Retrospective Study Of Jiedu Tongluo Tiaogan Formula In Treating Type2 Diabetes Mellitus And Its Mechanism Of Regulating Endoplasmic Reticulum Stress-Autophagy

In this paper,the clinical efficacy of Jie du Tong luo tiao gan formula（JTTF）improving patients with type 2 diabetes mellitus（T2DM）was evaluated by retrospective study,component analysis and vitro cell experiment.Material basis and biological connotation of the drug efficacy were explored to provide data support,scientific basis for the clinical application and mechanism research of JTTF in the treatment of T2DM.Objective:1 Through retrospective research methods,observed T2DM patients who met the inclusion criteria,evaluated the improvement of clinical symptoms and glucose and lipid metabolism indexes by JTTF,and evaluated its clinical efficacy,so as to provide objective basis for clinical application,in order to provide reference for the ideas and methods of JTTF in the treatment of T2DM.2.Established the quality control method of JTTF,clarify the main composition and stability of its pharmacodynamic components,and provided a reliable experimental basis for the follow-up study of biological mechanism.3.The cell model was used to study the protective effect of JTTF on rat islet tumor cells（INS-1 cells）,as well as the effects on endoplasmic reticulum stress and excessive autophagy,so as to systematically clarify the molecular mechanism of JTTF in protecting islet function.It provided theoretical and experimental basis for the molecular mechanism of JTTF in treating T2DM.Methods:1.Through retrospective analysis of 283 patients who met the inclusion criteria,the basic information of patients at the time of entry and exit the group,as well as the fasting blood glucose,postprandial blood glucose,glycosylated hemoglobin,BMI,lipid metabolism and main symptoms were recorded respectively to comprehensive analysis its effectivenes,so as to explore the efficacy of JTTF in the treatment of T2DM.2.UPLC-Q-TOF-MS method was used to analyze the essential composition and stability of JTTF;then,JTTF traditional Chinese medicine lyophilized powder preparation was used to interfere with high glucose-induced INS-1 cells,and MTT,GSIS,Western Blot,q-PCR and other methods were used to analyze the protective effect of this prescription on cell glycotoxicity;Finally,the effects of the prescription on endoplasmic reticulum stress and excessive autophagy of INS-1 cells induced by high glucose were studied by Western blot,Q-PCR,flow cytometry and fluorescence chemistry,so as to systematically clarify the molecular mechanism of the prescription to protect islet function.Results:1 Through the observation of glucose and lipid metabolism,BMI and clinical symptoms of patients who took JTTF after 3 months of treatment,the data analysis shows that JTTF can improve FBG,2h PBG、Hb A1c of patients,and also reduce body weight.At the same time,it can better improve the clinical symptoms of patients,especially the main symptoms of toxic damage to Liver Collaterals and stagnation of heat in liver and stomach.It provide clinical evidence for the promotion of JTTF.At the same time,it also enriches the clinical experience of traditional Chinese medicine in the treatment of T2DM.2.The chemical components of JTTF were qualitatively analyzed by UPLC-Q-TOF-MS method,and a total of 28 chemical components were inferred,including 6 flavonoids,4phenylpropanoids,4 terpenes,3 saponins,and alkalis.3 kinds of alkaloids,2 kinds of anthraquinones,2 kinds of organic acids,2 kinds of amino acids,and 1 kind of aldehydes.At the same time,28 chemical components in JTTF were qualitatively identified.The results showed that flavonoids were mainly from Astragalus membranaceus and hazelnut flower,saponins were mainly from Salvia miltiorrhiza and Astragalus membranaceus,terpenoids were mainly from Salvia miltiorrhiza,phenylpropanoids were mainly from Astragalus membranaceus and bupleurum,organic acids were mainly from hazelnut flower,amino acids were mainly from Salvia miltiorrhiza and Astragalus membranaceus,and aldehydes were mainly from Bupleurum.In this study,UPLC-Q-TOF-MS technology was used to analyze the qualitative analysis of chemical components in JTTF.This method is efficient and convenient,and provides a scientific basis for the improvement of JTTF’s quality control method and drug stability research.3.When INS-1 cells were incubated with 25 to 500μg/m L of JTTF for 48 hours,the viability of INS-1 cells was not affected,indicating that JTTF had no toxic effect;through the screening of high glucose concentrations,the optimal modeling concentration was finally determined as 20m M;through GSIS experiments,it was concluded that JTTF can improveβ-cell insulin secretion and protect islet function;through the expression of insulin genes（INS1,INS2）and the genes and proteins of insulin gene regulators PDX1 and Maf A,the results showed that high glucose can inhibit the expression of its gene and protein,while JTTF can increase the expression of its m RNA and protein（P<0.01）,which indicates that JTTF has a protective effect on insulin synthesis and secretion of isletβcells induced by high glucose.4.Under hyperglycemic conditions,related persistent endoplasmic reticulum stress（ERS）can lead to Ca²⁺increased inβintracellular.The high glucose-induced Ca²⁺transport dysfunction in the endoplasmic reticulum lead to sustained ERS inβcells,it further leads to the dysfunction of isletβcells,and JTTF could inhibit the release of intracellular Ca²⁺（P<0.01）and maintain intracellular homeostasis;At the same time,in a high-glucose environment,Ca²⁺transport dysfunction,a large amount of Ca²⁺efflux,Camkkβwas activation led to the damage of isletβcells,which could be inhibited by JTTF（P<0.05）.Finally,stimulated by high sugar,isletsβCells produce ERS and made PERK,IRE1α,ATF-6 and GRP78 separated and triggered the long-term strong expression of GRP78,PERK,IRE1αand ATF-6 proteins which can lead to pancreaticβ-cell dysfunction.JTTF intervention can significantly reduce the protein expressions of GRP78,p-IRE1α,ATF6,and p-PERK（P<0.01）and then decreased the isletβCell dysfunction.5.Under the condition of persistent hyperglycemia,persistent ERS in pancreatic beta cells is induced,leading to excessive autophagy.By monitoring the levels of several autophagy-related proteins（m TOR,ULK1,Beclin-1,LC3,p62）inβcells using Western blot analysis,the results showed that JTTF treatment was able to inhibit excessive autophagy and protectβcells from high glucose induction damage（P<0.01）;then,the level of OA-stained autophagosomes inβcells was assessed using fluorescence chemistry.The results of autophagosome AO staining showed that compared with the control group,the autophagic vacuoles observed in the cells of the model group exposed to high glucose for 48hours were significantly increased,and the red fluorescence was enhanced.After JTTF treatment,with the increase of JTTF concentration,the autophagic vacuoles decreased in a dose-dependent manner,and the red fluorescence decreased（P<0.05）.At the same time,the use of AICAR,an activator of autophagy,also verified the effect of this prescription on inhibiting excessive autophagy.Conclusion:1.The retrospective analysis of this study clarified that JTTF is a clinically effective prescription for the treatment of T2DM,and it is worthy of popularization and application.2.A total of 28 medicinal components of JTTF were identified,which further clarified the medicinal components of the formula and the stability of medicinal action.3.In vitro experiments,the protective effect of the prescription on the glucotoxicity of pancreatic beta cells was verified.4.The protective effect of JTTF on the glucotoxicity of isletβcells may be through inhibiting ERS and excessive autophagy,maintaining cellular homeostasis.