| ObjectiveA randomized controlled trial was used to observe the clinical efficacy of Bushen Qiangjin capsule in the treatment of knee osteoarthritis(KOA),and its effect on synovial lesions of KOA was preliminarily observed by musculoskeletal ultrasound;and then animal experiments were conducted to verify its effect on KOA synovitis and fibrosis and explore its mechanism of effect,and further elucidate its molecular mechanism of regulating fibroblast-like synovial cell pyroptosis and improving KOA synovitis and fibrosis through cell experiments.Methods1.Clinical research sectionSixty-four patients with KOA were randomly divided into experimental group and control group according to the proportion of 1:1.On the basis of basic treatment,Bushen Qiangjin capsule and celecoxib capsule were given orally for 4 weeks.The VAS score,WOMAC index,the quantitative score of traditional Chinese medicine(TCM)syndrome and the evaluation of syndrome curative effect were compared between the two groups before and after treatment.The changes of suprapatellar bursa effusion depth and synovium thickness were observed by musculoskeletal ultrasound before and after treatment.Additionally,the adverse events in the two groups were recorded detailedly.2.Animal experiment sectionAccording to body weight distribution,forty-eight male SD rats were randomly divided into blank group(Control),model group(Model),Bushen Qiangjin capsule group(BSQJ)and celecoxib capsule group(Celecoxib)with twelve rats in each group.The animal model of KOA was established by anterior cruciate ligament transection(ACLT).Four weeks after the establishment of the model,BSQJ group and Celecoxib group were given corresponding doses of drugs for intragastric intervention.The general conditions of rats in each group were observed after intervention,such as fur color,mental state,reaction sensitivity,diet,activity,walking gait and body mass,the transverse diameter of right knee joint of rats in each group was measured once a week,the behavior of rats in each group was evaluated by Lequesne MG score,and the histopathological changes of synovium of rats in each group were observed by HE and Sirius red staining,the expressions of NLRP3,Caspase-1,GSDMD,TGF-β and COL1A1 in synovium of rats in each group were observed by immunohistochemical staining,and the contents of interleukin-1β(IL-1β)and interleukin-18(IL-18)in serum and knee joint lavage fluid of rats in each group were detected by ELISA.3.Cell experiment sectionThe rat fibroblast-like synoviocytes(FLS)were randomly divided into four groups:blank group(Control),model group(Model),Bushen Qiangjin capsule group(BSQJ)and positive control group(MCC950).BSQJ group and MCC950 group were pretreated with corresponding drugs for 24 hours(normal culture in Control group and Model group),and then the model of pyroptosis was induced by lipopolysaccharide(LPS)combined with adenosine triphosphate(ATP)stimulation(except Control group).The levels of IL-1β and IL-18 in the culture supernatant of cells in each group were detected by ELISA,and the protein expressions of pyroptosis-related molecules NLRP3,Caspase-1,GSDMD and fibrosis-related molecules TGF-β,COL1A1,PLOD2 and TIMP1 of cells in each group were detected by immunofluorescence staining and WB.The mRNA expression of pyroptosisrelated molecules NLRP3,Caspase-1,GSDMD and fibrosis-related molecules TGF-β,COLA1,PLOD2 and TIMP1 of cells in each group were detected by RT-qPCR.Results1.Clinical research(1)Primary outcomes①VAS score:compared between groups,there was no statistical difference in VAS scores and differences between the two groups before and after treatment(P>0.05);compared within groups,VAS scores were significantly lower in both groups after treatment than before treatment,with statistically significant differences(P<0.01).②WOMAC index:compared between groups,there was no statistical difference in WOMAC total index,pain index,stiffness index,physical function index and differences between the two groups before treatment(P>0.05),and there was a statistical difference in WOMAC total index,stiffness and physical function index and differences between the two groups after treatment(P<0.05),and the test group was better than the control group in terms of WOMAC total index,stiffness and physical function index improvement,and there was no statistical difference in WOMAC pain index and difference between the two groups after treatment(P>0.05);compared within groups,WOMAC total index,pain index,stiffness index and physical function index were lower in both groups after treatment than before treatment,and there was a statistical difference(P<0.05).③Depth of effusion in suprapatellar bursa:compared between groups,there was no statistical difference in the depth of suprapatellar bursa and difference between the two groups before and after treatment(P>0.05);compared within groups,the suprapatellar bursa fluid depth was significantly lower in both groups after treatment than before treatment,and there was a statistically significant difference(P<0.01).④Synovial thickness:compared between groups,there was no statistical difference in synovial thickness and difference between the two groups before and after treatment(P>0.05);compared within groups,synovial thickness was significantly lower in both groups after treatment than before treatment,and there was a statistically significant difference(P<0.01).(2)Secondary outcomes①TCM syndrome score:in comparison between groups,there was no statistical difference between the two groups in the total TCM symptom score,TCM symptom element joint pain score,joint swelling score,joint fever score,joint chill score,waist and knee soreness and fatigue score before treatment(P>0.05),and there was a statistical difference between the two groups in the total TCM symptom score,TCM symptom element joint chill score,low back and knee weakness score and tiredness score after treatment(P<0.05),and the test group was better than the control group in terms of the improvement of total TCM symptom scores,TCM symptom element joint chilliness scores,waist and knee soreness and fatigue scores;compared within groups,the total TCM symptom scores,TCM symptom element joint pain scores,joint swelling scores,joint fever scores,joint chilliness scores,waist and knee soreness and fatigue scores in both groups were significantly lower than those before treatment.There was a statistically significant difference(P<0.01).②Therapeutic effect of TCM syndrome:there was a statistically significant difference in the TCM symptoms efficacy between the two groups after treatment(P<0.01),the effective rate of the test group was 86.67%and the effective rate of the control group was 60.00%,the effective rate of the test group was higher than that of the control group,and there was a statistical difference(P<0.05).③Adverse event occurrence:the incidence of adverse events during treatment was lower in both groups,1/30 in the test group and 1/30 in the control group.2.Animal experiment(1)Lequesne MG score:there were statistically significant differences in the Lequesne MG scores and differences between groups at 4 weeks after modeling and 4 weeks after intervention in each group(P<0.01),and the scores in the BSQJ group,Celecoxib group and Model group were significantly higher than those in the Control group at 4 weeks after modeling(P<0.01);after 4 weeks of intervention,the scores in the BSQJ group,Celecoxib group and Model group were significantly higher than those in the Control group(P<0.01).group and the Model group were significantly greater than the Control group(P<0.01),and the scores of the BSQJ group and the Celecoxib group were significantly lower than the Model group(P<0.01).Meanwhile,the difference of scores in both BSQJ and Celecoxib groups was significantly greater than that in Control and Model groups(P<0.01).(2)Transverse diameter of the right knee joint in rats:compared between groups,there was a statistically significant difference in the transverse diameter of the right knee joint in each group 4 weeks after modeling(P<0.01),and the transverse diameter of the right knee joint in the BSQJ.Celecoxib and Model groups was significantly larger than that in the Control group(P<0.01);there was a statistically significant difference in the transverse diameter of the right knee joint in each group 1 week after intervention(P<0.01),and the transverse diameter of the right knee joint in the BSQJ,Celecoxib and Model groups was significantly larger than that in the Control group(P<0.01).The transverse diameter of the right knee in the BSQJ,Celecoxib and Model groups was significantly larger than that in the Control group(P<0.01);the transverse diameter of the right knee in the Celecoxib and Model groups was larger than that in the Control group after 2 weeks of intervention(P<0.01);the transverse diameter of the right knee in the BSQJ,Celecoxib and Model groups was significantly larger than that in the Control group after 3 weeks of intervention(P<0.05).After 3 weeks of intervention,there was a statistically significant difference in the transverse diameter of the right knee joint in each group(P<0.01),and the transverse diameter of the right knee joint in the Model group was significantly larger than that in the Control group(P<0.01),and that in the Celecoxib group was smaller than that in the Model group(P<0.05);after 4 weeks of intervention,there was a statistically significant difference in the transverse diameter of the right knee joint in each group(P<0.01),and the transverse diameter of the right knee joint in the Model group was significantly larger than that in the Control group(P<0.01).The transverse diameter of the right knee joint in the BSQJ and Celecoxib groups was smaller than that in the Model group(P<0.05).In the intra-group comparison,there was a significant difference in the transverse diameter of the knee joint in all groups measured multiple times(P<0.01).Compared with 4 weeks after modeling,there were statistically significant differences in the blank group after 2,3 and 4 weeks of intervention(P<0.05),and compared with 1 week after intervention,there were statistically significant differences after 3 and 4 weeks of intervention(P<0.05).There were statistical differences in the model group after 2,3 and 4 weeks of intervention compared with 4 weeks after modeling(P<0.05);after 3 and 4 weeks of intervention compared with 1 week of intervention(P<0.05);and after 4 weeks of intervention compared with 2 weeks of intervention(P<0.05).There were statistically significant differences in the BSQJ group after 3 and 4 weeks of intervention compared to 4 weeks after modeling(P<0.01);and after 4 weeks of intervention compared to 1 week of intervention(P<0.01).There were statistically significant differences in the Celecoxib group after 2,3 and 4 weeks of intervention compared with 4 weeks after modeling(P<0.05);and after 3 and 4 weeks of intervention compared with 1 week of intervention(P<0.05).(3)Pathomorphological scores of synovial tissue:compared with the Control group,the histomorphological scores of synovial tissue were significantly higher in the BSQJ,Celecoxib and Model groups(P<0.01).The scores were significantly lower in the BSQJ and Celecoxib groups compared with the Model group(P<0.01).(4)Immunohistochemical staining results of synovial tissue:NLRP3,Caspase-1,GSDMD,TGF-β and COL1A1 positive cell rates were increased in the BSQJ group,Celecoxib group and Model group compared with the Control group(P<0.05).The rates of NLRP3,Caspase-1,GSDMD,TGF-β and COL1A1 positive cells were decreased in the BSQJ and Celecoxib groups compared with the Model group(P<0.05).(5)Serum and knee lavage fluid IL-1β and IL-18 levels in rats:compared with the Control group,serum and knee lavage fluid IL-1β and IL-18 levels were significantly higher in the BSQJ,Celecoxib and Model groups(P<0.01).Serum and knee lavage fluid IL-1 β and IL-18 levels were significantly lower in the BSQJ and Celecoxib groups compared with the Model group(P<0.01).Serum and knee lavage fluid IL-1β and IL-18 levels were significantly higher in the BSQJ group compared with the Celecoxib group(P<0.01).3.Cell experiment(1)Levels of IL-1β and IL-18 in cell culture supernatants:IL-1β and IL-18 expressions were significantly higher in the BSQJ,MCC950 and Model groups compared with the Control group(P<0.01).The expression of IL-1β and IL-18 was significantly lower in the BSQJ and MCC950 groups compared with the Model group(P<0.01).The expression of IL-1β and IL-18 were significantly higher in the BSQJ group compared with the MCC950 group(P<0.01).(2)Effect of the medicated serum of Bushen Qiangjin capsule on mRNA expression of FLS-related genes①Results of pyroptosis-related gene mRNA expression:compared with the Control group,the relative expression of NLRP3 mRNA was significantly higher in both Model and BSQJ groups(P<0.01),the relative expression of Caspase-1 was significantly higher in the Model,BSQJ and MCC950 groups(P<0.01),the relative expression of GSDMD mRNA was significantly higher in the Model and BSQJ The relative expression of GSDMD mRNA was significantly increased in both Model and BSQJ groups(P<0.01),and the relative expression of GSDMD mRNA was also increased in the MCC950 group(P<0.05).The relative expressions of NLRP3,Caspase-1 and GSDMD mRNA were significantly lower in both BSQJ and MCC950 groups compared with the Model group(P<0.01).Compared with the MCC950 group,the relative expression of Caspase-1 mRNA was significantly higher in the BSQJ group(P<0.01),and the relative expression of NLRP3 and GSDMD mRNA was increased in the BSQJ group(P<0.05).②Results of fibrosis-related gene mRNA expression:compared with the Control group,the relative expression of TGF-β mRNA was significantly higher in both Model and BSQJ groups(P<0.01),the relative expression of TGF-β mRNA was also increased in the MCC950 group(P<0.05),the relative expression of COL1A1 mRNA was significantly increased in the Model and BSQJ groups(P<0.01),and the relative expression of COL1A1 mRNA was also increased in the MCC950 group(P<0.05),the relative expression of PLOD2 mRNA was significantly increased in the Model group,BSQJ group and MCC950 group(P<0.01),the relative expression of TIMP1 mRNA was significantly increased in the Model group and BSQJ group(P<0.01),and the relative expression of TIMP1 mRNA was also increased in MCC950 group(P<0.05).Compared with the Model group,the relative expressions of TGF-β,COL1A1,PLOD2 and TIMP1 mRNA in the BSQJ group and MCC950 group were significantly decreased(P<0.01).The relative expression of PLOD2 mRNA in the BSQJ group was significantly higher than that in MCC950 group(P<0.01).(3)Effect of the medicated serum of Bushen Qiangjin capsule on FLS-related protein expression(immunofluorescence method)①Results of pyroptosis-related protein expression:compared with the Control group,the relative fluorescence expressions of NLRP3,Caspase-1 and GSDMD in the Model group,BSQJ group and MCC950 group were significantly increased(P<0.01).Compared with the Model group,the relative fluorescence expressions of NLRP3,Caspase-1 and GSDMD in the BSQJ group and MCC950 group were significantly decreased(P<0.01).The relative fluorescence expressions of NLRP3,Caspase-1 and GSDMD in the BSQJ group were significantly higher than those in the MCC950 group(P<0.01).②Results of fibrosis-related protein expression:compared with the Control group,the relative fluorescence expressions of TGF-β in Model group was significantly increased(P<0.01),and the relative fluorescence expressions of COL1A1 and PLOD2 in the Model group,BSQJ group and MCC950 group were significantly increased(P<0.01),and the relative fluorescence expression of T1MP1 in the Model group was also significantly increased(P<0.01).Compared with the Model group,the relative fluorescence expressions of TGF-β,COL1A1,PLOD2 and TIMP1 in the BSQJ group and MCC950 group were significantly decreased(P<0.01).Compared with the MCC950 group,the relative fluorescence expression of COL1A1 and PLOD2 in the BSQJ group were both increased(P<0.05).(4)Effect of the medicated serum of Bushen Qiangjin capsule on FLS-related protein expression(WB method)①Results of pyroptosis-related protein expression:compared with the Control group,the relative expression of NLRP3,Caspase-1 and GSDMD proteins were significantly higher in the Model and BSQJ groups(P<0.01),and the relative expression of NLRP3 and Caspase1 protein was also increased in the MCC950 group(P<0.05).The relative expressions of NLRP3,Caspase-1 and GSDMD protein were significantly lower in both BSQJ and MCC950 groups compared with the Model group(P<0.01).The relative expression of Caspase-1 protein was increased in the BSQJ group compared with the MCC950 group(P<0.05).②Results of fibrosis-related protein expression:compared with the Control group,the relative expressions of TGF-β and TIMP1 protein in the Model group were significantly increased(P<0.01),the relative expressions of COL1A1 and PLOD2 protein in the Model group and BSQJ group were significantly increased(P<0.01),and the relative expression of PLOD2 protein in the MCC950 group was also increased(P<0.05).The relative expressions of TGF-β,COL1A1,PLOD2 and TIMP1 protein in the BSQJ group and MCC950 group were significantly lower than those in the Model group(P<0.01).The relative expressions of COL1A1 protein in the BSQJ group were higher than those in the MCC950 group(P<0.05).Conclusions1.Bushen Qiangjin capsule could effectively improve the joint pain,stiffness,function and the scores of TCM syndrome in patients with KOA,especially in improving joint chills,waist and knee weakness and fatigue,and the effective rate of TCM syndrome is higher than that of the control group.Additionally,it could significantly reduce the depth of suprapatellar bursa effusion and synovium thickness,and the safety is execllent.2.Bushen Qiangjin capsule could significantly improve synovitis and synovial fibrosis in KOA rats,and its mechanism may be related to inhibiting the activation of NLRP3 inflammasome in synovium,and then regulating pyroptosis and inflammatory cascademediated by NLRP3 inflammasome.3.The medicated serum of Bushen Qiangjin capsule could reduce the the inflammation and fibrosis by regulating FLS pyroptosis induced by LPS and ATP,and its mechanism may be related to the inhibition of NLRP3 inflammasome activation. |
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