Experimental Study Of SiRNA Interference Combined With BMSCs In The Treatment Of Rheumatoid Arthritis Based On The NF-κB Signaling Pathway

Rheumatoid arthritis(RA)is an autoimmune disease,and there is currently no cure.The disease seriously affects human health,and animal RA reduces the efficiency of the livestock and poultry breeding industry.Studies have shown that small interfering RNA(siRNA)can effectively inhibit the expression of target genes and can effectively treat related diseases.Bone marrow mesenchymal stem cells(BMSCs)can target and migrate damaged tissues,and their multidirectional differentiation potential can repair damaged tissues,mediate and regulate inflammation in contact with inflammatory cells,and can effectively treat autoimmune diseases.Studies have found that the NF-κB signaling pathway is closely related to inflammation and autoimmune diseases.In this study,the RA rat model was used to explore the role of NF-κB signaling pathway in the pathogenesis of RA,and to study the pathogenesis of RA.In vitro transplantation of siRNA combined with BMSCs was used to explore the feasibility of combined treatment of RA based on NF-κB signaling pathway.1.The dynamic changes of NF-κB signaling pathway in the pathogenesis of RA rats1.The dynamic changes of NF-B signaling pathway in the pathogene-sis of RA ratsIn the experiment,chicken type II collagen(CIA group)emulsion was used as a sensitizer to establish an RA rat model to study the inflammatory effects and dynamic changes of the NF-κB signaling pathway in the pathogenesis of RA.The experiment was divided into a control group and a CIA group.In the CIA group,chicken type II collagen was injected subcutaneously from the root of the tail to the back of the rat.The control group was replaced with normal saline.The rat’s growth status,behavior,pathology,imaging,and foot Toe swelling and spleen index confirmed successful RA rat model;through the detection of interleukin-1β(IL-1β),tumor necrosis factor-p(TNF-α),NF-κB-P65,p-NF-κB-P65 to study NF-The dynamic changes of κB signaling pathway in the pathogenesis of RA,in-depth study of the mechanism of NF-κB signaling pathway in the pathogenesis of RA.The results showed that the expression of IL-1β and TNF-α in the serum of the CIA group was significantly higher than that of the control group at 1 week(P<0.01).The toe swelling value of the rats in the CIA group was extremely significantly higher than that in the control group(P<0.01)after the model was established 2 weeks.The body weight of the rats in the CIA group was significantly lower than that of the control group(P<0.01),and the immobility time was significantly higher than that of the control group(P<0.01).The mRNA expression levels of IL-1β,TNF-α,and NF-κB-P65 in the CIA group were significantly higher than those in the control group(P<0.01);NF-κB-P65,p-NF-κB in the CIA group-P65 protein expression was also extremely significantly higher than that of the control group(P<0.01);the spleen index of the CIA group was extremely significantly higher than that of the control group(P<0.01);DR-X-ray imaging showed that bone hyperplasia and bone formation occurred in the CIA group The slices show that the articular cartilage is infiltrated by inflammatory cells,leading to cartilage destruction,joint synovitis,etc.,indicating that the rat RA model is reliable.It is suggested that with the development of inflammation,the NF-κB signaling pathway is overactivated,and the interaction between IL-1β,TNF-α and other factors aggravates the inflammatory response.2.In vitro study of siRNA interference with the expression of IL-1βand TNF-α and the regulation of inflammatory cells by BMSCsThe increased expression of IL-1β and TNF-α in RA rats leads to aggravation of inflammation,suggesting that inhibiting the expression of IL-1β and TNF-α may inhibit the development of inflammation,so as to achieve the purpose of treating RA.In this study,LPS stimulated inflammatory cells RAW264.7(monocyte macrophages)to establish an in vitro cell inflammation model,and interfered with the expression of IL-1β and TNF-α through siRNA technology.And using BMSCs and inflammatory cell co-cultivation technology to detect its regulatory effect on inflammation,and explore the feasibility of siRNA interference combined with BMSCs in the treatment of RA in vitro.The experimental results showed that the expression of IL-1β and TNF-α in the culture supernatant and cell lysate of the inflammatory cell model established with 500ng/ml LPS stimulation was significantly higher than that in the normal treatment group(P<0.01);In the screening experiment,it was found that the combination of 50 pmol siRNA and 1 μl transfection reagent ratio was the best transfection combination,and 48 h was the best transfection time;after siRNA transfection,siRNA+LPS+RAW264.7 in the cell culture supernatant The expression of IL-1β in the group was significantly lower than that in the LPS+RAW264.7 group(26.75±2.15 pg/ml vs 56.15±2.49 pg/ml,P<0.01);the expression of TNF-α in the siRNA+LPS+RAW264.7 group The amount was significantly lower than that in the LPS+RAW264.7 group(223.37±6.43 pg/ml vs 371.42±5.48 pg/ml,P<0,01).It suggests that siRNA can effectively inhibit the secretion of IL-1β and TNF-α by inflammatory cells,and can inhibit the inflammatory response of inflammatory cell models.The results of the scratch experiment showed that the scratch surface of the LPS+RAW264.7+BMSCs group was significantly smaller than that of the LPS+RAW264.7(P<0.01).It suggests that BMSCs have a good regulatory effect on inflammatory cell models and regulate cell proliferation.3.Experimental study of the therapeutic effect of siRNA combined with BMSCs on RA Based on the NF-κB signaling pathwaysiRNA combined with BMSCs was used to treat RA rats in vivo,with changes in body weight,toe swelling,behavioral changes,the expression of IL-1β and TNF-α in serum,and the level of IL-1β,TNF-α,and NF-κB-P65 in spleen tissue.The mRNA expression level,the ankle and knee joint NF-κB-P65,p-NF-κB-P65 protein expression changes,to explore the effect and mechanism of gene interference combined with BMSCs in the treatment of RA based on NF-κB signaling pathway.The experimental results showed that the weight growth rate of the methotrexate group,BMSCs group,siRNA group,and siRNA+BMSCs group was higher than that of the PBS group,and the weight growth rate of the siRNA+BMSCs group was significantly higher than that of the PBS group(P<0.01).The toe swelling value,spleen index,swimming immobility time,and serum IL-1β and TNF-α expression in the above treatment groups were all lower than those of the PBS group,and the siRNA+BMSCs group were significantly lower than the PBS group(P<0.01).Cell labeling experiments showed that exogenous BMSCs were found in joint osteogenesis,cartilage,and synovium,indicating that BMSCs can home to damaged joint tissues.Except that the p-NF-κB-P65 protein expression in the ankle joint of the methotrexate group was not significantly different from the PBS group,the expression of NF-κB-P65 and p-NF-κB-P65 protein in the knee and ankle joints of each treatment group The levels were lower than those in the PBS group,and the siRNA+BMSCs group was significantly lower than the PBS group(P<0.01);the mRNA expression levels of IL-1β,TNF-α,and NF-κB-P65 in the spleen tissues of each treatment group were all lower than those of PBS The siRNA+BMSCs group was significantly lower than the PBS group(P<0.01).It is suggested that the combination therapy can effectively alleviate the onset of RA and inhibit the activation of the NF-κB signaling pathway,and BMSCs can target and repair damaged tissues,providing a theoretical basis and experimental basis for subsequent gene interference combined with BMSCs to treat RA.