To Explore The Molecular Mechanism Of Inhibition Of Renal Tubular Epithelial Cell Proliferation By Gansongyin Regulation Of MiR-21-5p In Adipocyte Exosomes Based On The "Yibing Tonifying Kidney Method"

Objective1.The mechanism of DN was verified by the co-culture system of 3T3-L1 and TCMK-1cells.2.To verify the effect of GSY on DN by interfering miRNA-21-5p in exosomes secreted by adipocytes.3.The mechanism of GSY on the expression of miRNA-21-5p secreted by adipocytes and lipid changes.Methods(1)TCMK-1 cells were divided into blank group(NC),glucose model group(MOD)and Gan Song Yin low-medium-high dose group.CCK-8 and flow meter were used to detect the cell activity,cycle and apoptosis level of TCMK-1 cells.Western blot and q RT-PCR were used to detect the protein content and gene expression.(2)Induce 3T3-L1 preadipocytes;The stability of IR model was tested by establishing IR model.The co-culture system of3T3-L3 adipocytes and TCMK-1 cells was established,the IR model was established,the glucose consumption was calculated,and the stability of the IR model was tested.;Adipocytes and co-culture system,blank group(NC),IR model group(MOD),Gan Song Yin group(GSY-H),oil red O to detect the change of lipid droplets,GOD-POD method to detect glucose consumption and triglyceride content.Western blot and q RT-PCR were used to detect the protein content and gene expression.(3)Exo and TEX were extracted from the supernatant of the co-culture system by differential centrifugal method to observe the morphology of Exo,and the particle size and concentration of Exo were detected by NTA.Western blot and q RT-PCR were used to detect the content of related proteins and gene expression.The co-culture system was divided into blank group(NC),IR model group(MOD)and Gansong Yin low-medium-high dose group.The activity,cycle and apoptosis levels of TCMK-1 cells were detected by CCK-8 and cell cycle and flow meter.The contents of related proteins and gene expression were detected by Western blot and q RT-PCR.Results(1)GSY interferes with TCMK-1 cells.Compared with MOD,GSY reduces the viability of TCMK-1 cells(P<0.01),apoptosis showed dose-dependent tolerance reduction(P<0.01);Cell cycle arrest occurred in G0/G1 phase and S phase.Western blot results showed that GSY could up-regulate the protein expressions of SMAD7,p-SMAD2,p-SMAD3 and p-SMAD7,and down-regulate the protein expressions of TGF-β1,SMAD2 and SMAD3 in IR state(P<0.05 or P<0.01).q RT-PCR results: GSY up-regulated the expression of TGF-β1,SMAD2,SMAD3 and SMAD7 genes in IR(P<0.05 or P<0.01);(2)Induction results: the "ring" shaped lipid drops were dyed red;The IR model of adipocytes at 48 h was stable,and the IR model of co-culture system at 48 h was stable(P<0.01);Results of adipocyte and co-culture system: compared with MOD group,the decomposition of lipid droplet and glucose consumption increased after GSY intervention(P<0.01);TG content of adipocytes increased,TG content of co-culture system decreased(P<0.01).Western blot results:Compared with MOD group,GSY could up-regulate the expression of PPARγ and down-regulate the expression of GLUT4 and FABP4(P<0.05 or P<0.01);(3)Exo appeared as spherical vesicles under TEM.The particle size of Exo was 130.2nm and the concentration was 4.8E+12/ml.The expressions of CD9,CD63 and TSG101 were detected by Western blot.The expression of PPARγ,GLUT4 and FABP4 genes was detected by q RT-PCR.CCK8 results: Compared with MOD,GSY decreased the activity of TCMK-1 cells(P<0.01),apoptosis showed a dose-dependent tolerance reduction(P<0.01);Cell cycle arrest occurs in the G0/G1 phase.Western blot results showed that GSY up-regulated the expressions of SMAD3,SMAD7 and p-SMAD2 proteins in IR state,and down-regulated the expressions of TGF-β1、SMAD2、p-SMAD3 and p-SMAD7 proteins(P<0.05 or P<0.01).The results of q RT-PCR showed that GSY up-regulated the expression of SMAD2 gene and down-regulated the expression of TGF-β1,SMAD3 and SMAD7 genes(P<0.05 or P<0.01);Compared with the MOD group,the expression of miRNA-21-5p in TCMK-1 cells decreased after GSY intervention,while the expression of miRNA-21-5p in Exo and TCMK-1 cells increased(P<0.05 or P<0.01).Conclusion1.GSY has a protective effect on renal tubular epithelial cells in IR mice.2.GSY improves glycolipid metabolism by regulating the expression levels of PPARγ,GLUT4 and FABP4 proteins and the expression of PPARγ,GLUT4 and FABP4 genes.3.GSY protects renal tubular epithelial cells by regulating TFG-β 1/SMAD signaling pathway through miR-21-5p secreted by adipocytes.