The Protective Effect Of Nrf2 Regulating NLRP3 Inflammasome In The Glomerular Endothelial Cell Of Lupus Nephritis

BackgroundSystemic lupus erythematosus(SLE)is a kind of common autoimmune disease which causes multiple organ damage.Almost all patients of SLE have kidney damage.Lupus nephritis(LN)is the most common and serious clinical manifestation of SLE.The pathogenesis of SLE and LN is complex.The exact mechanisms are still unclear.It is generally accepted that SLE is caused by genetic or endocrine factor,environmental factors(infection,ultraviolet radiation,etc.)and dysregulation of immune system.In addition,inflammation plays a critical role in Lupus nephritis.Recent studies have shown that NLRP3 inflammation is involved in the initiation and progression of SLE.Among mammals,a typical representative of the early innate immune response to pathogens or endogenous damage signals is NLRP3 inflammation.NLRP3 inflammation is a multiprotein complex mainly consist of NLRP3,ASC and Caspase-1.The oligomerization of NLRP3 inflammation leads to autocatalytic activation of Caspase-1,which converts inactive pro-IL-1? and pro-IL-18 into bioactive form and promote cell necrosis.One of the crucial elements for NLRP3 activation is the generation of reactive oxygen species(ROS).In recent years,a number of studies have found that oxidative stress is also an important factor in the pathogenesis of SLE.Patients with lupus nephritis showed obvious oxidative stress disorder.Nrf2,a basic leucine zipper transcription factor that regulates redox balanceand stress response,has attracted much attention in recent years.Drugs targeting on Nrf2 gains the FDA's approval to be used in the treatment of recurrent multiple sclerosis.The study about the effect of Nrf2 on inflammation will be of great clinical significance.Recent studies suggest that Nrf2 has a protective effect in many inflammatory diseases,such as sepsis,asthma,infectious diseases and emphysema caused by smoking etc.However,the specific role and mechanism of Nrf2 in lupus nephritis is poorly understood.ObjectiveThis study aims to explore whether and how the regulation of Nrf2 on NLRP3 inflammasome signalling pathway contributes to the pathogenesis of lupus nephritis.Firstly,we examine the relationship between serum concentration of IL-18,IL-1?and disease activity of lupus nephritis.Secondly,we explore the effect and molecular mechanism of Nrf2 regulating NLRP3 inflammasome signalling pathway in peripheral blood mononuclear cells(PBMCs)from SLE patients and its effect on human umbilical vein endothelial cells(HUVECs).Materials and Method1.40 SLE patients and 30 age-and sex-matched healthy controls were included in the study.Both IL-1? and IL-18 were measured by ELISA in all serum samples.2.Freshly isolated PBMCs from SLE patients were stimulated by LPS?ATP to activate NLRP3 inflammation,and pre-incubated with Nrf2 agonist or inhibitor.The change in the gene expressions of Nrf2,NLRP3,Caspase-1,IL-1?,IL-18 were detected by real time PCR and IL-1? and IL-18 in supernatant were detected by ELISA.Whether Nrf2 regulates NLRP3 inflammation pathways was analyzed.On the basis of this,we established the co-culture system of PBMCs from SLE patients and HUVECs.Analyze whether Nrf2 regulating NLRP3 inflammation pathways affects the growth of HUVEC in the co-culture system and its role in protect endothelial cells.Results1.The association between serum level of IL-18 and IL-1? in SLE patients and disease activity indexes:1)Serum IL-18 and IL-1? were significantly higher in SLE patients compared with HC(p <0.001).2)IL-18 level in SLE patients was positively associated with LN,SLEDAI and24 h urinary protein(p <0.05).3)IL-1? level in SLE patients was positively associated with LN,SLEDAI,serositis,ESR and Ig M,whereas reversely related to C3,Hb,albumin,albumin/globulin(p <0.05).4)IL-1? levels in SLE patients with varying degrees of activity were significantly higher than that in stable patients(p=0.02,p=0.007 and p=0.002,r-espectively).2.The role of Nrf2 on regulating NLRP3 Inflammasome in PBMCs from SLE patients and its protective effect on HUVECs was explored.1)High expression of NLRP3 signalling pathway mRNA was observed in PBMCs from SLE patients.After pre-incubated with Nrf2 agonists,expression of NLRP3 inflammation and its downstream inflammatory factors m RNA was reduced.After pre-incubated with Nrf2 inhibitor,expression of NLRP3 inflammation and its downstream inflammatory factors mRNA was increased.2)Establish co-culture system of PBMCs and HUVECs,the overall viability of cells in it decreased after activation of NLRP3 inflammation.The overall viability of the cells in the co-culture system incraesed after pre-incubated with Nrf2 agonists,whereas decreased after pre-incubated with Nrf2 inhibitor.Under the same conditions as co-culture system,there was no difference in cell viability between PBMCs and HUVECs cultured alone.Conclusions1.Elevated serum IL-1? and IL-18 levels in SLE patients were found in correlation with disease activity.2.High expression of NLRP3 inflammasome and its downstream inflammatory factors was observed in PBMCs from SLE patients,which would inhibit the proliferation of HUVECs can be inhibited.Nrf2 may play a protective role on endothelial cells in LN by regulating NLRP3 inflammasome signalling pathway.