| Objective:1.To observe and evaluate the clinical effect of Sishenjian decoction on knee osteoarthritis provide reference for the clinical treatment of KOA.2.To observe the intrinsic relationship between macrophage polarization and macrophage pyroptosis in the course of KOA.3.To explore the therapeutic mechanism of Sishenjian in treating KOA based on the synovial macrophage polarization and macrophage pyroptosis,which willl provide the oretical basis for TCM prevention and treatment of KOA.Methods:1.Through clinical experiment,the patients were divided into Sishenjian group and Etoricoxib group.The knee pain was assessed by WOMAC score before medication at week 4 and 8 after medication.Blood samples were collected and serum systemic inflammatory factors(IL-6,IL-18,IL-1β),synovial fibrosis indexes(TIMP1,VEGF,TGF-β),and cartilage degeneration indexes(MMP-1,MMP-13)were detected by ELISA.2.The rats were divided into sham operation group and KOA group.KOA model was established by DMM method.After the treatment,the cartilage was stained with Safranine solid green staining,the synovial tissue was stained with HE,the damaged cartilage tissue was scored by OARSI score,and synovial inflammation was scored by Krenn score.The polarization of macrophages was observed by immunofluorescence double staining of synovial tissue.WB was used to detect the expression levels of pyroptosis markers such as NLRP3,GSDMD,ASC and IL-1β.The protein expression levels of synovial fibrosis markers such as TIMP1,VEGF and TGF-β were detected by WB.The expression of pain related markers in the serum such as SP and CGRP were detected by ELISA.3.The rats were divided into sham operation group,KOA group,low-concentration Chinese herbal medicine group,medium-concentration Chinese herbal medicine group and high-concentration Chinese herbal medicine group.KOA model was established by DMM method.After the treatment,WB was used to detect the expression levels of pyroptosis markers such as NLRP3,GSDMD,ASC and IL-1β in synovial tissue,and the protein expression levels of synovial fibrosis markers such as TIMP1,VEGF and TGF-β were detected by WB.The expression of pain related markers in the serum such as SP and CGRP were detected by ELISA.4.The rats were divided into sham operation group,KOA group,inhibitor group and Sishenjian group.KOA model was established by DMM method.The KOA model was established by DMM.NS8593 was used to inhibite the polarization of macrophages.The polarization of synovial macrophages was observed by immunofluorescence double staining.The cartilage was stained with Safranine solid green staining,the synovial tissue was stained with HE,the damaged cartilage tissue was scored by OARSI score,and synovial inflammation was scored by Krenn score.WB was used to detect the expression levels of pyroptosis markers such as NLRP3,GSDMD,ASC and IL-1β in synovial tissue,and the protein expression levels of synovial fibrosis markers such as TIMP1,VEGF and TGF-β were detected by WB.The expression of pain related markers in the serum such as SP and CGRP were detected by ELISA.The mechanical pain sensitivity of lower limbs was detected.Results:1.At the 4th week after medication,the expression levels of inflammatory indicators in Sishenjian group and Etoricoxib group such as IL-18,IL-6 and IL-1β,the synovial fibrosis indicators such as TIMP1,VEGF and TGF-β,and the cartilage degeneration indicators such as MMP-1 and MMP-13 and WOMAC score were obviously lower than those before medication(P<0.05).All inflammatory indexes in The Sishenjian group were lower than those in the Etoricoxib group,but the difference was not obvious(P>0.05).The WOMAC score in the Sishenjian group was not significantly different from that in the Etoricoxib group(P>0.05).The total effective rate in the treatment of KOA was 92.5%in the Sishenjian group and 82.5%in the Etoricoxib group.The total effective rate in the treatment of KOA in the Sishenjian group was significantly higher than that in the Etoricoxib group(P<0.05).At week 8,The expression levels of inflammatory indicators in Sishenjian and Etoricoxib group such as IL-6,IL-18,IL-1β,the synovial fibrosis indicators such as TIMP1,VEGF,TGF-β,the cartilage degeneration indicators such as MMP-1 and MMP-13 were obviously lower than those before medication(P<0.05),but there was no obvious difference compared with the fourth week(P>0.05).The WOMAC score of the Etoricoxib group was lower than that before treatment,but significantly higher than the follow-up results of the Sishen decoction group in the 8th week(P<0.05),and lower than that of the 4th week in this group,but the difference was not statistically significant(P>0.05).2.Compared with sham operation group,OARSI score of cartilage tissue and Krenn score of synovial tissue in KOA group were obviously increased(P<0.05),the expression of CD68 and iNOS in synovial tissue were obviously increased(P<0.05),the expression of CD206 in synovial tissue were not obviously regulated(P>0.05).The pyroptosis protein expression levels of NLRP3,GSDMD,ASC and IL-1β were obviously increased(P<0.05).The expression levels of synovial fibrosis indexes such as TIMP1,VEGF and TGF-β were obviously increased(P<0.05).The pain related protein expression levels of SP and CGRP in the serum were obviously increased(P<0.05).3.Compared with sham operation group,the pyroptosis protein expression levels of NLRP3,GSDMD,ASC and IL-1β in KOA group were obviously increased(P<0.05).The expression levels of synovial fibrosis indexes such as TIMP1,VEGF and TGF-β were obviously increased(P<0.05).The pain related protein expression levels of SP and CGRP in the serum were obviously increased(P<0.05).Compared with the KOA group,all indexes above in the low concentration group were obviously decreased(P<0.05).Compared with the low concentration group,all indexes above in the medium concentration group were obviously decreased(P<0.05).Compared with the medium concentration group,all indexes above in the high concentration group were obviously decreased(P<0.05).4.Compared with sham operation group,the OARSI score of cartilage tissue and Krenn score of synovial tissue in KOA group were obviously increased(P<0.05),the expression of CD68 and iNOS in synovial tissue in KOA group were obviously increased(P<0.05),the expression of CD206 in synovial tissue in KOA group were not obviously regulated(P>0.05),the pyroptosis protein expression levels of NLRP3,GSDMD,ASC and IL-1β in KOA group were obviously increased(P<0.05),the expression levels of synovial fibrosis indexes such as TIMP1,VEGF and TGF-β in KOA group were obviously increased(P<0.05),the pain related protein expression levels of SP and CGRP in the serum in KOA group were obviously increased(P<0.05).Compared with KOA group,the OARSI score of cartilage tissue and Krenn score of synovial tissue in the inhibitor and Sishen groups were obviously increased(P<0.05),the expression of CD68 and iNOS in synovial tissue in the inhibitor and Sishen groups were obviously increased(P<0.05),the expression of CD206 in synovial tissue in the inhibitor and Sishen groups were obviously decreased(P<0.05),the pyroptosis protein expression levels of NLRP3,GSDMD,ASC and IL-1β in the inhibitor and Sishen groups were obviously increased(P<0.05),the expression levels of synovial fibrosis indexes such as TIMP1,VEGF and TGF-β in the inhibitor and Sishen groups were obviously increased(P<0.05),the pain related protein expression levels of SP and CGRP in the serum in the inhibitor and Sishen groups were obviously increased(P<0.05).On the 7th day after medication,the foot threshold of the Sishenjian group and inhibitor group were significantly higher than that of the KOA group(P<0.05),but lower than that of the sham operation group(P<0.05),and there was no significant difference between the Sishenjian group and the inhibitor group(P>0.05).On the 14st and 21st day after medication,the foot threshold of the Sishen group and the inhibitor group were significantly higher than that of the KOA group(P<0.05),and there was no significant difference in the foot threshold between the Sishen group,the inhibitor group and the sham operation group(P>0.05).Conclusion:1.Sishenjian decoction has a significant effect on improving synovial inflammation and relieving joint pain in KOA patients.2.M1-type polarization of macrophages in the synovial tissue of KOA can significantly activate the NLRP3/ASC signaling pathway,induce pyroptosis of macrophages and aggravate synovial inflammation in KOA.3.Sishenjian can inhibit the M1-type polarization of macrophages,inhibit the pyroptosis of macrophages and the downstream inflammatory response,which will significantly improve the synovial inflammation and relieve the joint pain of KOA. |
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