Experimental Study On The Regulation Of β Cell Function By Islet Paracrine In Tibetan Medicine Triphala

Objective:In this paper,the contents of gallic acid,corilagin and ellagic acid in the lyophilized Phyllanthus emblica,Terminalia chebula,T.Bellirica and the powder of Triphala(THL)were determined.Cell and animal experimental systems were used to evaluate the function of Tibetan medicine THL in regulating the secretion of parislet secretion factors Insulin(INS),Glucagon(GC)and Somatostatin(SST)to regulate the function of islet βcells in diabetic condition.To provide scientific basis for the prevention and treatment of diabetes.Methods:1.Determination of gallic acid,corilagin and ellagic acid in Tibetan medicine THL by HPLC.2.β and α cells were cultured in different glucose concentration medium to induce hyperglycemia model.The levels of INS,GABA,GLP-1 and c AMP in the supernatant ofβ-TC-6 cells,GC and c AMP in the supernatant of α-TC1-6 cells,and GLP-1,GC and INS in the supernatant of TC1-6+TC-6 cells were detected by ELISA after the combined culture of THL containing serum.The expression of INS,GC,PKA and CREB genes in β,α and α+β cells were detected by q RT-PCR.3.Use STZ intraperitoneal injection to replicate the diabetes model in rats.After success,the rats were divided into model,THL(low,medium,and high)dose and sitagliptin positive administration group,and they were administered intragastrically for 8 weeks.Record rat body weight and blood glucose;ELISA method to detect peripheral blood serum INS,GC,SST and other factors and pancreatic tissue c AMP level,immunohistochemistry to detect rat pancreatic tissue INS,GC,SST,and calculate HOMA-β index;Westrn Blot method was used to detect the expression of IRα,GCGR,GLP-1R,GABRB2,SSTR5,PKA,p-PKA,CREB and Akt protein in rat pancreatic tissue,and q RT-PCR method was used to detect INSR,GCGR,GLP-rat pancreatic tissue 1R,SSTR5,GABAAR,PKA,CREB,Akt and other gene expression.Results:1.The HPLC method for the determination of the quality markers of Tibetan medicine THL met the requirements.The contents of gallic acid in Phyllanthus emblica,Terminalia chebula,T.Bellirica and THL freeze-dried powder are 1.0661,1.1353,1.3273,6.2351mg/g,respectively;the contents of corilagin were 0.2926,3.496 6,0.3546,and 3.6007mg/g;the contents of ellagic acid were 0.1702,0.3180,0.1051,and 1.2313mg/g respectively.The sample recovery rate and RSD value met the requirements.2.Cells cultured in DMEM high glucose medium for 48 h could induce high glucose model.ELISA results showed that compared with high glucose group,the levels of INS and GLP-1,GABA and c AMP in β-cell supernatant of 10% drug-containing serum were increased(P<0.01)after 24 h cell culture.The GC level in supernatant of α cells was decreased(P<0.05),and the c AMP level in α cells was decreased(P<0.01).The level of INS in the supernatant of β+α cells increased,the level of GC decreased(P<0.05)and the level of GLP-1 increased(P<0.05).q RT-PCR results showed that compared with the high glucose group,the expression of INS,PKA and CREB genes in β cells were up-regulated after 24 h co-culture of 2.5% and 10% THL drug-containing serum and cells(P<0.05,P<0.01).The expressions of GC,PKA and CREB genes in α cells were down-regulated(P<0.05).In β+α cells,the expression of INS was up-regulated(P<0.01),and the expression of GC was down-regulated(P<0.01).PKA and CREB were up-regulated.3.THL can reduce blood glucose and increase body weight in diabetic rats.ELISA results showed that compared with model group,the serum levels of INS,GLP-1,GIP,GABA and SST in THL administration group were increased(P<0.01),and the level of c AMP in pancreatic tissue was increased(P<0.01).The levels of GC and 5-HT decreased(P<0.01);He staining showed that the islet area of rats in the administration group gradually increased,especially in the high-dose THL group.Immunohistochemical results showed that compared with model group,the secretion expression of INS,GC and SST in pancreatic tissue of rats in administration group was increased,while the secretion expression of GC and SST was decreased.HOMA-β index was increased in rats(P<0.01).Western blot results showed that,compared with model group,the expressions of IRα,GABRB2,SSTR5,PKA,p-PKA,Akt and other proteins in the pancreatic tissue of rats in the positive drug group and THL group were up-regulated(P<0.01),and the expression of GCGR protein had a downward trend.q RT-PCR results showed that compared with model group,In SR,GLP-1R,GLP-1R,PKA,CREB,Akt and other genes in sitagliptin group and THL high-dose group were up-regulated(P<0.01,P<0.05),and the expression of SSTR5 was down-regulated,while the expression of GCGR was down-regulated(P<0.01).There was no significant trend in the expression of GABAAR gene.Conclusion: Tibetan medicine THL can improve the function of pancreatic β-cells in diabetic conditions through islet paracrine.Its mechanism is related to the binding of islet paracrine factor and its receptor,there by enhancing the c AMP/PKA/CREB signaling pathway.