The Therapeutic Effects Of Rat IL-10 Plasmid DNA Tail Vein Injection On Hepatic Fibrosis In Rat And Its Mechanisms

Aims: to investigate the therapeutic effects of rIL-10 plasmid DNA in large volume in short time tail vein injection on hepatic fibrosis in rat and its mechanisms.Methods: RT-nest-PCR and gene recombination technique were used to construct rat interleukin-10 eukaryotic expression vector (pcDNA3-rIL-10). The reconstituted plasmid pcDNA3-rIL-10 and the empty vector pcDNA3 were transfected respectively into BRL cells by asialoglycoprotein receptor mediated liposome PEIjet-gal. The expression of rIL-10 gene was detected by RT-PCR and ELISA analysis. The influence of the transfection of reconstituted plasmid pcDNA3-rIL-10 on BRL cells was detected by MMT and flow cytometric analysis. Reconstituted plasmid pcDNA3-rIL-10 was rapidly injected into rat in large volume in a short time through tail vein. RT-PCR, ELISA and immunohistochemistry analysis were used to detect the expression and distribution of rIL-10 gene in rat. Thirty SD rats were divided randomLy into control group (group N), fibrosis model group (group M), rIL-10 gene therapy group (group I) and empty vector control group (group P). Rats in group N were treated with 0.5mL normal sodium twice a week for 8 wk via intraperitoneal injection, and rats in other groups were treated with 0.5mL pig serum twice a week for 8 wk. At the beginning of the 5th wk, rats in group N and M were injected weekly with Ringer,s solution, rats in group I were injected weekly with the rIL-10 recombinant plasmid pcDNA3-rIL-10 and rats in group P were injected weekly with empty vector pcDNA3 via tail vein. At the end of the 8th wk, all rats were put to death and then collected sample of liver tissue and serum. The grading and staging of hepatic fibrosis were measured by HE staining. The change of ALT and AST in serum was measured by biochemistry analysis. The change of collagen type I,III was measured by Picrosirius staining and the expression ofα-SMA,MMP-13 and TIMP-1 in liver tissue were measured by S-P immunohistochemistry. Results: DNA sequencing and restriction endonuclease digestion confirmed that eukaryotic expression vector pcDNA3-rIL-10 was constructed successfully. RT-PCR and ELISA assay confirmed that the BRL cells transfected with plasmid pcDNA3-rIL-10 expressed highly secretary type rIL-10 protein and the expression of rIL-10 show a few protection effect on RBL cells transfected. RT-PCR and immunohistochemistry confirmed the presence of the rIL-10 transcription and expression mainly in liver and ELISA assay confirmed maintaining a stable expression of rIL-10 in serum can be assessed by repeated administration. The liver pathohistology analysis confirmed experimental rat hepatic fibrosis model induced by pig serum was established successfully. Compared with the rats in group M and P, rats in group I showed significant therapeutic effects: decreased the degree of hepatocyte necrosis and degeneration and the number of inflammatory cells around lobular veins, reduced the disposition of collagen fibers and reduced the levels of ALT and AST in serum(P<0.01). Picrosirius staining confirmed the expression of collagen type I,III was significantly suppressed (P<0.01)and the immunohistochemistry confirmed positive levels forα-SMA and TIMP-1 were decreased significantly in group I (P<0.01).Conclusions: rIL-10 plasmid DNA tail vein injection can prevent efficiently the progression of experimental rat hepatic fibrosis induced by pig serum, the therapeutic effects of rIL-10 plasmid injection on hepatic fibrosis may be primarily exerted by making rIL-10 transcription and expression mainly in liver, inhibiting the expression ofα-SMA and TIMP-1 and promoting collagen type I,III resolution.