| Background:Systemic lupus erythematosus,occurred mainly in women of childbearing age,was a chronic autoimmune disease with multisystem damages,the main pathological changes were inflammatory reaction and vascular abnormalities.Variety of mechanisms involved in the development of the disease,including environmental factors,genetic factors,hormone levels and immune regulatory factors,both innate and adaptive immunity were involved in the pathogenesis of SLE.As the first barrier system,the innate immune system plays an important role in the removal of foreign pathogens and activation of the effective adaptive immune responses.Innate immune distinguishes pathogen associated molecular patterns(PAMP)by pattern recognition receptors(PRR).Till the present moment,three kinds of PRR have been found.of which,some NOD like receptors(NLRs)can be activated,forming a large protein complex named "Inflammasome".lnflammasome can activate the inflammatory caspase protein kinase(Caspase-1)and produce the active forms of the inflammatory cytokines interleukin-1 beta(IL-1b)and interleukin-18(IL-18),which play an important role in the process of inflammation and immune response.There are four main reported inflammasomes:Nod-like receptor protein1(NLRP1),Nod-like receptor protein3(NLRP3),ice-protease-activating factor(IPAF)and absent in melanoma2(AIM-2).of which NLRP3 is currently the most studied inflammasome and can be activated by many pathogens such as bacteria,viruses,metabolite and crystal.In our previous study,we found that NLRP3 inflammasomes expression were lower in SLE patients compared with healthy controls,the expression of inflammatory components increased after the drug treatments,suggesting that it may play a protective role in the pathogenesis of SLE,but the specific mechanism still unknown.Recent researches reveal that NIMA-related kinase 7(NEK7).a serine and threonine kinase involved in cell cycle and mitosis,acts as a key protein in the activation of NLRP3 inflammasome signal pathway.Objective:To investigate the expression of NEK7-NLRP3 inflammasome signaling pathway in the PBMC of SLE patients and its clinical significance.Methods:A total of 38 SLE patients(SLE group)were recruited from the Department of Rheumatology and Immunology in Provincial Hospital Affiliated to Shandong University.At the same time,33 healthy volunteers were selected as the healthy controls(healthy control group).Quantitative real-time transcription PCR and Western blotting methods were performed to determine mRNA and protein levels of NEK7,NLRP3 inflammasome components(NLRP3,ASC and Caspase-1)and its downstream cytokines(IL-1b.IL-18)in PBMC from the two groups.Enzyme linked immunosorbent assay(ELISA)was used to detect the expression levels of IL-1b and IL-18 in serum.The same methods were used to detect the changes of the above indexes in 25 SLE patients after treatments.Correlations between clinical and laboratory parameters were analyzed.Results:1.Compared with healthy controls.NEK7.NLPR3 and ASC mRNA expression levels were lower in the PBMC from patients with SLE(P=0.0004,P=0.0001 and P=0.0005;respectively).But the Caspase-1,IL-1b and IL-18 mRNA expression levels were higher in SLE patients(P=0.0001.P=0.0001 and P=0.0001;respectively).2.Western blotting results proved that NEK7and NLRP3 protein expression levels were lower in the PBMC from SLE patients than healthy controls(P=0.0317 and P=0.0079).But there was no difference in the expression of ASC between the two groups(P=0.8413).The protein levels of Caspase-1.IL-1b and IL-18 were higher in patients with SLE(P=0.0159.P=0.009 and P=0.016;respectively).3.Serum levels of IL-1b and IL-18 were significantly higher in SLE patients than in healthy controls(P=0.000).After drug treatments,serum IL-1b and IL-18 levels decreased significantly(P=0.000).and IL-1b level was still higher than those in controls(P=0.002),but there was no significant difference between the expression of IL-18 in the SLE patients after treatments and healthy controls(P=0.495).4.Correlation analysis between NEK7 and NLRP3 inflammasome components and their downstream cytokine mRNA levels in SLE patients.4.1 The results of correlation analysis showed that there was a positive correlation of mRNA levels between the NEK7 and NLRP3,ASC(r=0.3728,P=0.0274:r=0.4454,P=0.0073,respectively),but the NEK7 mRNA levels had no correlation with Caspase-1,IL-1b and IL-18(r=-0.2930,0.06625,-0.1063;P=0.0876,0.7053.0.5433,respectively).4.2 There was a positive correlation of mRNA levels between the NLRP3 and ASC(r=0.7157,P=0.0001),but the NLRP3 mRNA levels had no correlation with Caspase-1?IL-1b and IL-18(r=-0.1521,0.05126,0.1441;P=0.3831,0.7699.0.4088.respectively).4.3 Correlation analysis showed that the mRNA expression levels of ASC had no correlation with Caspase-1.IL-1b and IL-18(r=-0.2000,-0.02479.0.1583:P=0.2494.0.8876,0.3638,respectively).4.4 Correlation analysis showed that there was a positive correlation between the Caspase-1 and IL-1b,IL-18 mRNA levels(r=0.4790.0.7090;P=0.0036,0.0001.respectively);so as IL-18 and IL-1b mRNA levels(r=0.4996,P=0.0022).5.Correlation analysises with clinical datas(anti-dsDNA.anti-AnuA and SLEDAI)5.1 The mRNA expression levels of NEK7 were negatively correlated with anti-AnuA(r=-0.5630,P=0.0006)and had no correlation with anti-dsDNA.SLEDAI(r=-0.2701,-0.2692;P=0.1166.0.2034.respectively).5.2 The mRNA expression levels of NLRP3 were negatively correlated with anti-dsDNA.anti-AnuA and SLEDAI(r=-0.5453,-0.7087,-0.7430:P=0.0007.0.0001.0.0001,respectively).5.3 The mRNA expression levels of ASC were negatively correlated with anti-AnuA and SLEDAI(r=-0.3722,-0.7667;P=0.0277.0.0001.respectively),but had no correlation with anti-dsDNA expression levels(r=-0.3261.P=0.0559).5.4 The mRNA expression levels of Caspase-1 had no correlation with anti-dsDNA and SLEDAI(r=0.2281,0.1533;P=0.1876.0.3794.respectively),but positively correlated with anti-AnuA(r=0.4361.P=0.0088).5.5 The mRNA expression levels of IL-1b had no correlation with anti-dsDNA and anti-AnuA(r=-0.0230,0.1888;P=0.8956.0.2774,respectively),but positively correlated with SLEDAI(r=0.7953.P=0.0001).5.6 The mRNA expression levels of IL-18 were positively correlated with anti-dsDNA,anti-AnuA and SLEDAI(r=0.4177,0.3498,0.5259;P=0.0125,0.0394,0.0012,respectively).6.Serum levels of IL-1b in SLE patients was positively correlated with anti-dsDNA,anti-AnuA,ESR and SLEDAI(r=0.584,0.504,0.566,0.624;P=0.002,0.010,0.003,0.001;respectively)and was inversely associated with C3(r=-0.398,P=0.049).Serum levels of IL-18 in SLE patients was positively correlated with anti-dsDNA,ESR and SLEDAI(r=0.544,0.512,0.612;P=0.005,0.009.0.010,respectively).7.After drug treatments,the mRNA expressions of NEK7 and NLRP3 increased obviously:1.1189 ± 0.5017 vs 2.4685±0.9712(P=0.025)for NEK7 and 0.5726±0.1718 vs 0.9682±0.2675(P=0.024)for NLRP3.But there was no significant difference for ASC mRNA levels in the SLE patients before and after treatment:1.0350±0.6351 vs 1.2686±0.8319(P=0.634).The expressions of mRNA for Caspase-1.IL-1b and IL-18 decreased significantly after treatments:1.6419±0.3514 vs 0.7687±0.2223(P=0.002)for Caspase-1;7.1753±2.2051 vs 3.2899±1.3161(P=0.010)for IL-lb and 3.3933±0.8431 vs 1.4175±0.2151(P=0.001)for IL-18.8.After drug treatments,the protein levels of NEK7 and NLRP3 increased significantly:1.9415±0.4547 vs 3.0900±0.8325(P=0.027)for NEK7 and 0.5056±0.3042 vs 1.0066±0.2865(P=0.046)for NLRP3;But there was no significant difference for ASC protein levels in the SLE patients before and after treatments:0.8152±0.2222 vs 0.9048±0.1032(P=0.437).The expressions of protein for Caspase-1.IL-1b and IL-18 decreased significantly after treatments:2.4149±0.6172 vs 1.2169±0.3724(P=0.006)for Caspase-1;4.0684±0.0855 vs 3.7134±0.1129(P=0.001)for IL-1b and 2.7540±0.8599 vs 2.5211±0.1986(P=0.043)for IL-18.9.Compared with SLE patients without renal damages,patients with lupus nephritis showed lower mRNA expression levels of NEK7,NLRP3 and ASC:1.8173±0.7953 vs 1.0187±0.3036(P=0.002)for NEK7,0.7432±0.2791 vs 0.4612±0.2317(P=0.004)for NLRP3 and 1.1426±0.4972 vs 0.7455±0.2536(P=0.012)for ASC,and higher protein expression levels of Caspase-1,IL-1b and IL-18:1.4763±0.5189 vs 2.5654±0.6186(P=0.000)for Caspase-1.3.0022±2.0904 vs 7.6056±4.7246(P=0.000)for IL-1b,2.6668±1.4962 vs 5.9491±3.3549(P=0.000)for IL-18.10.Compared with SLE patients without renal damages,patients with lupus nephritis showed lower protein expression levels of NEK7,NLRP3 and ASC:2.4661 ±0.8916 vs 1.7945±0.9244(P=0.010)for NEK7,0.8669±0.1290 vs 0.4779±0.1400(P=0.001)for NLRP3 and 1.1628±0.2963 vs 0.7191±0.1434(P=0.008)for ASC,and higher protein expression levels of Caspase-1,IL-1b and IL-18:2.1471 ±0.9455 vs 3.5734±0.9081(P=0.024)for Caspase-1,3.1865±1.0688 vs 4.8724±0.8605(P=0.013)for IL-1b,2.6391±0.4480 vs 3.8800±0.3223(P=0.000)for IL-18.Conclusions:1.The low expression of NEK7-NLRP3 complex is negatively correlated with disease activity of SLE and it may play a protective role in the pathogenesis of SLE and LN.2.In PBMC from SLE patients,the positive effect of NEK7 on NLRP3 was observed,and the low expression of NLRP3 in SLE patients may be related to the low expression of NEK7.3.High expression of Caspase-1 in PBMC from SLE patients had no correlation with NEK7,NLRP3 and ASC,prompted that expression of Caspase-1 in patients with SLE not only affected by the NLRP3.there may also had other signaling pathways regulating the expression of Caspase-1.4.Overexpression of Caspase-1 in SLE patients.mediated the maturation and release of IL-1b and IL-18,contributes to the pathogenesis of SLE and LN. |
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