Study On The Intervention Effect Of "Xibining" On Fibrosis Of Knee Osteoarthritis

Objective:To explore the mechanism of synovial tissue hypoxia in knee osteoarthritis to aggravate synovial fibrosis via fibroblast-like synoviocytes pyroptosis,and to explore the possible mechanism of Xibining in the treatment of knee osteoarthritis.M ethod:1.The rat model of KOA was established,and the synovial tissue of rats was observed by immunofluoresce nce,whic h was labeled with pimonidazo le,an anoxic marker in syno vial tiss ue.Both mRNA and protein expression of HIF-la in synovial tissue were detected.After drugs were used to inprove hypoxia in synovial tissue of KOA rats,the transverse diameter of knee joint and anatomical observation were performed to estimate the degree of synovial fibrosis in rats.Pathological changes of synovial fibrosis were observed by HE and Sirius red staining.Both mRNA and protein expression of TGF-?,COL1A1,PLOD2 and TIMP1 were detected.2.Isolated primary rat KOA-FLS,LPS/ATP was used to simulate KOA inflammatory environment and induced pyroptosis.Transfecting with siRNA to silence HIF-la,both mRNA and protein level of Caspase-1,ASC,NLRP3,GSDMD were meansured.They acted a core role in the activation of pyroptosis.The content of IL-1? and Il-18 in supernatant were detected by elisa,and the apoptosis rate was detected by flow cytometry.3.KOA anoxic cell model was constructed and confirmed by pimonidazole staining.siRNA transfection silenced GSDMD and detected the mRNA and protein expression of TGF-?,COL1A1,PLOD2 and TIMP1 in FLS.4.The rat model of KOA was established and the hypoxia status of synovial tissue of knee joint was observed by pimonidazole staining after oral administration of Xibining.Both mRNA and protein expression of HIF-1? and Caspase-1,ASC,NLRP3,GSDMD in synovial tissue were meansured.The transve rse diamete r of knee joint and anatomical observatio n we re performed to estimate the degree of synovial fibrosis in rats.Pathological changes of synovial fibrosis were observed by HE and Sirius red staining.Both mRNA and protein expression of TGF-?,COLTA1,PLOD2 and TIMP1 were detected.Result:1.Pimonidazole staining showed that the synovial tissue of KOA rats was more hypoxic than that of normal rats.Digoxin(100 ug/kg),an inhibitor of HIF-la,could inhibit this situation,and the gene and protein expression of HIF-la in synovial tissue of KOA group was higher than that of Normal group,and the Digoxin group had inhibitory effect.The difference was statistically significant(p<0.05).The transverse diameter of knee joint and anatomical observation showed that the degree of synovial fibrosis in KOA rats was aggravated,and that in Digoxin group was improved.In HE staining,the synovial lining cells of Digoxin group were arranged neatly,the connective tissue was loose,and the inflammatory cells were less infiltrated.Under Sirius red staining,collagen deposition increased in KOA group,but decreased in Digoxin group.The mRNA expression of TGF-?,COL1A1,PLOD2 and TIMP1 were detected by PCR.Compared with the Normal group,the expression of TGF-?,COL1A1,PLOD2 and TIMP1 in the KOA gro up was significantly increased(p<0.01).Digoxin attenuated the ip-regulation of these genes(p<0.01).WB to detect the protein expression of these above-mentioned fibrosis marker found the same trend.2.In FLS of KOA rats,both mRNA and protein expression of Caspase-1,ASC,NLRP3,GSDMD increased in LPS/ATP group(p<0.05 vs Normal group),but decreased in HIF-la siRNA group(p<0.05 vs LPS/ArTP group).The content of IL-1? and IL-18 in culture supernatant was determined by ELISA.The content of IL-1? and IL-18 in HIF-la siRNA group was tower than that in LPS/ATP group(p<0.05)and higher than that in Normal group(p<0.05).Flow cytometry showed that the apoptosis rate of HIF-la siRNA group was significantly higher than that of Normal gro up(p<0.01)and significantly lower than that of LPS/ATP group(P<0.01).3.After exposed to hypoxia(1%O2)for 24 hours,pimonidazole staining of the Vehicle group and the GSDMD siRNA group showed a similar anoxic state.However,the expression of both mRNA and protein of TGF-?,COL1A1,PLOD2 and TIMP1 were down-regulated in GSDMD siRNA group compared with the Vehicle,and the difference was statistically significant(p<0.05).4.The synovial tissue hypoxia in Xibining group was less than that in the KOA group.Both mRNA and protein expression of HIF-la and Caspase-1,NLRP3,GSDMD in Xibining group was tower than that in KOA group(p<0.05).The anatomical observation showed that the degree of synovial fibrosis in the Xibining group was alleviated,and the transverse diameter of the knee joint in the Xibining group was tower than that in the KOA group on Day 42 and Day 56(p<0.05).He staining and Sirius red staining showed that the synovial lining cells were arranged neatly,the connective tissues were loose,the inflammatory cells were less infiltrated,and the amount of collagen deposition was less in Xibining group.Both mRNA and protein expression of TGF-?,COL1A1,and TIMP1 were down-regulated in Xibining group corpared with the KOA group(p<0.05).Conclusion:1.The synovial tissue was in anoxic state and caused the increase of HIF-1? in the synovial tissue.2.In KOA,increased HIF-1? induced FLS pyroptosis.3.In KOA,FLS pyroptosis may aggravate the progression of synovial fibrosis.4.The oral administration of Xibining impro ved the hypoxic state of synovial tissue of the knee joint,decreased the expression of HIF-1?,and may alleviate synovial fibrosis by inhibiting the cell pyroptosis of the synovium.