Research On The Combined Efficacy Of Intervention And GBE On Lower Extremities Vascular Disease In Diabetes Mellitus

Diabetes mellitus is one of the chronic diseases which pose serious threat to people's health at present, its complications negatively affect the life quality and life expectancy of diabetes patients. Low limb extremities diseases is one of the complications of diabetes, it will result in acrogangrene and finally give rise to amputation.. One report from Europe showed that among the non-trama amputation patients, 35-50 percent were caused by diabetes.So low limb extremities is the major cause of disabilities derived from diabetes. Therefore, it is of great significance to pay more attention to early diagnosis to ameliorate the complications and reduce the disabilty rate.The strategies utilized in traditional treatment of low limb extremities diseases derived from diabetes are to control the risk factors such as blood sugar, blood pressure and blood lipid level and promote blood flowing with medication, local application of medication and antibiotics. But in patients with severe condition, the traditional treatment will not work and may result in amputation without effetive therapy in time. So far, vesseal intervention therapy is the most effective way for lower extremilties derived from diabetes, it can improve symptoms and life quality of diabetes patients.The findings from researches on the mechanisms of diabetes development showed that oxidative stress was one of the major mechanisms which caused the complications of diabetes. Hyperglycemia stimulated surplus production of reactive oxygen species, oxidative stress induced the abnormal changes of proteins, lipids and DNA in cells, and resulted in pathological metabolic pathway and damages to cell and finally diabetes developed. Therefore, it is necessary to implicate antioxidation therapy in therapy of diabets complications. The present research is to compare the efficacy between combination of intervention therapy with antioxidation and intervention alone to explore the function of antioxidation in intervention therapy. The present research comprise two parts, one is basic research on GBE function, the other one is clinical study.In basic research, the protective effects of GBE on the endothelial cells exposed to high level of glucose and diabetes rats model were studied.In animal experiments, WISTAR rats, body weight(200±20)g,half were femal and half male. Rats were fed for a whole week to watch therir healthy state and who were active and healthy were to be used for experiments. The experiment rats fasted for 12 hours before STZ were injected through peritoneal injection at the dose of 50 mg?kg-1; the rats in normal control group were injected citrate buffer at the dose of 50 mg?kg-1. On day 3, 5, 7 after STZ administration, the blood sugar level were tested in all experiment rats and those which possessed blood sugar level more than 16.7 mmol?L-1 for three times were classified as diabets rats.The experiment design is as followed: normalcontrol group(C), diabets mellitus group(DM), Ginkgo biloba extract group( GBE). Rats in DM were injected with saline at the dose of 0.83 ml?kgbody weigt-1 and rats in GBE were injected with GBE at the dose of 0.83 ml?kg-1 through peritoneal injection every day continuously up to 30 days. On day 15 and 30, the rats from all groups were sacrificed to the collect the blood for the test of blood sugar level, triglyceride(TG), high density lipoprotein(HDL), low density lipoprotein(LDL) and insulin level. Each time, five rats from each group. The blood were collected into a centrifuge tube and standed in the refrigerator at 4℃for 1 hour and centrifuged at 2500 rpm for 10 minutes to obtain the serum and the samples would be tested in the biochemistry machine.Human umbilical vein endothelial cells(ECV 304) were used to test the protective effects of GBE on the cells and its mechanisms of action.Prior to treatment of cells with RPMI supplemented with 10% FCS containing high glucose (35 mM) concentrations and combined high glucose concentration and GBE(500μg/ml ) , ECV 304 cells were synchronized by serum deprivation for 48 h in RPMI containing normal glucose levels supplemented with 0.5% FBS. ECV 304 cells were subsequently exposed to high glucose concentration(35 mM) and combined high glucose concentration(35 mM) and GBE(500μg/ml ) conditions in RPMI supplemented with 10% FBS for 24 hours. An osmotic control of 25 mM mannitol was included in all experiments. Intracellular calcium concentration was determined by Fluo-3/AM Ester. The production of MDA and activities of SOD were determined by reagent kit.In clinical investigation, 60 diabetes patients with lower extremities disease admitted between January 2006 and December 2007 were enrolled as subjects, male 38, female 22, the average age was(62.31±4.50)years old, the duration of diabetes was (20.0±4.9)years.Based on their willingness, the subjects were classified into three groups, PTA+ GBE, PTA alone and internal medication, each group 20 subjects. Patients in PTA alone and internal medication group were given Ligustrazine,0.24g Ligustrazine in 0.9% 250mL saline through vein injection once a day for two weeks. B12 500μg once a day, intramuscularly injection for two weeks.Patients in PTA+GBE were given GBE 20ml in 0.9% saline by in vein in jection for two weeks besides the same medication with the above two groups. After PTA operation, the patients received GBE continuously for 2 weeks besides the same medication with the other two groups in which the patients were given. The symptoms of patients from the three groups were monitored and coldness scores were estimated. The blood sample were collected from all the patients from the three groups to test the level of ROS, MDA, GSH-PX and SOD.The results from animal experiment showed the level of blood glucose, TG, LDL, TC in rats from GBE group was significanlty lower than those from DM group(P<0.05), the level of insulin, HDL was significantly higher(P<0.05) when the diabetes rats were treated with GBE for 30 days. Compared with normal rats, the rats from GBE groups had higher blood glucose, TG, TC, LDL level and lower insulin and HDL level. There was no difference in the index of blood glucose, TC, TG, HDL, LDL, and insulin between rats in GBE group and DM groups(P>0.05)when the rats in GBE group were treated by GBE for 15 days.The ECV 304 cells were exposed to high glucose concentration for 24 hours had higher level of intracellular calcium compared with those exposed to combined high glucose concentration and GBE(500μg)(P<0.05). MDA level produced by ECV 304 cells exposed to combined high glucose concentration and GBE was significantly lower than those exposed to high glucose concentration(P<0.05).SOD activities in cells exposed to combined high glucose concentration were significantly higher than those exposed to high glucose concentration(P<0.05).Clinical investigation results showed that MDA and ROS level in diabetes patients were significantly higher than those in healthy adults(P<0.05), GSH-PX and SOD level were significantly lower(P<0.05). The oxidative condition in diabetes patients with lower extremities disease was worse.Intervention operation with antioxidative therapy produced better therapeutic effects on diabetes patients with lower extremities diseas than intervention operation alone; the patients treated by PTA+GBE had better outcome in lower extremity and feet pain scores, coldness scores, systolic pressure of ankles artery, ankle brachial index,peak changes of blood flow of the affected limb than those treated only by PTA. The efficacy of PTA was better than traditional internal medication.The results of the present research indicated that antioxidative therapy would enhance the efficacy of PTA, so it was necessary to implicate antioxidative therapy in the treatment of diabetes and its complications.