Mechanisms Of Total Flavonoids Of Litsea Coreana Leve. Var On Prevention Of Chronic Vascular Complications Of Diabetes

Diabetes Mellitus is a common multiple endocrine metabolic disease and has been a serious threat to human health. Patients with type 2 diabetes mellitus（T2DM）are the most common one among all diabetes mellitus. In the final analysis, all chronic complications of T2DM are induced by vascular lesions and endothelial dysfunction. For the past few years, many scholars at home and abroad extracted from plants and separated a variety of pharmacologically active compounds,of which the flavonoids was the most valued. Preliminary research from Anhui Medical University Pharmacy College indicated that Total Flavonoids of Litsea Coreana（TFLC） had hypoglycemic and hypolipidemic functions. This research firstly established one rat model of type 2 diabetes with insulin resistance by combining high-fat emulsion via gavage and injection of streptozotocin（STZ）, then treated with TFLC continuously for 6 weeks. The results demonstrated that TFLC had a protective effect on vascular complications in model rats. The mechanisms were possibly related to the reduction of JNK phosphorylation in renal and vascular endothelial cells, the improvement of vasomotoricity, the lower concentration of serum inflammatory factors, the enhancement of high glucose-stimulated vascular endothelial cell proliferation, the diminution of apoptosis and oxidative stress and so on. 1. Establishment and Evaluation of rat models of Type 2 Diabetes with insulin resistanceObjective:To establish a rat model with insulin resistance （IR） feature similar to the pathogenesis of human type 2 diabetes mellitus （T2DM）.Methods:SD rats（n=96） were randomized into normal control group（NC, n=20） and high-fat diet group（HF, n=76）,the latter fed with high-fat emulsion （10ml/kg/d） via gavage and the NC group treated with the same volume of physiological saline.The body mass （BM） were measured weekly. After 5 months feeding, the BM, systolic blood pressure （SBP）, serum triglyeride （TG）, fasting blood sugar （FBG）, fasting insulin （FIns） were measured, and insulin sensitivity index （ISI） was calculated. Insulin sensitivity of 6 rats randomly chosen from each group was evaluated with euglycemic hyperinsulinemic clamps technique by observing their glucose infusion rate （GIR）. Streptozotocin（STZ,12mg/kg） and complete Freund’s adjuvant （CFA, 0.5ml/kg） were administered to the rest of the HF rats via intraperitoneal injection once a week in no more than 3 times. The rats whose random blood sugar > 11.1mol/L were diagnosed as type 2 diabetes mellitus. The 24h urinary microalbumin （mALB） levels in the model rats were measured and the endothelial cell ultrastructures of kidneys and thoracic aorta vessels in model rats were observed with transmission electron microscope.Results:The BM, SBP, level of serum TG and FIns in HF group after 5 months feeding were significantly higher than those in the control group（P <0.05）, and ISI was significantly lower. FBG increased in HF group but there was no significant difference compared with NC group （P>0.05）. The HF rats acquired significant lower GIR compared with NC rats （P <0.05）. After the treatment of STZ and CFA, random blood glucose concentration of 53 rats in HF group were higher than 11.1mol/L, accounting for 85.5%of the total rats. Compared with that of NC group, endothelial cells of model rats were detached from vessel wall in thoracic aorta vessels, vacuolar degeneration occurred in some mitochondria, and mild extension occurred in rough endoplasmic reticulum of the endothelial cell from model rats. The basilar membrane was unevenly thicker in the capillary of renal glomerulus, the majority of foot process was fused, and mesenteria structure was increased and dimed. In addition, the electronic dense agents were seen in mesenteria thickened fraction, and vacuolar degeneration in some mitochondria was observed in vessel endothelial cells. The kidney hypertrophy index and 24h urine albumin levels in DM group also increased significantly as compared with those of NC group （P<0.05）.2. Effects of TFLC on metabolism index, inflammatory factors and thoracic aorta vessel endothelial function of obese type 2 diabetic rat with insulin resistanceObjective:To observe protective effects of TFLC on function of ultramicrostructure of vessel endothelium and inflammatory factors in T2DM model rats accompanying with insulin resistance, and to observe the influence of TFLC on vasomotion of ex vivo thoracic aorta rings in normal SD rats.Methods:SD rats with T2DM and IR were allocated into DM and PIO, TFLCL and TFLCH group randomly. The PIO group were given pioglitazone via gavage （10mg/kg·d）, and TFLC group were given TFLC via gavage （400mg/kg·d or 200mg/kg·d） respectively. After 6 weeks of treatment, body mass,heart mass /body mass ratio, systolic blood pressure, fasting blood glucose, glycosylated hemoglobin-A1c （HbA1c）, high sensitive C-reactive protein （CRP）, nitric oxide （NO）, endothelin-1（ET-1）, and fasting insulin levels were measured, and arteriae aorta from all groups were observed under transmission electron microscope. The dissociated thoracic aorta from normal SD rats were cut into thoracic aorta rings about 2mm to establish ex vivo thoracic aorta rings perfusion model. The rings were divided into NC, TFLC, and PIO group randomly. The TFLC group was added into organ bath in different concentration of TFLC （25mg /L、50mg/L、100 mg /L） one by one, and the PIO group was administrated with 10μmol /L PIO. Results:Compared with those of NC group, SBP, FBG, FIns, CRP, HbA1c, ET-1, homeostasis model asessment-insulin resistant index（HOMA-IR） in the DM group increased markedly, while NO and ISI decreased significantly （P <0.05）. Compared with those of DM group, FBG, HbA1c, FIns, HOMA-IR in PIO and TFLCH group decreased significantly. Endothelial cells of DM group were detached from vessel wall in thoracic aorta vessels. Vacuolar degeneration occurred in some mitochondria, and mild extension occurred in rough endoplasmic reticulum of the endothelial cells in model rats. Meanwhile, only minor pathological changes were detected in TFLCL, TFLCH, and PIO group. TFL of different concentrations can significantly decrease endothelial vascular ring tension（P<0.05） . Low concentrations of TFLC （25mg/L） can influence vasodilation, and showed dose-dependent manner in a certain concentration range （25-100mg/L）.3. Effects of TFLC on JNK signaling pathway in vascular endothelial cell from SD rat under high concentration of glucoseObjective:To observe the influence of TFLC on proliferation and apoptosis, oxidative stress and JNK signaling pathway of rat vascular endothelial cells under high concentration of glucose, so as to investigate the mechanism of TFLC protecting vascular endothelial cells.Methods:The VECs were divided into 6 groups randomly: Normal group （Glu 5.5mmol/L）, High glucose group （Glu 47.5mmol/l）, High glucose+PIO (1×10^-6mmol/L),High glucose + SP600125（1.0μmmol/L）, High glucose+TFLC （100mg/L）, High glucose+TFLC （50mg/L）. NO and ET-1 levels in the supernatant were detected with nitrate reductase and radio-immunity assay. Apoptosis and ROS in all groups were measured with flow cytometry. JNK and p-JNK expressions were checked with Western blot to observe the influence of different concentration of TFLC on vascular endothelial cells in high concentration of glucose. Results:NO levels decreased noticeably in supernatant of cell culture under high concentration of glucose. Inversely, ET-1 levels, ROS and apoptosis increased obviously. JNK phosphorylation was also notably higher when compared with cells under low concentration of glucose （P<0.01）. TFLC can significantly reverse the high glucose effect on various cells, which was in dose-dependent manner （P <0.05）.4. Effects of TFLC on kidney and c-Jun N-terminal kinase signaling pathway in obese type 2 diabetic mice with insulin resistanceObjectives:To observe the effect of TFLC on renal pathological changes of diabetic rats with insulin resistance and its effect on c-Jun N-terminal kinase （JNK） expression and phosphorylation, to probe the protective mechanism of TFLC on renal function of diabetic rats and to provide a new premise for cure of Diabetic Nephropathy （DN）. Methods:SD rats with T2DM and IR were allocated into DM, PIO, TFLCL and TFLCH group randomly. The PIO group were given pioglitazone via gavage （10mg/kg·d）, and TFLC group were given TFLC via gavage （400mg/kg·d or 200mg/kg·d） respectively. After 6 weeks of treatment, renal pathological changes in kidneys were observed by means of HE, PAS and PASM staining respectively. JNK or p-JNK expressions in kidneys were analyzed semiquantitatively by the methods of immunohistochemistry, Realtime quantitative RT-PCR and Western Blot. Results: 1. The pathological changes of rat kidney tissue in DM group were observed. Compared with those of NC group, the size of glomeruli and numbers of karyocyte increased, capsular space became narrower, basement membrane was irregularly thicker, and mesenteria matrix increased in DM group. Compared with DM group, above-mentioned pathological changes in TFLCH and PIO group improved to some degree. Compared with those of NC group, JNK expression levels in the kidneys of DM group increased significantly （P<0.05） according to immunohistochemistry （IHC）, real time RT-PCR and Western blot analysis. Compared with DM group, the level of JNK expression in PIO and TFLCH groups decreased significantly （P<0.05）. The result of immunohistochemistry also proved significant decrease of pJNK in TFLCH group.Conclusions:1)Intraperitoneal injection of low dose of STZ and CFA after 5 months of high-fat diet feeding could induce SD rats to develop type 2 diabetes mellitus accompanying with insulin resistance, aortic and renal vascular impairment changes,hyperlipidemia,hypertention,which is similar to human type 2 diabetes.2)TFLC treatment can significantly improve insulin resistance in the rats with T2DM, and decrease the level of FBG、HbA1c、TG、and. TFLC treatment can also reduce the morphologic lesion of thoracic aorta in the rats with T2DM, and Improved endothelium-dependent diastolic dysfunction.3)TFLC could protect the vascular endothelial cells and 25-100 mg / L TFLC can enhance viability of high glucose cultured vascular endothelial cell, increase secretion of NO, reduce ET-1, ROS levels and apoptosis, and significantly decrease p-JNK levels.4)TFLC had protective effects by improving the pathological changes of kidney structure of diabetic rats, as well as downregulating the expression of JNK and level of pJNK, The protective effect may be related to reduced JNK expression and phosphorylation.