The Role Of Nitric Oxide And Superoxide Playing In The Trauma Healing Of DM 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), nitric oxide synthase(NOS) and supreoxide (O2-).1. The role of NO playing in the trauma healing and experiment objectiveThere 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 NO donors or eNOS therapy to restore the diabetic wound healing impairment through improving NO level in cutaneous tissue. 2. The role of O2- playing in the trauma healing and experiment objectiveA 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 induces 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.So it's also rational to use O2- inhibitor therapy to speed up the diabetic wound healing through reducing O2- level in cutaneous tissue.Based on the above materials, 4 objectives of the present study were carefully set:To establish type I DM mice model with streptozotocin (STZ) and its wound healing impairment model, for future therapy study.With selective inhibitors and dominant-negative regulatory protein,to study the relationship between the mechanisms underlying elevated cutaneous O2- level in STZ DM mice and the O2- generating pathways of NAD(P)H oxidase, xanthing oxidase and uncoupled eNOS. To examine the levels of the endothelial nitric oxide synthase(eNOS) expression and Nitric Oxide(NO) generation in the DM mice and the normal mice before and after the trauma-forming operation and to study the effect to the growth of normal skin fibroblast by using different NO donors.With some drugs used on the trauma,to observe their roles in the STZ DM mice's wound healing. These objectives will help to reveal the pathogenesis of diabetic wound healing impairment and provide theoretical evidence for potential therapeutic application.The major experiment results and conclusions of the present study were manifested briefly in below 4 parts.1. Establishment of mice models1) Establishment of STZ induced type I DM mice model (STZ-DM Model)KM mice were injected intraperitoneally with STZ (45 mg/kg) for 5 consecutive days. Blood was collected and whole blood glucose levels were measured to assure the blood glucose level already exceeded 11.1 mmol/L. Then the blood glucose levels and body weights were monitored for 8 weeks. The results showed that, during the whole period, the blood glucose levels of STZ-DM model significantly increased (p<0.05) while the body weights significantly decreased (p<0.05), which is completely consistent with publications and implies that type I DM developed in the mice model.2) Establishment of STZ-DM wound healing impairment model (Impairment Model)8 weeks after STZ injection,STZ DM mice were anesthetized and a full-thickness excisional round wound whose diameter was 1cm was created with the perforex. The wound healing rates were determined and the results showed that the wound healing rates of STZ-DM mice were significantly delayed (p<0.05). It implied that the wound healing capability of mice was severely damaged by DM development.2. The mechanism study on cutaneous O2- generation pathwaysA total of 4 selective inhibitors were employed in the present study. Among them,allopurinol (ALLO) inhibits xanthine oxidase activity, L-NAME inhibits eNOS activity,the other 2 inhibit NAD(P)H oxidase activity through different ways: apocynin (APO) exerts suppression directly, chelerythrine (CHE) exerts suppession through inhibiting PKC activity.The experiment results showed that two NAD(P)H oxidase inhibitors significantly suppressed the O2- elevation in STZ-DM cutaneous samples (p<0.05),which implies that the NAD(P)H oxidase pathway is the major source of O2- in cutaneous tissue of STZ-DM Model. However, ALLO and L-NAME didn't show obviously effects on O2- levels, which implies that the xanthine oxidase pathway and uncoupled eNOS pathway didn't or only fractionally participate in the generation of O2- in cutaneous tissue of the STZ-DM Model.3. The examine of the levels of eNOS expression and NO generation in the DM mice before and after the trauma-forming operation and the study of the effect to the growth of normal skin fibroblast by using different NO donors1) Observation of the eNOS and NO levels before and after trauma-forming operationThere was no difference of the eNOS level expressed in the skin between the DM mice and the normal mice,while the NO level was higher in the normal mice than DM mice before the trauma-forming operation (P<0.05). The eNOS protein and NO level were significantly increased in the normal mice on the fifth day after the trauma-forming operation than before (p<0.05), whereas decreased in the DM mice (p<0.05).2) Cultivation of the normal skin fibroblastUsing the tissue cutting by circumcision cultured the fibroblast with enzyme digestion. Normal skin fibroblast could grow well after purification.Enough fibroblast was gained.3) Study of the effect to the growth of normal skin fibroblast by using different NO donorsWith three NO donors,SNP,GSNO and N-hydroxybenzenesulfonamide,their effects to the growth of fibroblast was investigated by the 3H-TdR incorporation method and flow cytometry.The results showed that SNP could significantly promote the growth of fibroblast in the concentration of 0.01 mmol/L and 0.1mmol/L (p<0.05).While it could vigorously decrease the fibroblast amount in the concentration of 1 mmol/L and 10mmol/L (p<0.05).GSNO could increase ibroblast amount in the concentration of 0.01 mmol/L,compared with the decrease effect in the concentration of 1 mmol/L and 10mmol/L (p<0.05). N-hydroxybenzenesulfonamide had a similar contribution to the fibroblast with GSNO.By cultured with the three NO doner in the concentration of 1 mmol/L,the fibroblast died in a large number examined by flow cytometry.4. The study of wound therapy on impairment Model with some drugsThe L-arg, eNOS, SNP and apocynin(APO) were delivered on the Impairment Model traumas.It showed that SNP could significantly speed up the DM mice trauma healing.It made no difference between the SNP group and the control group (p>0.05).The eNOS,L-arg and APO also play a positive role in the trauma healing (p<0.05).Based on the above experiment results, 4 brief conclusions can be drawn: 1) NAD(P)H oxidase pathway was the major source of accumulated O2- in STZ-DM or high glucose treated cutaneous tissue;2) The significantly decreased NO and eNOS levels and increased O2- level contributed a lot to the pathogenesis of wound healing impairment in STZ-DM Model;3)The low concentration NO donors could promote the fibroblast growth,while the high concentration donors might lead to the fibroblast death directly.The NO played an important role in the fibroblasts'growth;4) The use of SNP, eNOS,L-arg or APO significantly ameliorated the wound healing impairment in STZ DM Model through improving NO and eNOS level or decreasing O2- level in cutaneous tissue.