To Investigate The Effect And Mechanism Of SOCS3 On The Proliferation Of Human Mesangial Cells Stimulated By IgA1 In Patients With IgA Nephropathy

Objective:To investigate the effect of overpression SOCS3 on the proliferation of human mesangial cells stimulated by IgA1 from serum of patients with IgAN, and explore the possible mechanism. Method:1) Serum IgA1 from IgAN was isolated with jacalin affinity chromatography and Sephacryl S-200 HR gel filtration method then polymerized.2) Human glomerular mesangial cell was transfected by overexpression SOCS3 adenovirus then stimulated by aIgA1.3) Determine the time and concentration of aIgA1 and the transfection conditions, set up 4 experimental groups:a) Control group: HMC only with 10%FBS and 1% penicillin-streptomycin of 1640 culture medium for normal culture and timing change.b) IgA1 group: normal cultured HMC was stimulatedby aIgA1.c) IgA1+ Adv-EGFP group: normal cultured HMC was transfected with Adv-EGFP for 48 hours then stimulated by IgA1.d) IgA1+ Adv-SOCS3-EGFP group: normal cultured HMC was transfected with Adv-SOCS3-EGFP for 48 hours then stimulated by aIgA1.4) MTT to detected cell proliferation, western blot and Real-time PCR to observe the expression of SOCS3, TLR4, TGF-β1protein and mRNA of each group.5) The experimental data was expressed by sx ± and T-test was performed by statistical software of IBM SPSS Statistics 20. The results in p<0.05 showed that there was significant difference. Results:1) HMC was stimulated by aIgA1 for 12 h, 24 h and 48 h, cell proliferation was promoted significantly after stimulated for 24 h or 48 h, and the difference between 0.25, 0.5, 1.0mg/ml group and normal group was statistically significant(p<0.05), however 1.5mg/ml group and normal group was not statistically significant. Comparing IgA1 group with control group, the protein and mRNA expression of SOCS3, TLR4, TGF-β1 were significantly increased in IgA1 group(p<0.05).2) HMC was transfected by the Adv-EGFP and Adv-SOCS3-EGFP overexpression vector for 48 h, and was intervented by aIgA1. Use MTT to detecte cell proliferation and western blot and Real-time PCR to observe the expression of protein and mRNA of each group. At 24 h and 48 h, the difference between IgA1+ Adv-SOCS3-EGFP group and IgA1+ Adv-EGFP group was statistically significant(p<0.05). SOCS3 could inhibit the proliferation of HMC stimulated by aIgA1. Comparing IgA1+ Adv-EGFP group with IgA1+ Adv- SOCS3- EGFP group, the protein and mRNA expression of SOCS3, TLR4, TGF-β1 were significantly decreased in IgA1+ Adv-SOCS3-EGFP group(p<0.05). Conclusions:1) aIgA1 from serum in the patients with IgAN can promote the proliferation of HMC, and it has time-dose relationship between proliferation rate and IgA1.2) The expression of SOCS3, TLR4 and TGF-β1 was lower in normal HMC, and IgA1 from the patients can increase their expression in HMC.3) The expression of SOCS3 in HMC was increased by transfected of adenovirus. The overexpression of SOCS3 can inhibit the proliferation of HMC, and reduce the expression of TLR4 and TGF-β1. Therefore SOCS3 may play a protective role in kidney injury.