The Experimental Therapy On Collagen-induced Arthritis Mice With Lentiviral-mediated RNA Interference Targeting IL-6

Objective:To study the effect of gene therapy with lentivirus-mediated RNA interference targeting interleukin 6(IL-6)on collagen-induced arthritis(CIA)mice and to further explore the potential mechanism of IL-6 in arthritis.Methods:Part 1:Quantitative real-time polymerase chain reaction(RT-qPCR)was used to test IL-6 mRNA expression in J774A.1 murine cell line to screen siRNAs with the highest interference efficiency by transfecting technique.Lentivirus targeting IL-6(LV-IL6-RNAi)particles were built based on siRNA sequence,and infected J774A.1 cells.The best titer of infection was determined by fluorescence microscope,and the interference efficiency was detected by RT-qPCR.DBA/1 mice were used to establish CIA model.The experiment was divided into LV-IL6-RNAi group,the empty virus(LV-NC-RNAi)group as the negative control,and the methotrexate(MTX)group as the positive control.Arthritis scores were used to value those groups.Serum IL-6,interleukin 1β(IL-1β)and tumor necrosis factorɑ(TNF-ɑ)in mice were detected by enzyme-linked immunosorbent assay(ELISA).Inflammatory cell infiltration in ankle arthritis was observed by Hematoxylin-eosin staining(HE staining).Part 2:Immunohistochemistry was used to observe the IL-6 of ankle joints in healthy mice,LV-NC-RNAi group,and LV-IL6-RNAi group.Western blotting(WB)was used to detect NLRP3,caspase-1 in ankle synovium.In vitro,J774A.1 cells were stimulated by different concentrations of IL-6,adenosine triphosphate(ATP),IL-6/ATP,lipopolysaccharide(LPS)/ATP as positive control,and PBS as the negative control.The proportion of caspase-1 activated cells was detected by flow cytometry.Then,J774A.1cells were divided into PBS,IL-6,ATP,and IL-6/ATP groups.WB was used to test caspase-1 activation in cells,living cell workstation was used to observe the cell morphology,flow cytometry was used to distinguish the type of cell death,ELISA was used to test IL-1βin the supernatant of J774A.1 cells.Results:Part 1:The interference efficiency of siRNA-1 was 80.23%,which was significantly higher than that of other siRNAs.When viral titer was 1×10~7TU/ml,the proportion of J774A.1 cells infected by LV-IL6-RNAi was 85.89%±2.45%,which was significantly higher than that in other groups(F=70.57,P<0.01),and the interference efficiency was about 86.15%.The score of CIA arthritis in LV-IL6-RNAi group was significantly lower than that in LV-NC-RNAi group and MTX group(F=22.49,P<0.01).IL-6 level in serum of LV-IL6-RNAi group was significantly lower than that of LV-NC-RNAi group and MTX group(F=40.97,P<0.019).The levels of IL-1βand TNF-ɑin serum of mice in LV-IL6-RNAi group and MTX group were significantly lower than those in LV-NC-RNAi group(F=19.24,P<0.01;F=9.52,P<0.01).At 400×magnification,the number of inflammatory cells in LV-NC-RNAi group,LV-IL6-RNAi group,and MTX group were 213.80±6.76,113.30±6.70,117.20±6.23,respectively(F=74.75,P<0.01).Part 2:Compared with LV-NC-RNAi group,IL-6 infiltration decreased significantly in LV-IL6-RNAi group and healthy mice.NLRP3 and caspase-1 content of ankle joint in LV-IL6-RNAi group and healthy mice decreased significantly(F=47.14,P<0.01;F=33.53,P<0.01).The percentage of caspase-1 cells in 774A.1 activated by IL-6/ATP was 93.10%,which was significantly higher than that of IL-6 or ATP(F=1432.00,P<0.01).Caspase-1 activation in IL-6/ATP group was significantly higher than that in other groups(F=17.18,P<0.01).IL-6/ATP co-stimulation caused obvious nuclear fragmentation and death of J774A.1 cells.The type of cell death was inflammatory necrosis represented by double-positive PI/FAM-FITC.IL-1βin the supernatant of IL-6/ATP group was significantly higher than that of PBS group,IL-6 group,and ATP group(F=987.00,P<0.01).Conclusion:LV-IL6-RNAi could relieve arthritis in CIA mice effectively,and the therapeutic efficacy of single located injection was similar to that of multiple defuse injection of MTX.In addition,NLRP3 inflammasome activation in synovium decreased with IL-6infiltration falling down.In vitro,it was found that IL-6 might activate NLRP3inflammasome via ATP pathway,inducing inflammatory necrosis and releasing a large amount of IL-1β,and finally promoting inflammation.