Study Of Anti-Diabetic Effect Of Oleanolic Acid And Its Mechanism

Diabetes mellitus (DM) is one of the most common endocrine diseases. The mortality is only inferior to cardiovascular disease and malignancy in the developed country, and becoming the third fatal pathogeny. The World Health Organization has estimated that the total number of diabetic patients is not less than 171 million presently, and expected that it will rise to 366 million in 2030. The incidence of diabetes will be increased to 4.4% in 2030 from 2.8% presently. In despite of the application of plentiful hypoglycemic agents in clinic, there is not effective drug for diabetes due to the severe toxicity and side effects, inconvenience and instable efficacy. Insulin is an effective hypoglycemic drug, but it only alleviates hyperglycemia of diabetic patient. And diabetes can not be cured completely by insulin. Natural anti-diabetic pharmaceutical are attracting attention in the medicinal field by the less toxicity and no-drug-resistance. The natural drugs are exploited rapidly in the developed country. Some new agents that were rooted in natural products frequently appeared. Ligustrum lucidum Ait has been used in traditional Chinese medicine due to its antitumor and hepatoprotective properties. There are a few reports on hypoglycemia of Ligustrum lucidum Ait. However, the treated mechanisms of Ligustrum lucidum Ait in diabetes are not reported yet. In order to develop new anti-diabetic pharmaceutical, the systematic and deep research on hypoglycemia of Ligustrum lucidum Ait is highly desired.In this paper, the principal active compound-oleanolic acid (OA) was extracted from Ligustrum lucidum Ait by multi-crystal method. The extract was isolated, purified and analyzed by thin layer chromatography (TLC) and mass spectra (MS) technology. OA was ground to powder with 200-700 nm diameters. Diabetic rats and mice were induced with STZ or alloxan intraperitoneally. Diabetic animals were randomly divided into normal control group (NC), diabetic control group (DM), DM+OA low dose group (DM+OA LD) and DM+OA high dose group (DM+OA HD). The genes of PPARα, PPARγ, PKB and PKC were relative with metabolizing of saccharide and lipid. The expression of PPARα, PPARγ, PKB and PKC in the OA-treated animals was analyzed by immunohistochemical stain, single cell gel electrophoresis, ELISA, Westhern Blot and RT-PCR and so on. Meanwhile, the effect of OA was studied systematically on cell apoptosis, antioxidant ability and hormone level which are related with saccharide metabolizing. The results were listed as follows.When diabetic rats were treated with OA for 40 days, the decreasing rates of the plasma glucose levels were 32.4% (in DM+OA LD group) and 46.4% (in DM+OA HD group), respectively. The levels of TG, TC, LDL-c in DM+OA HD group were significantly lower, while their level of HDL-c increased significantly (P <0.05, 0.01), which implied that OA has the function of hypolipidemia. The levels of serum ALP, AST and ALT of OA-treated rats approached to normal value. The results indicated that OA could protect the liver of the diabetic rats. Furthermore, OA also strengthened the antioxidant ability by increasing the activities of SOD and GSH-px.The toxicity and side effect of OA was examined by HE stain, acute toxic test and single cell gel electrophoresis. Results showed that OA could increase the quantity of the pancreatic islets and the cells in pancreatic islet; OA has the ability to amend the impairment on liver and kidney of STZ-induced diabetic rats. In acute toxic test, the mice were fed OA 1000 mg/kg, as a result, there is not death of mouse. Moreover, the result of single cell gel electrophoresis indicated that OA did not destroy DNA of the lymphocytes basically.The levels of insulin and thyroid hormones of the diabetic rats were analyzed, and the results indicated that OA could increase significantly the insulin secretion of diabetic rats (P <0.05), but the changes of serum T3, T4 and TSH levels were insignificant. Furthermore, OA did not affect significantly the weight of liver, lung, kidney and pancreas of the diabetic rats (P >0.05), but the weight of the thymus was significantly higher (P < 0.05). It is tempting to speculate that OA might stimulate the growth of thymus, consequently increasing the immune function of animals.Effect of OA on apoptosis was studied; the results showed that expression of Bax mRNA in pancreas and kidney tissues was reduced after OA-treated, while expression of Bcl-2 was enhanced. The ratio of Bcl-2/Bax approached to normal level, and there was significant difference with diabetes control mice (P <0.05, 0.01). The research indicated that OA might restrain the apoptosis of pancreas and kidney. By immunohistochemical analysis, the results implied that OA also ameliorated expression of Bax and Bcl-2 protein in liver, and restore the function of saccharide and lipid metabolizing, consequently, which avoid diabetic complication.The mechanisms of OA on anti-diabetes were investigated by determining expression of some cytokines. The results indicated that PPARαlevel was up-regulated in pancreas and kidney of OA-treated diabetic mice after OA treatment for 8 weeks, but the difference was insignificant with control mice (P >0.05). The expression of PPARγmRNA was significantly up-regulated in OA-treated diabetic mice kidney and pancreas tissue (P >0.05). Westhern Blot determining indicated that the expression of PKB protein was increased significantly in OA-treated diabetic mice kidney (P <0.05). The present study provided further evidence in support of OA on stimulating secretion of insulin, amending the impairment on liver, kidney and pancreas of STZ-induced diabetic rats, hypoglycemic and hypolipidemic activity. The study also found that OA did not affect the expression of PKC in diabetic mice liver and kidney (P >0.05).In the study, the effects of extract of Ligustrum lucidum Ait-oleanolic acid on diabetic rats were investigated systematically, and the action mechanisms were discussed on hormone, protein and molecule levels. It will provide a scientific proof for development of Ligustrum lucidum Ait as a suitable natural anti-diabetic agent.