Effect Of Sparstonin B On NLRP3 Iinflammasome And α1,3 Fucosyltransferase Activity In Rheumatoid Arthritis

Objective: Rheumatoid Arthritis is an autoimmune,chronic and inflammatory disease.Among the various rheumatic diseases,RA incidence rate is the highest,causing serious social impact.Sparstolonin B is a natural product isolated from Sparganium Vitex,which is widely used in the treatment of many diseases.Many studies have shown that Sparstolonin B has significant anti-inflammatory and anti-angiogenic effects.Based on the establishment of rat model of collagen induced arthritis,this study first observed whether Ssnb has protective effect on the inflammatory response of RA rat,At the same time,using NLRP3 inflammasome as the hub,we observed the effect of Ssnb on Fuc T-V activity,which provided a new theoretical basis for the treatment of RA.Methods: 1.SD male rats were randomly divided into Control group,RA model group and RA + Ssnb(300 mg/kg)group.The RA group was established by multi-point injection of Freund’s complete adjuvant and bovine type II collagen.The activity of rats was observed and the arthritis score of each group was calculated according to the joint symptom score standard;The pathological changes of rat joints were observed by HE staining;the expressions of ROS,Fuc T-V,CD3,neutrophil and Macrophages were detected by immunofluorescence;the changes of serum cytokines IL-17,IL-6,TNF-α and TGF-βwere detected by ELISA.The protein expression levels of TLR2,TLR4,My D88,NF-κB,Fuc T-Ⅴ,E-selectin and P-selectin were detected by Western blot;RT-PCR was used to detect the expression of TLR2,TLR4,My D88 and NF-κB.2.The expression levels of GSDMD-N,GSDMD-C,pro-Caspase-1,pro-IL-1β and NLRP3 were detected by Western blot;The expression levels of ASC,Fuc T-Ⅴ and NLRP3 were detected by immunofluorescence;The m RNA expressions of NLRP3,Caspase-1,IL-1β and IL-18 were detected by RT-PCR.3.Human rheumatoid arthritis fibroblast like synoviocytes were divided into four groups: Control group,LPS group,Ssnb group and Ssnb + NLRP3 agonists group.The expression of IL-17,IL-6,SOD,MDA,Fuc T-V and TGF-β in cell supernatant was detected by ELISA;The expression of ASC spots and ROS were detected by immunofluorescence;The levels of E-selectin and P-selectin were detected by western blot.Results: 1.compared with RA group,the score of arthritis index in Ssnb group was significantly lower(P<0.05);The results of histopathology showed that the proliferation of synovial cells was not obvious and the infiltration of inflammatory cells in the stroma was decreased in the Ssnb group;The release of ROS in the joints of rats decreased significantly(P<0.05);The levels of IL-17,IL-6 and TNF-α in serum decreased significantly(P<0.05),and the expression of TGF-β increased significantly(P<0.05);The expression of TLR2,TLR4,My D88,NF-κB protein and gene decreased significantly(P<0.05);In addition,the co expression of Fuc T-Ⅴ and CD3,neutrophil and Macrophages was significantly decreased in the Ssnb group;The expression levels of Fuc T-Ⅴ,E-selectin and P-selectin were significantly decreased in rats(P<0.05).2.Compared with RA group,the contents of GSDMD-N and GSDMD-C in Ssnb group decreased significantly(P<0.05);In the Ssnb group,ASC aggregated around the nucleus and formed large protein spots,which were significantly reduced;The contents of pro-Caspase-1,pro-IL-1β and NLRP3 decreased significantly(P<0.05);Meanwhile,the expression levels of NLRP3,Caspase-1,IL-1β and IL-18 were significantly decreased(P<0.05);In addition,the levels of IL-1β and IL-18 in serum of Ssnb group decreased significantly(P<0.05);The co expression of Fuc T-Ⅴ and NLRP3 decreased significantly after treatment with Ssnb(P<0.05).3.1)The expression of IL-17 and IL-6 in Ssnb group was significantly lower than that in LPS group,while the expression of TGF-β was significantly increased(P<0.05).The expression of IL-17 and IL-6 in Ssnb + NLRP3 agonist group was significantly higher than that in Ssnb group,while the expression of TGF-β was significantly lower than that in Ssnb group(P<0.05);2)A large amount of ROS was released in LPS group,and Ssn B could effectively inhibit the expression of ROS,but after administration of NLRP3 agonist,the inhibitory effect of Ssn B on ROS was down-regulated and the expression of ROS was increased;3)Compared with LPS group,the content of SOD in Ssn B group increased and the content of MDA decreased.Compared with Ssn B group,the content of SOD in Ssn B+NLRP3 activator group decreased and the content of MDA increased(P < 0.05);4)In the Ssnb group,ASC spots around the nucleus were significantly reduced.In the group of Ssnb + NLRP3 agonist treated with NLRP3 agonist,a large number of ASC aggregation spots were observed around the nucleus;5)Compared with LPS group,the content of Fuc T-Ⅴ in Ssn B group decreased,and the expression of Fuc T-Ⅴ increased in Ssn B+NLRP3 activator group compared with Ssn B group;6)Compared with LPS group,the levels of E-selectin and P-selectin in Ssn B group decreased significantly,but the expression of E-selectin and P-selectin in Ssn B+NLRP3 activator group treated with NLRP3 agonist was significantly higher than that in Ssn B group.Conclusion: Ssnb can alleviate joint injury and inhibit the inflammatory response of RA model rats.The mechanism may be that Ssn B down-regulates the activity of Fuc T-Ⅴ by down-regulating NLRP3 inflammatory body pathway,and then inhibits the occurrence and development of inflammatory response.