Studies On The Molecular Mechanisms Of Gegen Qinlian Decoction Regulated Dm Rats Intestinal Metabolite SCFAs Via GPR43-AMPK Pathway In The Prevent & Treatment Of DN

Diabetes Mellitus(DM)is a metabolic disease caused by complex pathogenes and characterized by chronic hyperglycemia.The progression of diabetic nephropathy(DN)in DM patients is closely associated with intestinal flora disorders.At present,pharmacological experiments have revealed that Gegenqinlian Decoction(GQD)has good anti-inflammatory and antiviral effects.GQD can also improve the composition of intestinal flora.In addition,GQD also has a long clinical history for the prevention and treatment of DN,and it also shows certain advantages in the treatment of DM.However,the molecular mechanism effect of GQD on the prevention and treatment of DM remains unclear.In this study,the expression of GPR43 mediated by intestinal short-chain fatty acids in rats with GQD were evaluated.Our results provides a new strategy and target for the prevention and treatment of DN.Objective: To study the effect of GQD on intestinal short-chain fatty acids in DM rats,and to further investigate its molecular mechanism.Methods: The intestinal short-chain fatty acids were determined by Gas Chromatographic-Mass Spectrometry(GC-MS).Hematoxylin-eosin staining(HE)was used to observe the pathological changes of colonic staining of rats.The swelling and dilatation of rat intestinal epithelial cells were observed by transmission electron microscopy.The gene expression levels of GPR43 and GPR41 in kidney and liver were detected by real-time quantitative PCR.The protein expression levels of GPR43,GPR41,AMPK,P-AMPK and β-actin were detected by Western blot.The contents of TNF-α,MCP-1,IL-6,IL-1β and D-lactic acid in serum were determined by ELISA.Molecular docking was performed to verify the combination of puerarin,baicalin,berberine and glycyrrhizic acid with GPR43.In the cell experiment,GPR43 was knocked out and the protein expression levels of GPR43 and AMPK were detected by Western blot.Real-time quantitative PCR was used to detect AMPK gene expression levels.Results: GQD increased the content of intestinal short-chain fatty acids,especially with the presence of acetic acid.GQD can reduce the content of D-lactic acid in serum,reduce the levels of TNF-α,MCP-1,IL-6 and IL-1β in serum,and improve the swelling and dilation of intestinal epithelial cells.Molecular docking showed that the main active small molecule compounds in GQD had good binding effect with GPR43.GQD can increase the expression of GPR43 protein in liver and kidney of DM rats,and GQD can also increase the expression of AMPK and P-AMPK protein in DM rats.In cell experiments,GQD increased the expression of AMPK in 293 T cells with GPR43 knockout.Conclusions: GQD regulated the content of intestinal SCFAs in DM rats.GPR43-AMPK is an important protein target in the prevention and treatment of DN by GQD.