Mechanism Of SPLA₂-IB And PLA₂R In Podocyte Injury

Objective: Phospholipase A₂ receptor（PLA₂R）is a surface antigen of primary membranous nephropathy,which is also a receptor for secretory phospholipase A₂（sPLA₂）and one of the proteins involved in cell senescence.At present,little is known about the physiological function of PLA₂ R,and most of studies are related to anti-PLA₂ R antibody.Therefore,this study focuses on the mechanism of sPLA₂-IB and PLA₂ R in podocyte injury.Methods: 1.The human podocytes are placed in 33 ℃ and 5% CO₂ incubator for proliferation culture.Cell morphology and growth were observed under inverted microscope.When the cells were fusion to 50%-70% to replace 37 ℃ medium,and then placed in 37℃ and 5% CO₂ incubator were cultured 7-10 days.Cells were synchronized for follow-up experiment.2.respectively 10-9M,10^-7M and 10^-5M sPLA₂-IB stimulate the differentiation and maturation of podocytes in 2 hours;the wound healing assay to determine cell migration rate;cell nucleus morphology was observed by Hoechst 33342 staining;Western blot was used to analyze the protein expression of p-cPLA_2α,cPLA_2α,p-p38,p38 and p53;HPLC was used to determination of arachidonic acid（AA）in the cell culture supernatant.3.According to PLA₂ R mRNA in vitro chemical synthesis of siRNA,using the RNAi transient transfection of podocytes,checking the PLA₂ R gene silencing efficiency by RT-PCR,with 10^-5M sPLA₂-IB to stimulate the differentiation and maturation of podocytes in 2 hours,detecting the protein expression of p-cPLA_2α,cPLA_2α,p-p38,p38 and p53 by western blot after gene silencing,cell nucleus morphology was observed Hoechst 33342 staining;HPLC method was used to quantitated the expression of AA in cell culture supernatant.Results:（1）Compared with the control group,the migration ability of podocytes decreased when the concentration of sPLA₂-IB(10-9M-10^-5M)was increased,and the 10^-5M sPLA₂-IB group can significantly inhibit the ability of cell migration.Hoechst staining showed nuclei were densely stained,or a chunky hyperchromatic,suggesting that the number of apoptotic cells increased gradually,which 10^-5M sPLA₂-IB group could significantly promote cell apoptosis.The expression of p-cPLA_2α,p-p38 and p53 protein in a dose dependent increase,and 10^-5M sPLA₂-IB group could significantly increase the expression of p-cPLA_2α,p-p38 and p53.The expression of AA in cell supernatant increased with the increase of sPLA₂-IB concentration,and the expression level of AA increased significantly under 10^-5M sPLA₂-IB induction.（2）The expression level of PLA₂ R mRNA decreased significantly after transfection with 150 ng PLA₂R siRNA for 48 hours.When the expression of PLA₂ R decreased,the cells were stimulated by 10^-5M sPLA₂-IB for 2 hours,compared with the control group,the apoptosis rate of podocyte decreased,as well as the expression level of p-cPLA_2α,p-p38 and p53 protein,the expression of AA also decreased.Conclusion: The results of this study show the sPLA₂-IB acting as a PLA₂ R ligand to activate c PLA_2α,which in turn produce enzymatic activity,hydrolysis of the endoplasmic reticulum or nuclear membrane to provide AA,and the latter is associated with podocyte apoptosis.