| Background: Lupus nephritis(LN)is an important prognostic factor in patients with systemic lupus erythematosus(SLE).Renal podocyte injury is the key link of glomerular injury.The abnormal structure and function of podocytes play an important role in the pathogenesis of lupus nephritis.Interferon(IFN)pathway is an important pathogenic pathway of SLE.IFIT1(interferon-inducible gene)is the most abundant gene in the type I interferon-inducible gene family.It has been found that the expression of IFIT1 in peripheral blood samples from patients of SLE is elevated,similarly,the same elevation is observed in the renal tissue of LN patients.IFIT1 expression levels can be used as a new indicator of lupus disease activity candidate.What are changes will occur when IFIT1 are highly expressed in podocytes? Is IFIT1 involved in podocyte injury in lupus nephritis?No reports have been reported at home and abroad.This is the focus of this study.If the relationship between the expression of IFIT1 and the damages of podocyte and filtration barrier in LN has been ascertained,that not only helps to further improve the pathogenesis of IFN-I pathway in SLE,but also can be used as a new target for specific intervention treatment of LN.Methods: The study was divided into two parts.1.Animal experiment: 18 female MRL/lpr mice as the study group,the control group was made up of 18 female BALB/c mice.24 hours urine protein quantitative analysis,complement C3,C4 and anti-ds-DNA antibody determination.HE staining,PAS staining and transmission electron microscopy were used to observe the pathological changes of renal tissue.The expression of Nephrin,Podocin,F-actin,synaptopodin and IFIT1 were observed by IF.The correlation between IFIT1 and the optical density of four podocyte proteins was analyzed by linear regression analysis.2.Cell experiment: Lentivirus transfected mouse immortalized podocytes to establish mouse podocytes which overexpress IFIT1.The expression of IFIT1 was detected by RT-PCR and Western blot.CCK8 assay was used to detect the proliferation of podocytes,the apoptosis rate was detected by flow cytometry,and the expression levels of Bcl-2,Bax,clevaed,p38 and p-p38 were detected by Western blot.Gene chip technology for the over expression of IFIT1 damage podocyte conduction pathway.Results: 1 Animal experiments: MRL/ lpr mice compared to the same month BALB/c mice 24 hours urine protein(3B1.01±0.10 vs 3M 2.11±0.29,P<0.01;4B1.04±0.30 vs 4M3.56±0.26,P<0.01;5B0.99±0.24 vs 5M6.42±0.57,P<0.01),anti ds-DNA antibody(3B37.65±1.75 vs 3M125.93±7.50,P<0.01;4B38.62±3.17 vs 4M185.79±8.95,P<0.01;5B37.05±2.32 vs 5M208.39±21.16,P<0.01)significantly increased,Complement C3,there was no significant difference between 3 month old BALB/c mice and MRL / lpr mice(3B98.86±15.92 vs 3M86.39±5.46,P>0.05).Compared with BALB/c mice at 4 and 5 months of age,MRL/lpr mice were significantly reduced(4B103.50±16.55 vs 4M78.42±3.26,P<0.01;5B101.73±18.93 vs 5M57.09±1.82,P<0.01).Compared with group BALB/c,the complement C4 in the same age group of MRL/lpr mice was significantly reduced(3B26.23±4.58 vs 3M17.66±1.07,P<0.01;4B28.76±2.60 vs 4M15.31±0.47,P<0.01;5B28.76±3.51 vs 5M13.73±1.04,P<0.01).HE staining and PAS staining,transmission electron microscope were used to observe the changes of renal pathology.The pathological changes of MRL / lpr mice were compared with BALB / c mice in the same age group,and the pathological changes were increasing with the increase of age.Linear regression analysis showed that the IFIT1 optical density was negatively correlated with Nephrin,Podocin and F-actin(P < 0.05).2.Cell experiments: RT-PCR(Transfection0.51±0.12 vs Control0.24±0.03 P < 0.05)and Western blot(Transfection3.47±0.26 vs Control0.83±0.04 P<0.01)confirmed the successful establishment of over expression IFIT1 mouse podocytes.CCK-8 detected absorbance of control group,control lentivirus group and IFIT1 overexpression group shows 24h(0.278±0.007 vs 0.278±0.007 vs 0.240±0.006)、48h(0.358±0.010 vs 0.356±0.006 vs 0.262±0.008)、72h(0.482±0.011 vs 0.483±0.003 vs 0.287±0.009 P<0.05),CCK-8 assay showed that over expression of IFIT1 could significantly inhibit the proliferation of mouse podocytes.Flow cytometry analysis showed podocyte apoptosisof control group,control lentivirus group and IFIT1 overexpression group(3.47±0.68vs3.73±0.31vs22.70±0.89),that means overexpression of IFIT1 significantly induced podocyte apoptosis.WB detection showed that the expression level of Bax(0.81±0.04 vs 0.57±0.01 P<0.01),cleaved caspase3(0.64±0.02 vs 0.21±0.01 P<0.01),p-p38/p38(0.88±0.04 vs 0.46±0.16 P<0.05)in IFIT1 group was significantly higher than that in control group,and the expression level of Bcl-2(0.51±0.10 vs 0.80±0.05 P<0.05)was significantly lower than that of control group.Gene chip technique was used to detect the gene F2 r and Eral1(relate to the proliferation and apoptosis)expression of control group,control lentivirus group and IFIT1 overexpression group,F2r(4.54±0.80 vs 4.62±1.49 vs 8.11±0.68)and Eral1(9.10±1.50 vs 10.68±1.28 vs 3.66±1.00),the IFIT1 overexpression group was significantly up-regulated and down-regulated(P <0.01)compared with the control group and the control lentivirus group.Conclusion:1.The enhanced expression of interferon-inducible gene IFIT1 in the MRL / lpr of spontaneous lupus mice negatively correlate with F-actin protein,Nephrin protein and Podocin protein expression which are essential for maintaining normal glomerular filtration function,possibly involved in the injury of podocytes in lupus nephritis,thus promoting the occurrence of proteinuria.2.IFIT1 can inhibit the proliferation and promote the apoptosis of podocytes,reduce the number of podocytes,damage the glomerular filtration barrier,and damage the renal function.3.IFIT1 may increase the expression of Pro apoptotic protein Bax,activate Caspase3 and p38,decrease the expression of Bcl-2,and finally induce podocyte apoptosis through F2 R and Eral1 signaling pathway. |
PDF Full Text Request