Study On Effect Of Tibetan Medicine Triphala On Apoptosis And Proliferation Of Islet β Cells In Diabetic Rats

Diabetes mellitus is an endocrine disease with a rising incidence.Apoptosis and proliferation of islet beta cells are one of the important causes of diabetes mellitus.It has been found that high glucose can induce apoptosis of islet beta cells by activating mitochondrial pathway and inflammasome pathway,thereby inhibiting insulin secretion and eventually leading to diabetes.Therefore,inhibiting the apoptosis of islet beta cells is of great significance for delaying the occurrence of diabetes mellitus.Triphala is a prescription commonly used to treat high altitude polycythemia.Foreign studies have found that Triphala can reduce fasting blood sugar by inhibiting alpha-glucosidase.However,there is no report that the Tibetan medicine Triphala can treat diabetes by inhibiting the apoptosis of islet beta cells.Therefore,this study is to investigate whether different doses of Tibetan medicine Triphala can affect incretin signaling pathway to inhibit apoptosis of pancreatic islet beta cells induced by high glucose and promote proliferation of pancreatic islet beta cells,so as to provide certain basis and reference for the research and development of Tibetan medicine Triphala in the treatment of diabetes mellitus.Part Ⅰ Determination of gallic acid in Tibetan Medicine TriphalaObjective To establish an HPLC method for the determination of gallic acid in the water extract of Tibetan medicine Triphala.Methods The mobile phase consisted of methanol-0.3% phosphoric acid(4:96);detection wavelength: 273 nm;column temperature: 25℃;flow rate:1 m L/min Chromatographic conditions for content determination.Result The chromatographic peaks of gallic acid and impurities in the water extract of Tibetan medicine Triphala were well separated,and the chromatographic peaks did not tail.The linear relationship was good in the range of 0.16-0.960μg(r2=0.9997).The average content was 13.515 mg/g.The average recovery rate was 97.15% and RSD was 1.78%.Conclusion The method has the advantages of short peak time,simple mobile phase composition and good reproducibility.The content of gallic acid measured by this method is high.It can also provide a reference for the control of water extraction of Tibetan medicine Triphala.Part Ⅱ Effect of Tibetan Medicine Triphala on Apoptosis and Proliferation of Islet β Cells in STZ Diabetic RatsObjective To explore the effect of Tibetan medicine Triphala on apoptosis and proliferation of pancreatic islet beta cells in diabetic rats.Methods The model of diabetic rats was established by single intraperitoneal injection of STZ.The blood sugar was measured 3 days later.If the blood sugar was more than 16.7 mmol/L,the model was successful.The model was treated with Triphala at doses of 0.43 g/kg,0.86 g/kg and 1.72 g/kg.The positive drug was given by intragastric administration of Sitagliptin.After 6 weeks of administration,blood was taken and pancreatic tissue was stripped.The levels of GLP-1 and GIP in rat serum and c AMP in pancreatic tissue were detected by ELISA.The changes of islet,islet beta cells and islet alpha cells were observed by HE staining,aldehyde fuchsin-orange G staining and buffered silver nitrate staining.The expressions of TXNIP,GLP-1R,insulin,TCF7L2 and AKT in pancreatic tissue were measured by Western blot.Result 1.After 6 weeks of administration,the levels of serum GLP-1,GIP and c AMP in pancreas tissue were measured.Compared with model group,the levels of serum GLP-1 and GIP in Triphala group increased(p<0.01),and the levels of c AMP in Triphala group increased(p<0.01).3.Pathological section results:(1)HE staining showed that the pancreatic islets in the normal group were larger and more islet cells were in the central position under microscopy;the number of islet cells in the model group was smaller,and the boundary between islets and peripheral cells was not obvious;(2)The orange G staining of islet cells showed that the number of islet beta cells in the model group decreased and the number of intracytoplasmic granules in islet beta cells was less.The number of islet beta cells in positive group and Triphala group was relatively large,and the morphology of islet was also restored.(3)Buffered silver nitrate staining showed that in the model group,the number of islet alpha cells increased and the content of granules in the cytoplasm increased.The number of isletalpha cells in positive group and Triphala group was relatively small.4.Western results showed that the expression of TXNIP and TCF7L2 protein was up-regulated,while the expression of GLP-1R,insulin and AKT protein was down-regulated in the model group(p<0.01).Compared with the model group,the expression of TXNIP and TCF7L2 protein in the Triphala group was down-regulated(p<0.05),and the expression of GLP-1R,insulin and AKT protein was up-regulated(p<0.01).Conclusion: Tibetan medicine Triphala can increase the activity of Wnt/beta-catenin pathway by inhibiting the expression of TXNIP,thereby increasing the level of serum incretin(GLP-1 and GIP),increasing the level of intracellular c AMP,promoting insulin secretion,inhibiting the apoptosis of beta cells,promoting the proliferation of beta cells.