The Role And Mechanism Of Kruppel Like Factor 15 In Rat Peritoneal Mesothelial Cell Senescence

Objective To investigate the role of Kruppel like factor 15 in the aging of rat peritoneal mesothelial cells and the role and the possible regulatory mechanism of KLF15 in TGF-?1 induced peritoneal mesothelial cell aging model of rats and the possible regulatory mechanism,we used liposomes to transfect the rat primary peritoneal mesothelial cells to construct a model of the overexpression of KLF15 in the cells,and given TGF-?1 treatment after overexpression.After intervention with TGF-?1,we detected the mRNA and protein expression of each signal pathway factor and the effect on cell senescence.Methods The mesentery and spleen gastric ligament of SD male rat were isolated,and primary peritoneal mesothelial cells of rats were cultured.Peritoneal mesothelial cells of the third generation were used as an experimental object.The eukaryotic overexpression plasmid pcDNA3.1-KLF15 was constructed.The plasmid pcDNA3.1(empty plasmid)and pcDNA3.1-KLF15(overexpressed plasmid)were transfected into the rat peritoneal mesothelial cells respectively with liposomes.The expression of KLF15 was verified by the expression of KLF15 mRNA and protein by Western Blot and Real-Time PCR.Given TGF-?1 intervention,cells were divided to 6 groups : control group(group control),(group pcDNA3.1)empty plasmid transfection group(group pcDNA3.1),KLF15 overexpression group(group pcDNA3.1-KLF15),TGF-?1 treatment group(control+TGF-?1)and TGF-?1 treatment of empty plasmid transfection group(pcDNA3.1 group + TGF-?1 group).TGF-?1 treatment KLF15 The expression group(group pcDNA3.1-KLF15+ TGF-?1).The concentration of TGF-?1 was 2ng/mL,and 24 h was treated.Western Blot was used to detect the expression of p-Smad2/3 and p21 protein.Real-Time PCR was used to detect Smad2,Smad3 and p21 mRNA expression.Cell ?-galactosidase staining kit was used to detect cellular senescence.Results The primary rat peritoneal mesothelial cells were identified by flow cytometry.The purity of peritoneal mesothelial cells of primary rat was over 90%,which could be used in experiments.The transformed Escherichia coli strains were sequenced and had sequence alignment with DNAman software.The synthesized plasmids were the correct plasmid containing KLF15 gene.After transfection of overexpression plasmid 36 h,the expression of KLF15 protein and mRNA increased significantly in pcDNA3.1-KLF15 group compared with that of control group(P<0.05),but there was no significant difference in the expression of KLF15 protein and mRNA in the empty plasmid transfected group(P>0.05),suggesting that the primary rat peritoneal mesothelial cells were successfully overexpressed in KLF15.The expression of mRNA and protein of Smad2,Smad3 and p21 in pcDNA3.1-KLF15 group was significantly lower than that in control group(P<0.05).After TGF-?1 treatment,the expression of mRNA and protein in Smad2,Smad3 and p21 was significantly higher than that of group control(P<0.05),pcDNA3.1-KLF15+ TGF-?1 group was significantly higher than that of pcDNA3.1-KLF15 group(P<0.05),and pcDNA3.1-KLF15+TGF-?1 group was significantly lower than that of control+TGF-?1 group.There was no significant difference between control group and pcDNA3.1 group(P>0.05).There was no significant difference between control+TGF-?1 group and pcDNA3.1+TGF-?1 group(P>0.05).The rate of ?-galactosidase positive cells in group pcDNA3.1-KLF15 was significantly lower than control group(P<0.05).After TGF-?1 treatment,the rate of cell senescence ?-galactosidase positive cells was significantly higher in control+TGF-?1 group than in control group(P<0.01),and pcDNA3.1-KLF15+TGF-?1 group was significantly higher than pcDNA3.1-KLF15 group(P<0.05),pcDNA3.1-KLF15+TGF-?1 group was significantly lower than control+ TGF-?1 group.There was no significant difference between control group and pcDNA3.1 group(P>0.05).There was no significant difference between control+TGF-?1 group and pcDNA3.1+TGF-?1 group(P>0.05).Conclusions After the treatment of TGF-?1,the protein expression of p-Smad2/3 and p21 increased,and the expression of Smad2 and Smad3 increased,the rate of senescent cells increased,suggesting that TGF-?1 participated in the aging of peritoneal mesothelial cells by regulating Smad2 and Smad3.After overexpression of KLF15 decreased the expression of p21,KLF15 can delay the senescence of rat peritoneal mesothelial cells,decrease the expression of Smad3 Smad2,increase the rate of senescent cells,suggesting that KLF15 may be through regulating TGF-?1/Smad signaling pathways involved in senescence of peritoneal mesothelial cells,contribute to the clinical development of drugs for prevention and treatment of peritoneal aging.