| Objectives: 1.To study the expression of Interleukin-38(IL-38)in peripheral blood and the activation of NF-κB signaling pathway in patients with systemic lupus erythematosus(SLE),and analyze the correlation between IL-38 and NF-κB signaling pathway and Clinical indicators.2.Culture PBMC of SLE patients in vitro,using recombinant IL-38 protein stimulation and IL-38 si RNA gene interference technology to clarify the effect of IL-38 on IKK/NF-κB signaling pathway in SLE patients.3.In animal experiments,study the expression of IL-38 and the activation of IL-36R/IL-1RAc P/NF-κB pathway in peripheral blood and kidney tissues of MRL/lpr lupus mice,using recombinant IL-38 protein and IL-38 through in vivo intervention,to explore the regulatory mechanism of IL-38 on the NF-κB pathway of MRL/lpr lupus mice and the influence of kidney disease.Methods: A total of 60 patients with SLE were enrolled in the study,and their condition met the classification criteria of the American College of Rheumatology in 1997.30 healthy people served as normal controls.In the animal experiment,20 female MRL/lpr lupus mice aged 12 weeks were randomly divided into PBS intervention group,recombinant mouse IL-38 protein intervention group,sh RNANC intervention group and IL-38 sh RNA intervention group.The normal control group was 5mice for 8 weeks.BALB/c mice of age.Use IL-38 recombinant protein and RNA interference technology to interfere in PBMC of SLE patients and lupus mice in vivo and in vitro.Enzyme-linked immunosorbent assay was used to detect IL-38,IL-36 R,IL-1RAc P,TNF-α,IFN-γ levels.Reverse transcription polymerase chain reaction method was used to detect IL-38,IL-36 R,IL-1RAc P,and IKKα/ βm RNA level.Nuclear factor-κB kit to detect nuclear factor-κB activity.Western blot analysis of IKKα/β and p-IKKα/β protein expression levels.Pathological section PAS staining method to detect renal tissue lesions in MRL/lpr lupus mice.Immunohistochemistry was used to detect the expression of IL-38,IL-36 R,IL-1RAc P and NF-κB in the kidney.Results: 1.(1)The levels of IL-38 m RNA and protein in peripheral blood of SLE patients were significantly lower than those of the normal control group.Among them,the level of SLE patients with active disease decreased more markedly(P<0.05).(2)The level of ds DNA antibody in SLE patients was greater than that of the healthy control group.Elevated,in which patients with active SLE are higher compare with those in remission.IL-38 levels are negatively correlated with ds DNAIg G antibodies,urine protein quantification and SLEDAI scores,and positively correlated with complement C3(P < 0.05).(3)SLE patients have significantly higher levels of IKKα,IKKβ,NF-κB and TNF-α in healthy controls,the increase in active patients was more significant than that in remission patients.The expression level of IL-38 was negatively related to the levels of IKKα,IKKβ,NF-κB and TNF-α(P<0.05).2.(1)In vitro,after the PBMCs of the active and remission SLE patients were co-cultured with recombinant IL-38,the levels of IKKα/β,NF-κB,TNF-α,ds DNAIg G antibody between the two groups of SLE patients compared with the untreated group Significantly reduced(P<0.05).(2)In vitro,after IL-38 si RNA was transfected into the PBMC of SLE patients in active and remission phases,compared with the untreated group,the expression levels of IKKα/β,NF-κB,TNF-α,ds DNAIg G antibody between the two groups of SLE patients Significantly increased(P<0.05).3.(1)In peripheral blood of MRL/lpr lupus mice,IL-38 m RNA and protein expression decreased,and IL-36 R and IL-1RAc P m RNA and protein levels increased(P<0.05).(2)The expression of IKKα,IKKβ,NF-κB,TNF-α,IFN-γ in peripheral blood of MRL/lpr lupus mice increased,and renal function,ds DNAIg G antibody,urine protein quantitative and urine white blood cell number increased(P <0.05).(3)After recombinant mouse IL-38 protein intervention,the levels of IL-36 R,IL-1RAc P,IKKα/β,NF-κB,TNF-α and IFN-γ in peripheral blood of MRL/lpr lupus mice were significantly reduced,and renal function,ds DNA antibody,urinary protein quantification and the number of urinary white blood cells were significantly reduced(P < 0.05).(4)After IL-38 sh RNA intervention in MRL/lpr lupus mice,the levels of IL-36 R,IL-1RAc P,IKKα/β,NF-κB,TNF-α,IFN-γ in peripheral blood increased,renal function,ds DNA antibody,urine protein quantification and the number of urinary white blood cells increased(P<0.05).(5)Renal pathology results show that the glomerular mesangial cells of MRL/lpr lupus mice proliferate,the matrix is widened,and lymphocyte infiltration can be seen in the renal interstitium.After the intervention of recombinant mouse IL-38 protein,the MRP/lpr lupus mouse glomerular membrane cells are slightly proliferated,with a small amount of lymphocyte infiltration.After IL-38 sh RNA intervention,MRP/lpr lupus mice glomerular mesangial cells proliferated,the matrix widened,and a large number of lymphocytes infiltrated.(6)The results of immunohistochemistry showed that the expression of IL-38 in the kidney tissue of MRP/lpr lupus mice was significantly reduced,and the levels of IL-36 R,IL-1RAc P and NF-κB increased.After the intervention of recombinant mouse IL-38 protein,the expression of IL-38 in the kidney of MRP/lpr lupus mice increased,and the levels of IL-36 R,IL-1RAc P and NF-κB decreased.After IL-38 sh RNA intervention,the expression of IL-38 in the kidney of MRP/lpr lupus mice decreased,and the levels of IL-36 R,IL-1RAc P and NF-κB increased(P < 0.05).Conclusion: The expression of IL-38 decreased in SLE,which caused the secretion of the corresponding receptor IL-36 R to increase,thereby recruiting a large amount of IL-1RAc P to form trimers,promoting the activation of NF-κB signaling pathway and inducing the transcription of TNF-α and IFN-γ and other inflammatory factors expression and production of various autoantibodies such as ds DNA,cause pathological damage to the kidney,leading to the onset of SLE.It shows that IL-38 participates in the occurrence and development of SLE by regulating the NF-κB signaling pathway. |
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