The Effective Preparation Of The Recombinant VIP And Their Effects On Streptozotocin-induced Diabetic Models In Mice

Objetive:VIP (vasoactive intestinal polypeptide) has a lot of biological functions, such as immunoregulatory role and profound anti-inflammatory effect. VPAC1 receptor mediates the most anti-inflammatory role of VIP. Diabetes mellitus are often associated with elevation the state of inflammatory. The recombinant VIP was obtained efficiently by using genetic engineering principle and technology. Then the recombinant VIP and VPAC1 agonist were employed to evaluate effects on diabetes mellitus in diabetic mice model induced by injection of streptozotocin. The mechanism of these effects mediated by the recombinant VIP and VPAC1 agonist was explored.Method:According to the natural sequence of VIP, the gene coding VIP was synthesized by two steps PCR. Engineering bacteria ER2566/pTWIN/VIP was constructed, and high-efficient preparation of VIP was accomplished with the IMPACT (Intein Mediated Purification with an Affinity Chitin-binding Tag) protein expressing and purification system. After the induction of diabetes mellitus in mice by streptozotocin, recombinant VIP and VPAC1 agonist were intraperitoneally administered to normal and streptozotocin diabetic mice respectively.The long-term effects of VIP and VPAC1 agonist on water intake, food intake, body weight, levels of plasma glucose and glucose tolerance, plasma insulin, the weight of spleen and pancreas, pancreatic islet histological changes were assayed to confirm the role of VIP and VPAC1 agonist in treating diabetes. The levels of TAOC,catalase,H2O2,MDA were determined with commercially available kits to study the effects of VIP and VPAC1 agonist on the oxidative stress and the capability of antioxidant defense system in tissue of diabetic mice.Results:The engineering strain ER2566/pTWIN/VIP was constructed, and 0.2mg recombinant VIP with the purity of about 95%was obtained from 1g fermentation product with IMPACT system. The molecular weight of recombinant VIP is proved the same with natural VIP by SDS-PAGE and mass spectrum. In the experiment on the role of recombinant VIP and VPAC1 agonist in streptozotocin induced diabetic mice models, we found that:compared with the STZ group, the administration of VIP depressed food intake, increased body weight, decreased plasma glucose, enhanced glucose tolerance and decreased serum insulin, increased the weight of spleen and pancreas. VPAC1 agonist displayed more effective effects than VIP on body weight, plasma glucose, glucose tolerance, plasma insulin, the weight of viscera. Pancreatic islet histological changes showed that compared with the STZ group, the level of inflammation and necrosis was decreased, indicating the improvement in the islet histology in VIP and VPAC1-A group. The results indicated that both recombinant VIP and VPAC1 agonist can ameliorate diabetes mellitus effectively. The mechanism research showed that compared with the control group, the level of TAOC was significantly decreased in liver and spleen of STZ group, but VPAC1 agonist induced remarkably elevated this level. In the liver, CAT content was significantly decreased in diabetic mice, while VPAC1 agonist induced remarkably elevated this content, so VPAC1 agonist induced an antioxidant activity in diabetic mice. In liver of STZ group, the index of oxidative stress, H2O2 and MDA were remarkably elevated compared with the control group, while VIP and VPAC1 agonist downregulated these contents. VPAC1 agonist had more effective effects than VIP in increasing the level of antioxidant and decreasing lipid peroxidation.Conclusion:High-efficient preparation of recombination VIP was accomplished, with genetic engineering principle and technology, which laid the foundation for its further application. In the experiment on diabetic mice indicated that the treatment with recombination VIP and VPAC1 agonist ameliorated significantly the diabetes induced by STZ by elevating the capability of antioxidant defense system and decreasing the level of oxidative stress and lipid peroxidation. VPAC1 agonist showed more effective than VIP due to the role that VPAC1 receptor mediates the anti-imflammatory role of VIP in diabetic mice.