The Gene Therapy And Mechanism Study Of Wound Healing Impairment In STZ-Diabetic Mice

Diabetes Mellitus (DM) is a multipathogenic, chronic metabolic disease characterized by hyperglycemia accomitant with impaired metabolism of glucose, lipid and protein induced by insulin secretion deficiency or dysfunction. DM causes severe diabetic syndrome, which can be responsible for high disablity and death rate of diabetic patients. Among many diabetic pathophysiological changes, wound healing impairment underlies several cutaneous diabetic syndromes, such as diabetic ulcer, diabetic gangrene and repeated local infection. The etiology of wound healing impairment is multifactorial, with vasculopathy and neuropathy being major contributors. Its pathogenic mechanism is tightly bound to nitric oxide (NO) and supreoxide (O2.-). There is growing evidence that NO plays a pivotal role in the normal wound healing. It promotes processes including angiogenesis, matrix accumulation and remodeling, migration and proliferation of fibroblasts, epithelial cells, endothelial cells, and karetinocytes. It also participates in the regulation of cytokines in inflammation response during the post phase of wound healing. The NO level in cutaneous tissue is maintained mainly by the endothelial nitric oxide synthase (eNOS), not by the other two NOS isoforms, i.e. inducible NOS and neuronal NOS. It was reported that increasing eNOS protein expression level and activities in tissue or cells will enhance the synthesis of NO, thus promoting the process of wound healing. On the contrary, decreasing that will weaken NO synthesis and delay wound healing. So it's rational to use eNOS gene transferring therapy to restore the diabetic wound healing impairment through improving NO level in cutaneous tissue. A large amount of proofs demonstrated that the accumulation of reactive oxygen speicies (ROS) was a strong impact factor in the pathogenesis of many kinds of diseases including DM. ROS exerts damage to the homeostasis of oxidant/reductant system, and induce intra-and extracellular oxidative stress, which causes the oxidation and destruction of many important large biological molecules, such as DNA, protein, sugars and lipids. As an important member of ROS, O2.-possesses all the properties of ROS family as well as the capability of deriving other ROS members, which react with NO forming a potent oxidant ONOO.-. It has been revealed that synthesis of O2.-was enhanced by diabetic hyperglycemia and the elevated O2.-levels resulted in development of vasculopathy and neuropathy, which partially manifest as wound healing impairment. Studies showed that reducing or blocking the synthesis of O2.-or scavenging existing O2.-helped to ameliorate the damaged vascular function in diabetic hyperglycemia. Among the synthases of O2.-, NAD(P)H oxidase, xanthine oxidase and uncoupled eNOS were considered the most competent in vascular endothalial system. However in DM or hyperglucose environment, the increasing O2.-in variant types of cells of vascular endothalial system was believed to come from protein kinase C (PKC) dependent NAD(P)H oxidase pathway, whereas the mechanism of elevated cutaneous O2.-in the same environment was not specified. Superoxide dismutase (SOD) is able to catalyze the decomposing reaction of O2., in which O2.-is transformed into hydroperoxide and an oxygen molecule. One of SOD's isoforms, Manganese-containing SOD (MnSOD) was proved to exerts important scavenging O2.-effects in mitochondria, where the respiration chain generates a large amount of O2.-that is believed to be a major soure of intra and extracellular O2.-. It was also reported that increasing MnSOD protein expression level and activities under oxidative stress status will improve the cells viability and resistance to an oxidative environment through getting rid of O2.-. So it's reasonable to use MnSOD gene transferring therapy to restore the diabetic wound healing function through decreasing O2.-level in cutaneous tissue. The present study was going to be carried out in a laboratory in Michgan State University of USA, where the lab staff have been continuously researching gene transfer therapy for years and have accumulated abundant experience in animal model establisment, adenovirus preparation and sample manipulation, thus providing rich resources for the present study. Based on the above materials and the previous study, 3 objectives of the present study were carefully set:eNOS gene and MnSOD gene into STZ DM wound healing impairment mice model with adenoviral vectors, and evaluate the therapeutic effects through determining the eNOS and MnSOD protein level, enzyme activities, NO and O2.-level.the PCR products displayed the right sizes, and it implied the genes carried inadenoviruses were what we expected. 2. The study on eNOS or MnSOD gene therapy with Impairment Model The transferring of eNOS gene The eNOS gene was delivered into the Impairment Model by adenovirus infectionand the wound healing rates and several biological indicators were examined. The resultsshowed that the wound healing rates were significantly increased after eNOS genetransfer (p<0.05), and close to that of the normal control. It also showed that thecutaneous eNOS protein levels and enzyme activities augmented significantly after eNOSgene transfer (p<0.05), concomitant with significantly increased NO levels (p<0.05) anddecreased O2 levels (p<0.05). These results suggested that the eNOS gene transfer .-significantly ameliorated the DM wound healing impairment through improving theeNOS protein expression level and enzyme activities. Meanwhile, the results showed that the NO levels, BH4 levels and eNOS enzymeactivities were significantly decreased in STZ-DM cutaneous samples (p<0.05) as well asO2.-being levels significantly increased (p<0.05). The results also showed thattheexcisional wound itself further downregulated the NO levels and eNOS proteinexpression levels in STZ-DM cutaneous samples (p<0.05). These results suggested thatthe declined NO level and elevated O2 level contributed to the pathogenesis of wound .-healing impairment in STZ-DM model. The transferring of MnSOD gene MnSOD gene was delivered into the Impairment Model by adenovirus infection andthe wound healing rates and several biological indicators were examined. The resultsshowed that, the wound healing rates were significantly increased after MnNOS genetransfer (p<0.05), although still slower than that of the normal control. It also showedthat the cutaneous MnSOD protein levels and enzyme activities increased significantlyafter MnSOD gene transfer (p<0.05), concomitant with significantly decreased O2 levels .-(p<0.05) and increased NO levels (p<0.05). These results suggested that the MnSODgene transfer significantly ameliorated the DM wound healing impairment throughimproving the MnSOD protein expression level and enzyme activities. In vitro gene transfer studyTo further ascertain the efficiency of adenovirus mediated gene transfer, STZ-DM cutaneous samples were tested in vitro. The experiment results showed that, the NO levels were significantly increased after eNOS gene tranfer in vitro (p<0.05) and this subsequently was obviously blocked (p<0.05) by the treatment with Nw-L-arginine methyl ester (L-NAME), a selective eNOS inhibitor. It also showed that the O2.-levels were significantly decreased both in gene transfer of eNOS and MnSOD (p<0.05). These results suggested that the adenoviral vectors were efficacious in delivering and expressing genes in cutaneous tissue. 3. The mechanism study on cutaneous O2.-generation pathways source of accumulated O2.-in high glucose treated cutaneous tissue is the pathway of NAD(P)H oxidase, not that of xanthine oxidase or uncoupled eNOS. Based on the above experiment results, 4 brief conclusions can be drawn: 1) Type I DM was successfully developed in male C57/B6 mice by STZ injection; 2) The significantly decreased NO levels and increased O2.-levels contributed a lot to the pathogenesis of wound healing impairment in STZ-DM Model; 3) The eNOS and MnSOD gene transfer significantly ameliorated the wound healing impairment in STZ-DM Model through improving NO level and reducing O2.-level in cutaneous tissue; 4) NAD(P)H oxidase pathway was the major source of accumulated O2.-in STZ-DM or high glucose treated cutaneous tissue.