Establishment Of A Murine Induced Model With Lupus-Prone CGVHD And Intervention Study On Lupus Nephritis Mice Based On TLR9/NF-?B/Nrf2 Signal Pathway

Background & objectives Systemic lupus erythematosus(SLE)is a systemic inflammatory disease mediated by autoimmune.Lupus nephritis(LN)is one of the most common complications and it is a major predictor of poor prognosis.Therefore,the further research on the pathogenesis of LN will bring hope for the treatment of specific cells,autoantibodies,cytokines and chemokines in order to regulate inflammation and tissue injury in the future.At present,the pathogenesis of LN is still unclear.Generally,abnormal immune responses in the body induce immune complex formation and deposition in kidneys.Then they produce the release of proinflammatory cytokines,cell adhesion molecules,chemokines and etc.,cause inflammation,result in the chemotaxis of monocytes and polymorphonuclear cell and follow mesangial hyperplasia of endothelial injury with the subsequent release of protease.The pathogenesis of SLE mainly includes immune system abnormality,oxidative stress and inflammatory response.The present study has proved that toll like receptor(TLR)9 is one of the bridges linking innate and adaptive immune responses.Nuclear factor kappa B(NF-?B)is a key nuclear transcription factor regulating inflammation gene expression.Nuclear factor erythroid 2-related factor 2(Nrf2)is a universal regulator of cell antioxidant.They are crucial in three aspects of immune system abnormality,inflammatory response and oxidative stress.The role of TLRs in the pathogenesis of SLE is becoming more and more important,especially TLR9.The ligand of TLR9 is the Cytosine phosphate guanine DNA(CPG-DNA)of hypomethylation.TLR9 can specifically identify the CPG-DNA of hypomethylation in the DNA of virus and bacterial,or synthetic oligodeoxynucleotides(ODNs),start signal transduction pathways,activate Tlymphocytes,B lymphocytes and dendritic cells,induce a large number of cytokines,inflammatory factors and autoantibodies and then participate in immune response.NF-?B is a transcription factor regulating inflammatory cytokines in the cells,and plays an important role in immune inflammatory response of LN.It can efficiently induce gene expression of various inflammation cytokines,be activated in the inflammatory sites of various inflammatory diseases,and enlarge the cascade the inflammatory reactions.The expression of NF-?B in glomerular endothelial and mesangial cells of LN patients is increased and activated,which is accompanied by the release of various inflammatory cytokines.Nrf2,as an important transcription factor regulating the antioxidant cytokines of the cells,mediates the gene transcription that encodes antioxidants and other cytoprotective factors.Because of the ubiquitous expression of Nrf2,it plays a key role in protecting many cells and organs from oxidative stress and toxicity.Nrf2 plays a protective role in LN by inhibiting oxidative damage and activation of NF-?B.Therefore,the TLR9/NF-?B/Nrf2 signal pathway is essential to maintain the environment stability in the body.Iguratimod(IGU),as a new immunomodulator,and mesenchymal stem cells(MSCs)both have immune regulation,inhibition of inflammation,antioxidant activity and other effects.Therefore,in order to identify the role of IGU and MSCs in LN and the possible mechanisms,we have conducted the following experiment.MethodsPart?: Methods of establishing a chronic graft-versus-host disease(cGVHD)lupus-prone murine model were studied.The murine model of induced cGVHD was established by transferring 5×107 lymphocytes from female BALB/c mice into BALB/c(female)× C57BL/6(male)F1 hybrids via tail-vein injection of three or four times.The model was identified and the next part of the experiment was proformed after the model success was confirmed.Part ?: Effect of IGU on cGVHD LN-prone mice was studied.From 4 weeks after the induction of cGVHD mice,IGU(30mg/kg/d)was treated for 10 weeks.The weights were measured every 2 weeks.The levels of Urine protein and urine creatinine were detected every 4 weeks.Two weeks after the end of the treatment,mice were euthanized,weights of the organs were measured and ANA andanti-dsDNA antibodies in serum were detected.IgG immune complex deposition in the glomeruli was detected by direct immunofluorescence.Pathological changes of the kidney were evaluated by the observation through optical and electron microscopy.Detection of TLR9,NF-?B,Nrf2 and other cytokines at the mRNA and protein levels was perfomed by RT-qPCR,or Western blot methods,or both.Part ?: Effect of MSCs on MRL/MPJ-Faslpr/J(MRL/lpr)LN-prone mice was studied.At the 16,18 and 20 weeks of age in mice,1×106 placental MSCs were injected via the tail vein.Mice were euthanized two weeks after the end of the treatment.Residual experiment methods refer to the previous part.Results: Methods of establishing a model of cGVHD lupus-prone mice have been studied.From the survival rate of mice,occurrence of proteinuria,ANA,anti-dsDNA antibody in serum,renal pathological evaluation,and the protein level of TGF-?1 by Western blot analysis,Lupus-like changes were established in the two model groups according to three and four injection times,and 5×107 cells every time by tail-vein injection.Effect of IGU on the TLR9/NF-?B/Nrf2 signal pathway in cGVHD mice was investigated.Compared with cGVHD untreated group,weights of IGU group gradually rose,the level of proteinuria gradually descended,ANA,anti-dsDNA antibody in serum and IgG immune complex deposition in the kidneys were depleted,and the renal pathology was improved.At the mRNA level,the expression of TLR9 decreased.At the mR NA and protein levels,decreased expression and attenuated activity of NF-?B with the declined levels of downstream inflammatory factors were found,while Nrf2 expression was elevated with the level of downstream antioxidant factors increased.Effect of MSCs in MRL/lpr mice was investigated.Compared with untreated group,the survival rate of mice in MSCs intervention group went up,proteinuria declined,spleen and kidney coefficients were decreased,serum ANA,anti-dsDNA antibody and IgG immune complex deposition level in kidney were reduced,and renal pathology was ameliorated.At the mRNA level,the decrease of TLR9 expression was found.At the mRNA and protein levels,decreased expression and attenuated activity of NF-?B with the declined levels of downstream inflammatory factors were found,while Nrf2 expression was elevated with the level of downstream antioxidant factor s increased.Conclusion: The above results demonstrated that the mouse model with lupus-prone cGVHD can be successfully established by taking BALB/c(female)× C57BL/6(male)F1 hybrids mice as recipients,BALB/c female mice as donors,and injecting parental lymphocytes for three or four times.IGU and MSCs both exerted considerable influence on Immunoregulation,anti-inflammatory and anti-oxidant stress in two different LN-prone murine models.One of the mechanisms is to regulate TLR9/NF-?B/Nrf2 signal pathway.