Study On Epigenetic Regulation Of NPHS1 And NPHS2 Genes In Podocytes By LPS

Part ? Effects of LPS on Podocytes Objective:To compare the expression of podocyte-related genes NPHS1 and NPHS2 under basic transcription and inflammatory stimulation,and to explore the possible mechanism of podocyte injury.Methods:(1)Mature rat podocytes were cultured in subculture,and LPS was used as inflammatory stimulator.The experimental cells were blank control group and LPS group.LPS was used in three concentrations of 50g/ml,100g/ml and 200g/ml.After 24 hours of action on podocytes,CCK8 method was used to analyze the effects of different concentrations of LPS on the proliferation of podocytes;(2)According to the experimental results,the expression levels of podocyte related molecules NPHS1 and NPHS2 in 100?g / ml LPS group and blank group were detected by real-time fluorescence quantitative PCR and Western blot,and the expression levels of apoptotic proteins caspase3/9,Bax and bcl-2 were measured by Western blot;(3)Flow cytometry was used to observe the effects of 50g/ml,100g/ml and200g/ml LPS and its receptor inhibitor TAK242 on podocyte apoptosis after 24 hours.Results:(1)The cell proliferation in the LPS group was significantly lower than that in the control group(P < 0.05),and showed a dose effect: the 100g/ml LPS group decreased more significantly than the 50g/ml LPS group(P < 0.01).The effect of 200g/ml LPS group was slightly stronger than that of 100g/ml LPS group,but there was no statistical difference between the two groups(P > 0.05);The expression of NPHS1,NPHS2 mRNA and protein in rat podocytes in LPS group was lower than that in control group(P <0.05).(2)Compared with the control group,the apoptosis of cells in the LPS group showed a significant upward trend,and there was no significant change in the apoptosis rate between the 100g/ml and 200g/ml LPS treatment groups.The expression of anti-apoptotic protein bcl-2 was significantly down-regulated and lower than that of the control group(P < 0.01),the expression of pro-apoptotic protein Bax was significantly up-regulated and higher than that of the control group(P < 0.01),caspase-9 and caspase-3 proteins were activated,and the expression levels were significantly up-regulated compared with the control group(P < 0.01).(3)The apoptosis rate of TLR4 signal transduction inhibitor TAK242 group was significantly lower than that of LPS group(P < 0.01),and the degree of apoptosis was basically the same as that of the control group.Conclusion:(1)LPS can induce inflammatory injury of podocytes,which decreases cell proliferation and increases cell apoptosis.This effect is related to the activation of caspase3/9 pathway and the down-regulation of NPHS1 and NPHS2 expression.(2)TLR4 receptor blockers can antagonize the apoptosis induced by LPS,suggesting that TLR4 signaling pathway may be the potential mechanism of LPS-induced podocyte apoptosis in rats,and its inhibitors may effectively improve LPS-induced podocyte damage.Part ? Effect of histone methyltransferase inhibitor G9 a on podocytes induced by LPS protective effect of injury Objective:By analyzing the changes of methylation level of NPHS1 and NPHS2 gene promoter region H3K9 in podocytes under inflammatory stimulation,the changes of histone epigenetic information in podocytes during pathological injury were revealed.Methods:(1)The cultured rat podocytes were divided into control group,LPS group and LPS+G9a inhibitor BIX01294 group.The expression level of podocytes related molecules was detected by real-time fluorescence quantitative PCR and Western Blot technology;The apoptosis rate of podocytes was observed by flow cytometry.(2)The methylation level of H3K9 in the promoter region of NPHS1 and NPHS2 genes was detected by immunoprecipitation.Firstly,the cells of each group were lysed with lysate and then treated with ultrasound.After incubation with H3K9me2,H3K9me3,RNA Polymerase ? ChIP grade antibody and control IgG,the antibody was precipitated with Protein G-magnetic beads,and finally the upstream regions of NPHS1,NPHS2 and GAPDH genes were detected by QPCR after uncrosslinking.Results:(1)As a G9 a histone methyltransferase inhibitor,BIX01294 significantly restored the down-regulation of NPHS1 and NPHS2 in podocytes induced by LPS,and resisted LPS-induced podocyte apoptosis(P < 0.01).(2)The results of co-immunoprecipitation showed that the expression of H3K9me2 and me3 in the promoter region of NPHS1 and NPHS2 increased after LPS induced podocytes,mainly the dimethylation level increased significantly(P < 0.01),and then the damaged podocytes were treated with G9 a methylase inhibitor BIX01294,and it was found that the dimethylation level of H3K9 decreased significantly compared with LPS group(P < 0.01).Conclusion:(1)Histone methylation mediated the inhibition of LPS on the expression of NPHS1,NPHS2 and other genes.(2)G9a can play a role by binding H3K9me2 to the promoter regions of NPHS1 and NPHS2 of podocytes.BIX01294,an inhibitor of G9 a methylase,can resist LPS-induced H3K9 dimethylation levels in the promoter regions of NPHS1 and NPHS2 genes,thus playing a protective role on podocytes.(3)In addition to initiating caspase pathway,histone methylation also participates in podocyte injury mechanism in LPS-induced podocyte apoptosis injury,and inhibition of histone methylation can play a protective role on podocytes.