The Study On GLP-1R Agonistic Effect Of Cinchonine And Its Therapeutic Effect On Nonalcoholic Steatohepatitis

Objective:Type 2 diabetes mellitus(T2DM)and non-alcoholic fatty liver disease(NASH)are two common metabolic diseases that are strongly associated with each other.The glucagon-like peptide-1 receptor(GLP-1R)is a seven-membrane transmembrane receptor that is mainly found in tissues such as pancreatic β-cells,the central nervous system and the cardiovascular system.As a target receptor for GLP-1,GLP-1R agonists have emerged as effective agents for the treatment of type 2 diabetes and are also considered to have some potential for the treatment of NAFLD.Small molecule GLP-1R agonists are ease of use in oral preparations and improved patient compliance,however,no such drug is currently available in the market.In this study,we analyzed the transcriptome of geniposide,a recognized small-molecule GLP-1R agonist,and combined with the CMap database,a small molecule cinchonine with GLP-1R agonist-like effects was identified,its insulinotropic and hypoglycaemic effects were investigated in relation to GLP-1R,and explored in relation to its treatment of NASH,providing a viable idea for the repositioning of small molecule drugs.Methods:(1)To analyze the transcriptome data in the small intestine of db/db diabetes mice treated with geniposide,and determine the differentially expressed genes(DEGs).(2)The DEGs were entered into the CMap database to obtain small molecules with GLP-1R agonist-like effects.(3)Molecular docking of cinchonine to GLP-1R protein using SYBYL software to identify its possible binding sites.(4)Insulin secretion assay to observe the effect of cinchonine on insulin secretion in rat pancreatic islet tissue under different sugar concentration conditions.(5)The Oral Glucose Tolerance Test(OGTT)was used to observe whether oral cinchonine lowers blood glucose and its hypoglycemic characteristics.(6)In rat islet tissue,the effect of GLP-1R blocker Exendin(9-39)on the insulin secretion of cinchonine was observed.(7)GLP-1R knockout mice and humanized GLP-1R expressing mice were used to observe the hypoglycemic effect of oral cinchonine.(8)Leptin gene knockout mice(ob/ob mice)were given GAN diet for NASH model construction,and different doses of cinchonine were given for preventive administration.(9)The body weight and food intake of ob/ob mice were recorded daily to observe the effects of different doses of cinchonine administration.(10)Serum biochemical assays were performed to analyse whether liver injury in ob/ob-GAN-NASH mice was improved by different doses of cinchonine administration.(11)H&E staining of liver sections and quantification of whether the administration of different doses of cinchonine improved steatosis,fat ballooning,inflammatory infiltration and non-alcoholic fatty liver score(NAS)in ob/ob-GAN-NASH mice.(12)Liver sections were stained with oil red O and quantified to analyze whether different doses of cinchonine administration improved liver fat accumulation in ob/ob-GAN-NASH mice.(13)Sirius red staining of liver sections and quantification of whether liver fibrosis in ob/ob-GAN-NASH mice was improved by different doses of cinchonine administration.Results:(1)A total of 211 DEGs were screened from geniposide transcriptomics data,then entered into CMap database to obtain the compound cinchonine with the highest relevance score.(2)Molecular docking using SYBYL software showed that cinchonine molecules can form hydrogen bonds with GLP-1R protein glutamate(GLU)residues and glutamine(GLN)residues.(3)The results of the insulin secretion assay showed that cinchonine sugar concentration-dependently promoted insulin secretion in rat pancreatic islet tissue.(4)The OGTT results indicate that oral cinchonine reduces blood glucose in mice in a dose-dependent manner.(5)The promoted insulin secretion effect of cinchonine was lost after the addition of the GLP-1R blocker Exendin(9-39)to the insulin secretion assay.(6)After GLP-1R knockout mice were used in OGTT,the hypoglycemic effect of oral cinchonine disappeared.(7)The OGTT results using mice expressing humanized GLP-1R indicate that oral cinchonine still had hypoglycemic effect.(8)The observations of daily body weight and food intake in mice showed that cinchonine dose-dependently inhibits body weight gain and food intake in ob/ob-GAN-NASH mice.(9)The results of serum biochemical assays indicated that cinchonine improved liver injury in ob/ob-GAN-NASH mice.(10)H&E staining of liver tissue sections and its quantification showed that cinchonine improved hepatic steatosis,fat ballooning,inflammatory infiltration and non-alcoholic fatty liver score(NAS)in ob/ob-GAN-NASH mice.(11)Oil Red O staining of liver tissue sections and its quantification showed that cinchonine improved hepatic fat accumulation in ob/ob-GAN-NASH mice.(12)Sirius red staining of liver tissue sections and its quantification showed that cinchonine administration improved liver fibrosis in ob/ob-GAN-NASH mice.Conclusion:Cinchonine agonizes GLP-1R to exert promote insulin secretion and hypoglycaemic effect.In ob/ob-GAN-NASH,oral administration of cinchonine significantly inhibit weight gain,inhibit food intake,and improve liver injury,fat accumulation,and thus liver fibrosis.The drug repositioning of cinchonine provides a feasible approach and idea for the development of small molecule GLP-1R agonists.