Effects And Mechanisms Of CCL24 In Early And Mid-stage Diabetic Nephropathy

Objective: Screening of inflammatory factor CCL24 with abnormal expression in early and mid-stage diabetic nephropathy by human inflammation antibody array.Assessing effects and mechanisms of CCL24 in early and mid-stage diabetic nephropathy using gene knockdown technique in vivo and vitro.Methods:(1)Collecting serum of diabetes mellitus(DM)and mid-stage diabetic nephropathy(DN)patients and screening CCL24 with human inflammation antibody array.(2)In vivo experiments,establishing models of DN through streptozotocin(STZ)injection combined with high-fat diet.Recording physiological parameters of mouse;Obsering morphological changes of kidney in DN mice by HE?PAS?PASM ?Masson and immunofluorescence.Recording a variety of physiological parameters in mice.Using western blot to detect the changes of key proteins and CCL24 in kidney of DN mice.(3)In vitro experiments,establishing cell models on mouse glomerular primary cells by glucose stimulating.Using western blot to detect the changes of key proteins and CCL24 in podocytes.Using crispr/cas9 to knockdown CCL24 gene and detect the changes of inflammation by western blot.Results:(1)Antibody array showed the expression of CCL24 levels is higher in serum of early and mid-stage DN patiens.(2)DN mice model was constructed successfully.Morphological results showed mesangial matrix expansion,glomerular basement membrane thickening,collagen deposition increased and fibrosis levels increased.(3)In vivo levels,western blot showed the expression of CCL24 is higher in kidney of DN mice(P<0.05).The expression of CCL24 is lower in kidney than in liver?spleen?lung?intestines in healthy mice.Expression of podocin and GLUT4 in DN mice were lower than healthy mice(P<0.05)and expression of fibronectin and IL1? in DN mice were lower than healthy mice(P<0.05).In vitro levels,western blot showed the expression of CCL24 was higher in glucose-induced podocytes(P<0.05);the expression of fibronectin,Gsk3? and p IRS1Ser307 was increased(P<0.05)and inflammatory signaling pathways NF?B? MAPK and JAK/STAT were activated.After knockdowning CCL24 gene by crispr/cas9 in glomerular mesangial cells,the expression of fibronectin was reduced(P<0.05)and IL6?TNF? and IL 1? was increased(P<0.05).Inflammatory signaling pathway NF?B was activated.Conclusion:(1)The CCL24 levels is increased in early and mid-stage DN.(2)CCL24 participates in the occurrence and development of DN by activating inflammatory pathway NF?B.(3)Decreasing the expression of CCL24 can reduce the level of kidney fibrosis in DN.