Experimental Study Of MMP-8 Gene On Anti-liver Cirrhosis In Model Rats

Objective:Excess typeⅠ,Ⅲcollagen accumulation is a major feature of liver fibrosis and cirrhosis, reversion of this disease has not been fully accomplished. Neutrophil collagenase(MMP-8) can degrade typeⅠcollagen effectively. Introduction of MMP-8 gene into liver cells could be an advantageous tool to degrade collagen. In this study, we constructed a adenovirus expressing MMP-8 and intended to investigate which can degrade collagens and attenuate established hepatic fibrosis in the rat.Methods:We cloned full length MMP-8 gene (pro-MMP8) and truncated MMP-8 gene (tMMP-8) from plasmid pGW1GH-MMP8 by polymerase chain reaction(PCR) and enzyme-cutting recombination technique, and constructed the plas- mid pDC-CH-tMMP8, pDC-C-MMP8 which express MMP-8 in adenoviral vector carrying replication-deficient HBV vector , we have also constructed the control plasmid pDC-CH-RFP2. The constructed plasmids were conformed by enzyme-cutting technique and then co-transformed with pBHG1ox△E1,3Cre into 293 cells in order to package and expand recombinant adenovirus. Liver cirrhosis rats induced by TAA for ten weeks were infected once with recombinant adenovirus(3×1011 viral particles per kilogram) by intravenous injection through tail veins. At 2 or 4 weeks after the adenoviral infection, rats were killed. Animals were injected with 0.3% Pentobarbital and killed thirty minutes later. Blood samples were collected by puncture of the abdominal aorta, and serum was stored at -70℃until analysis.Samples of the liver were snap-frozen at -70℃or were fixed in 10%buffered formalin for subsequent histological analysis.Results:We have successfully constructed the recombinant adenovirus expressing MMP-8(Ad-CH-tMMP8, Ad-C-MMP8) and test the activity of MMP-8 in animal exprement, compared with model group and negative control group, the fibrosis was dramatically attenuated at two weeks after the infection, Compared with model group and negative control group,HE staining and bitter acid-sirius red staining showed that hepatocyte steatosis, necrosis and inflammation were significantly mild in treatment group, along with hepatocyte proliferation and recovery of hepatic lobules structure(P<0.05); compared with model group, the content of HYP diminished(28.97±2.36 VS 17.04±0.61, 17.62±1.30; P<0.05);compared with model group and normal group, measurement of serum ALT showed that adenovirus expression in the liver transiently induced a significant increase in serum ALT(P<0.05). However, compared with normal group,model group and negative control group, serum total bilirubin and serum albumin levels did not change significantly in treantment group(P>0.05). After 4 weeks, the proliferative effect almost disappeared, but the hepatic fibrosis remained attenuated and the hydroxyproline content remained diminished. Whereas, the fibrosis in Ad- CH-RFP2-treated rats persisted.Conclusion:Studying results shows that the MMP-8 gene can degrade typeⅠcollagen effectively and attenuate established fibrosis in rat induced by TAA. It is promising for use in a clinical setting and providing a novel therapy tool for liver fibrosis.