| ObjectiveThe clinical efficacy and safety of Knee Osteoarthritis(KOA)treated with Bushen Qiangjin capsule were observed through clinical trials to provide a clinical basis for the application of kidney tonic and blood-activating drugs to prevent and treat KOA and to investigate further the effect of Bushen Qiangjin capsule on the cycle and apoptosis of chondrocytes through animal experiments and cell experiments to explore the potential mechanism of action of Bushen Qiangjin capsule in treating KOA.Methods1.Clinical researchA total of 70 patients with KOA who met the diagnostic criteria were randomly divided into the test group(n=35)and control group(n=35).The control group was treated with acelecoxib capsule,and the test group was treated with a Bushen Qiangjin capsule on the basisof the control group.The visual analog score(VAS),Western Ontario and McMaster Universities Osteoarthritis Index(WOMAC),range of motion of knee joint,depth of suprapatellar bursa effusion,the quantitative score of TCM syndrome,and curative effect evaluation of TCM syndrome were recorded before and after treatment.2.Animal experimentsThirty SD rats were randomly divided into the blank group(Blank group),model group(Model group),and Bushen Qiangjin capsule group(BSQJ group),with ten 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,the BSQJ group was given Bushen Qiangjin capsule,while the Blank group and Model group were given equal amounts of saline.After intragastric administration,the abdominal aortic blood and knee cartilage tissue of SD rats in each group were collected for related detection.The pathological sections of the knee joint of SD rats were made,and HE staining and fuchsin-fast green staining was performed.The structural changes of knee cartilage tissue of rats in each group were evaluated by Mankin score,and the apoptosis of chondrocytes in cartilage tissue was evaluated by TUNEL apoptosis staining.The contents of pro-inflammatory factors such as IL-1β IL-6,and TNF-α in the serum of rats were detected by Elisa.The expressions of apoptosis-related proteins Bax,Bcl-2,Caspase3,and Caspase9 and cartilage synthesis and metabolism proteins Collagen Ⅱ and Aggrecan in cartilage tissues of rats in each group were detected by immunohistochemical staining.3.Cell experimentsBSQJ drug-containing serum and blank serum were prepared,and the culture system of SD rat knee chondrocytes in vitro was established.CCK8 screened the optimal intervention concentration of drug-containing serum of the Bushen Qiangjin capsule.After 48 hours of culture,the third-generation chondrocytes were divided into the blank group(Blank group),model group(Model group),control group(Control group),and Bushen Qiangjin capsule group(BSQJ group).After the intervention,the cell cycle and apoptosis of each group were detected by flow cytometry.The relative expressions of p53,p21,CyclinDl,CDK4,and apoptosis-related genes Bax,Bcl-2,Caspase3,and Caspase9 were detected by RT-qPCR.The relative expressions of p53,p21,CyclinD1,CDK4,and apoptosis-related proteins Bax,Bcl2,Caspase3,and Caspase9 were detected by Western-blot.Immunofluorescence staining observed the relative expressions of apoptosis-related proteins Bax,Bcl-2,Caspase3,and Caspase9 in each group.Results1.Clinical research(1)VAS score:There was no significant difference in VAS score between the test group and the control group before treatment(P>0.05).After treatment,there was a significant difference in VAS scores between the test group and the control group(P<0.05),and there was also a significant difference between the two groups before and after treatment(P<0.01).Compared within the group,the VAS scores of the test group and the control group after treatment were significantly different from those before treatment(P<0.05 or P<0.01).(2)WOMAC scores:There was no significant difference in the total WOMAC score,WOMAC(pain)score,WOMAC(stiffness)score,and WOMAC(function)score between the test group and the control group before treatment(P>0.05).The differences between the test group and the control group after treatment were significant in terms of total WOMAC score,WOMAC(pain)score,WOMAC(stiffness)score,and WOMAC(function)score(P<0.05),and there was also a significant difference between the groups before and after treatment(P<0.01).Within-group comparison,the differences were significant in the total WOMAC score,WOMAC(pain)score,WOMAC(stiffness)score,and WOMAC(function)score in the test and control groups after treatment compared with those before treatment(P<0.01).(3)Range of motion of knee joint:There was no significant difference in the range of motion of the knee joint between the test group and the control group before treatment(P>0.05).After treatment,there was a significant difference in the range of motion of knee joints between the test group and the control group(P<0.05),and there was also a significant difference between the two groups before and after treatment(P<0.01).For intra-group comparison,the differences in the range of motion of knee joints between the test and control groups after treatment were significant compared with those before treatment(P<0.01).(4)Depth of suprapatellar bursa fluid:There was no significant difference in the depth of suprapatellar bursa fluid between the test group and the control group before treatment(P>0.05).After treatment,there was a significant difference in the depth of the suprapatellar bursa between the test group and the control group(P<0.05),and there was also a significant difference between the two groups before and after treatment(P<0.01).For intra-group comparison,the difference in the depth of suprapatellar bursa effusion between the test and control groups after treatment was significant compared with that before treatment(P<0.01).(5)Quantification scores of TCM symptoms:There was no significant difference in the quantitative scores of TCM symptoms between the test group and the control group before treatment(P>0.05).After treatment,there was a significant difference between the test group and the control group in the quantitative scores of TCM symptoms(P<0.05),and there was also a significant difference between the groups before and after treatment(P<0.01).In the intra-group comparison,the differences in the quantitative scores of TCM symptoms between the test and control groups after treatment were significantly compared with those before(P<0.01).(6)Therapeutic effect of TCM syndrome:After four weeks of treatment,the total effective rate of the test group and the control group was 87.50%and 62.50%,respectively,and there was a significant difference between the test group and the control group(P<0.05).(7)Adverse events:Adverse events during the treatment were low,with an incidence of 1/35 in both the test and control groups.2.Animal experiments(1)Mankin score:Mankin score in the Model group and BSQJ group was significantly higher than that in the Blank group(P<0.05 or P<0.01),and that in the BSQJ group was significantly lower than that in the Model group(P<0.05).(2)TUNEL apoptosis staining:The TUNEL positive cell rate in the Model group and the BSQJ group was significantly higher than that in the Blank group(P<0.01),while that in the BSQJ group was significantly lower than that in the Model group(P<0.01).(3)ELISA:The contents of IL-1β,IL-6,and TNF-α in the Model group and BSQJ group were significantly higher than those in the Blank group(P<0.01),and the contents of IL-1β,IL-6,and TNF-α in BSQJ group were significantly lower than those in Model group(P<0.01).(4)Immunohistochemical staining:The positive rate of CollagenⅡ,Aggrecan,and Bcl2 in knee cartilage in the Blank group was significantly higher than that in the Model group(P<0.05)and BSQJ group(P<0.01),and that in the BSQJ group was significantly higher than that in the Model group(P<0.05 or P<0.01).The positive cell rates of Caspase3,Caspase9,and Bax in knee cartilage in the Blank group were significantly lower than those in the Model group(P<0.05)and BSQJ group(P<0.05 or P<0.01),and those in the BSQJ group were significantly lower than those in the Model group(P<0.05 or P<0.01).3.Cell experiments(1)Flow cytometry:①Cell cycle:Compared with the Blank group,the proportion of the G0/G1 phase in the Model group,BSQJ group,and Control group increased significantly(P<0.01),while the proportion of the S phase decreased significantly(P<0.01).Compared with the Model group,the proportion of the G0/G1 phase in the BSQJ and Control groups decreased significantly(P<0.05),while the proportion of the S phase increased significantly(P<0.05).Compared with the Control group,the proportion of the G0/G1 phase in the BSQJ group was significantly lower(P<0.05),and the proportion of the S phase was significantly higher(P<0.05).Compared with the Blank group,the proportion of the G2/M phase in the Model group and Control group was significantly lower(P<0.01).Compared with the Model and Control groups,the proportion of the G2/M phase in the BSQJ group was significantly higher than in the BSQJ group(P<0.01).②Proliferation index:Compared with the Blank group,the proliferation index of the Model group,Control group,and BSQJ group decreased significantly(P<0.01).Compared with the Model group,the proliferation index of the Control and BSQJ groups was significantly higher(P<0.01).Compared with the Control group,the proliferation index of the BSQJ group was significantly higher(P<0.01).③Apoptosis:Compared with the Blank group,the apoptosis rate of chondrocytes in the Model group,BSQJ group,and Control group increased significantly(P<0.01).Compared with the Model group,the apoptosis rate of chondrocytes in the BSQJ and Control groups was significantly lower(P<0.01).Compared with the Control group,the apoptosis rate of chondrocytes in the BSQJ group was significantly lower(P<0.01).(2)RT-qPCR:①Cell cycle:Compared with the Blank group,the relative expression of p53 and p21 mRNA was significantly higher(P<0.05 or P<0.01),and the relative expression of CyclinD1 and CDK4 mRNA was significantly lower(P<0.01)in the Model,BSQJ,and Control groups.Compared with the Model group,the relative expression of p53 and p21 mRNA was significantly lower(P<0.05 or P<0.01),and the relative expression of CyclinD1 and CDK4 mRNA was significantly higher(P<0.01)in the BSQJ and Control groups.Compared with the Control group,the relative expression of p53 and p21 mRNA was significantly lower(P<0.05),and the relative expression of CyclinDl and CDK4 mRNA was significantly higher in the BSQJ group(P<0.05 or P<0.01).②Apoptosis:Compared with the Blank group,the relative expression of Caspase3,Caspase9,and Bax mRNA was significantly higher(P<0.05 or P<0.01),and the relative expression of Bcl-2 mRNA was significantly lower(P<0.05 or P<0.01)in the Model,BSQJ and Control groups.Compared with the Model group,the relative expression of Caspase3,Caspase9,and Bax mRNA was significantly lower(P<0.05 or P<0.01),and the relative expression of Bcl-2 mRNA was significantly higher in the BSQJ and Control groups(P<0.05 or P<0.01).Compared with the Control group,the relative expression of Caspase3,Caspase9,and Bax mRNA was significantly lower(P<0.05 or P<0.01),and the relative expression of Bcl-2 mRNA was significantly higher in the BSQJ group(P<0.05).(3)Western-blot:①Cell cycle:Compared with the Blank group,the relative expression of p53 and p21 proteins was significantly higher(P<0.01),and the relative expression of CyclinDl and CDK4 proteins was significantly lower(P<0.05 or P<0.01)in the Model,BSQJ,and Control groups.Compared with the Model group,the relative expression of p53 and p21 proteins was significantly lower in the BSQJ and Control groups(P<0.01),and the relative expression of CDK4 proteins was significantly higher in the BSQJ group(P<0.01).Compared with the Control group,the relative expression of p53 and p21 proteins was significantly lower(P<0.05 or P<0.01),and the relative expression of CyclinD1 and CDK4 proteins was significantly higher in the BSQJ group(P<0.01).②Apoptosis:Compared with the Blank group,the relative expression of Caspase3 and Caspase9 proteins was significantly higher in the Model,BSQJ,and Control groups(P<0.01),while the relative expression of Bcl-2 protein was significantly lower(P<0.01).Compared with the Model group,the relative expression of Caspase3 and Caspase9 proteins was significantly lower in the BSQJ and Control groups(P<0.01),while the relative expression of Bcl-2 protein was significantly higher(P<0.01).Compared with the Control group,the relative expression of Caspase3 and Caspase9 protein was significantly lower in the BSQJ group(P<0.01),while the relative expression of Bcl-2 protein was significantly higher in the BSQJ group(P<0.01).The relative expression of Bax protein in chondrocytes was significantly higher in the Model and BSQJ groups compared with the Blank group(P<0.05).The relative expression of Bax protein in chondrocytes was significantly lower in the BSQJ group compared with the Model group(P<0.05).(4)Immunofluorescence stainingCompared with the Blank group,the fluorescence relative expression of Caspase3,Caspase9,and Bax was significantly higher(P<0.05 or P<0.01),and the relative fluorescence expression of Bcl-2 was significantly lower(P<0.01)in the Model,BSQJ and Control groups.Compared with the Model group,the fluorescence relative expression of Caspase3,Caspase9,and Bax was significantly lower(P<0.01),and the fluorescence relative expression of Bcl-2 was significantly higher(P<0.01)in the BSQJ and Control groups.Compared with the Control group,the relative fluorescence expression of Caspase3,Caspase9,and Bax was significantly lower(P<0.01),and the relative fluorescence expression of Bcl-2 was significantly higher in the BSQJ group(P<0.01).Conclusions1.Bushen Qiangjin capsule can improve the joint pain,function,and range of motion of patients with KOA,reduce the effusion depth of the suprapatellar bursa and improve the quantitative score of TCM syndrome.2.Bushen Qiangjin capsule can regulate the expression of apoptosis-related proteins in cartilage tissue,delay the degeneration of knee cartilage in KOA rats,and protect articular cartilage.3.The medicated serum of Bushen Qiangjin capsule can accelerate the switching of the checkpoint of the Gl/S phase of the chondrocyte cycle,reduce the arrest of the G1/S phase,promote the proliferation of chondrocytes,reduce the apoptosis of chondrocytes,and play a protective role on chondrocytes. |
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