed by fluorescence quantitative PCR. Results:(1) The transcriptions ofCTGF mRNA, TIMP-1mRNA, Col-Ⅰ mRNA and Col-Ⅲ mRNA and theexpression of CTGF, TIMP-1, Col-Ⅰ and Col-Ⅲ protein in liver tissue were notsignificantly different between the Group D and E. While the transcriptions ofCTGF mRNA, TIMP-1mRNA, Col-Ⅰ mRNA and Col-Ⅲ mRNA and theexpression of CTGF,TIMP-1, Col-Ⅰ and Col-Ⅲ protein in liver tissue weredecreased in Group A, B and C, compared with those in Group E.(2) Thedegree of inhibition of double RNA interference group (Group A) on theexpression of Col-Ⅰ mRNA and the content of type Ⅰ collagen protein in livertissue was superior to single interference groups (Group B or C, p<0.05orp<0.01). Conclusions:(1) The shRNAs targeting CTGF and/or TIMP-1cansignificantly inhibit the expression of CTGF, TIMP-1, Col-Ⅰ, Col-Ⅲ genes andthe secretion of associated proteins in the rats with hepatic fibrosis.(2) Thecombined double interference targeting CTGF and TIMP-1may be better thanthe separate interference either targeting CTGF or TIMP-1.