Effects Of Flavonoids Of Coreopsis Tinctoria Nutt.and Main Components On High Glucose Palmitate-induced Energy Metabolism Disorders In HBZY-1 Cells

Objective:High glucose palmitate induced rat Mesangium cells（HBZY-1）to establish an in vitro diabetic nephropathy metabolic disorder cell model,to investigate the protective effect and mechanism of（Flavonoids of Coreopsis tinctoria Nutt,FC）and Plains Coreopsis on energy metabolism disorder in model cells.Methods:1.The rat glomerular mesangial cells（HBZY-1）were induced by high glucose and palmitic acid to establish DN model in vitro.The model group was treated with glucose 100 mmol·L^-1+palmitic acid 250μmol·L^-1（HG+PA）;2.The HBZY-1 cells were divided into normal group（NC）,high glucose and palmitic acid model group（HG+PA）and FC+HG+PA（1,5,10,20,40,80,160,320μg/mL）.MTS was used to detect the effect of different doses of FC on the proliferation of model cells,and the morphological changes of each group were observed under inverted microscope;（2）cell scratch test was used to observe the effect of FC on ECM secretion of model cells;（3）western The expression of 5’adenosine monophosphate activated protein kinaseα（p-AMPKα）,acetylcoa carboxylase 1（ACC1）,sterol regulatory element binding protein 1（SREBP-1）,oxidative stress factors:nitric oxide synthetase（eNOS）,nicotinamide adenine dinucleotide phosphate oxidase 4（NOX4）in the cells of each group were detected by blotting.3.Marein was divided into three groups:normal control group（NG）,model group（HG+PA）and malidin groups(25,50,100,200μmol·L^-1).The effect of malidin on the proliferation of HBZY-1 cells was detected by MTS method,and the optimal concentration was selected;（2）Western blot method was used to detect the cells in each group;1)energy metabolism factors p-AMPKα,AMPKγ1,ACC1,SREBP-1,SIRT3;2)oxidative stress NOX4,inflammatory factors MCP-1,ICAM-1,fibrosis factors FN,α-SMA,Collagen VI;3)TGF-β1/Smads signaling pathway;4)expression level of RhoA/ROCK signaling pathway protein under oxidative stress.Results:1.FC can inhibit the proliferation of HBZY-1 cells induced by high glucose and high lipid,increase the intercellular space and significantly reduce the density;FC can promote the expression of p-AMPKα,p-ACC1,eNOS,and inhibit the expression of SREBP-1,NOX4 and ACC1 in model cells;scratch test results show that FC can significantly inhibit the secretion of ECM in model cells,and significantly reduce the expression ofα-smooth muscle actin（α-SMA）.2.Marein inhibited the proliferation of HBZY-1 cells induced by high glucose palmitic acid;AMPK signaling pathway was activated,down regulated the expression of NOX4,MCP-1,ICAM-1,α-SMA,FN,CollagenⅥ,energy metabolism pathway factors SREBP1,SIRT3,TGF-β1/Smads（Smad2,p-Smad2,Smad3,p-smad3,Smad4）,RhoA,p-MYPT1,and up regulated the expression of p-AMPKα,AMPKγ1,p-ACC1.Conclusion:1.Flavonoids of Coreopsis tinctoria Nutt may inhibit renal cell proliferation,oxidative stress and improve renal fibrotic lesions by regulating the energy metabolism pathway AMPK/SREBP-1/ACC1.2.Marein may inhibit oxidative stress,inflammatory response,lipid metabolism,and fibrotic protein expression in HBZY-1 cells by activating the AMPK signaling pathway,and delay DN kidney injury.