The Experimental Therapy On Collagen Induced Arthritis With RNA Interfence Targeting Nuclear Factor κB (NF-κB) Essential Modulator(NEMO)

Objective:The purpose of this study was to evaluate the effect of RNA interferring therapy of nuclear factor κB essential modulator(NEMO)on rheumatoid arthritis(RA)and to further explore the role that NEMO may play in the pathogenesis of arthritis.Methods:To explore the possibility of experimental gene therapy targeting NEMO in RA,we used the lipopolysaccharide(LPS)-induced inflammatory model on macrophages and collagen-induced arthritis on mice in vitro and in vivo.We built three pairs of small interfering RNAs(si-RNAs)targeting NEMO to screen the optimal interfering sequence,based on which the lentivirus targeting NEMO(LV-shNEMO)was built.The quantitative real-time polymerase chain reaction(qPCR)was used to evaluate the interference efficiency.The lentivirus infected RAW264.7 cells with the optimal multiplicity of infection(MOI)and the stable transfer strain was obtained,following with the qPCR to verify the virus interfering efficiency.In vitro,the RAW264.7 cells respectively infected with LV-shNEMO and LV-shNC and then the wild-type cells were killed by puromycin.Then we utilized LPS to induce inflammatory models,followed by qPCR to evaluate the relative expression of pro-inflammatory cytokines: interleukin 1β(IL-1β),interleukin 6(IL-6)and tumor necrosis factor α(TNF-α)and western blotting(WB)to detect the relative proteins involved in NF-κB signaling pathway(NEMO,p65,phosphorate-p65,IκBα,and phosphorate-IκBα).Immunofluorescence staining(IF)was used to assess the nuclear translocation of phosphorate-p65(p-p65).In vivo,5-week-old male DBA/1 mice were used to build the CIA models and post-successful modeling mice were divided into three groups: the knockdown group,the negative control group,and the PBS control group,treated with intra-articular injections of LV-shNEMO,LV-shNC,and PBS,respectively.The arthritis scores were assessed every other day to quantify the severity of the inflammation.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression of serum IL-1β,IL-6 and TNF-α in mice.Hematoxylin-erosin staining(HE staining)was used to assess the inflammatory infiltration in the ankle joint in mice.To further explore the mechanism by which LV-shNEMO alleviates arthritis,we used RAW264.7 cells infected with LV-shNEMO and LV-shNC with LPS induction to perform the transcriptome sequencing analysis.The differential genes(DEGs)were enriched and analyzed using the software of pathway analysis(IPA),and the enriched pathway was verified by a variety of experiments(qPCR,IF,flow cytometry,WB).Results:In vitro,the interference efficiency of si RNA-3 was the best,and the construction of lentivirus particles was successfully completed,whose optimal MOI was 25.The m RNA expression level of pro-inflammatory cytokines such as IL-1β、IL-6、TNF-ɑ was significantly lower than the other groups,even with a different time of LPS induction.Moreover,WB results proved that LV-shNEMO inhibited the activation of the NF-κB signaling pathway,and IF exhibited that the nuclear translocation of p-p65 in the KD group was less than in the NC group.In vivo,the incidence of CIA was 88%.The average arthritis score of CIA mice in the KD group was significantly lower than that in the other groups while HE staining showed that the inflammatory infiltration of ankle joint was remarkably less in KD group than that of the NC and CIA group.The serum levels of IL-1β,IL-6 and TNF-α in the KD group were lower than those the others,and the differences were statistically significant.Transcriptome sequencing enrichment analysis suggested that RNAi targeting NEMO may alleviate inflammatory reactions by inhibiting the macrophage classical activation signaling pathway.The M1 subtype macrophage-associated pro-inflammatory cytokine(IL-1β、IL-6、TNF-α)and M1 macrophages markers(GM-CSF,iNOS)were detected by qPCR,both of which were less expressed in the KD group than in the NC group.Moreover,IF(CD16/32),flow cytometry(CD86),and WB(iNOS)exhibited that the expression of M1 subtype marker in the KD group was significantly lower than that in the NC group.Conclusion:In vitro,LV-shNEMO alleviates the inflammation by suppressing the M1-type macrophage polarization.In vivo,LV-shNEMO relieves the severity of arthritis in CIA mice.Thus,lentivirus-mediated NEMO-targeted RNAi therapy can be utilized as one of the potential treatments for rheumatoid arthritis.