Chondroprotective Mechanism And Efficacy Of Bushen Qiangjin Capsule On Knee Osteoarthritis By Reducing Oxidative Stress

ObjectiveWe aimed to investigate the clinical efficacy of Bushen Qiangjin Capsule on KOA by a randomized controlled study,and then verify its antioxidant effect and clarify the antioxidant mechanism of Bushen Qiangjin Capsule in the treatment of KOA through animal and cell experiments.Methods1.Clinical research sectionA total of 70 patients were randomly divided into the Control group and the Experimental group in a 1:1 ratio.The experimental group was treated with basic treatment and Bushen Qiangjin Capsule orally(2 capsules each time,3 times a day),while the control group was prescribed with basic treatment combined with glucosamine hydrochloride orally(0.75g/time,twice a day).The treatment period of all subjects lasted for 4 weeks.The Visual analysis scale(VAS)in activity,the Western Ontario and McMaster Universities Osteoarthritis Index(WOMAC)score,the difficulty of ascending and descending stairs,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,and the total SOD activity and MDA level in joint fluid of KOA patients with positive floating patella test in the two groups were detected before and after treatment.In addition,the adverse events in the two groups were recorded detailedly.2.Animal experiment sectionForty Sprague Dawley(SD,female)rats were randomly divided into Control group(Control,n=10),model group(Model,n=10),glucosamine group(Glu,n=10)and Bushen Qiangjin Capsule group(BSQJ,n=10).Except for the control group,the other three groups of rats used anterior cross dissociation(ACLT)to establish KOA model.Glu and BSQJ groups were given corresponding doses of glucosamine and Bushen Qiangjin Capsule by gavage according to the equivalent drug dose conversion method.Lequesne MG score was used to assess the severity of KOA,and the knee joints of rats were sliced and stained,and the degeneration of articular cartilage was scored by Mankin score.The activities of total superoxide dismutase(SOD).catalase(CAT)and malondialdehyde(MDA)in the serum ofthe four groups were detected.Moreover,immunohistochemical staining was performed to examine the expression of NRF2 in the articular cartilage of rats in each group.3.Cell experiment sectionChondrocytes were isolated from rat articular to establish an in vitro culture system.Groups were defined as control group(Control),model group(Model),glucosamine group(Glu)and Bushen Qiangjin Capsule group(BSQJ)according the different interventions.Glu and BSQJ groups were pretreated with corresponding drugs for 24 hours,and Control and Model group were treated with normal medium.Then,the other three groups cultured in presence of H2O2 to cause oxidative stress,except for control group.The production of intracellular reactive oxygen species(ROS)was detected by using DCFH-DA fluorescent probes,and the apoptosis rate of chondrocytes was examined by flow cytometry.The nuclear factor erythroid 2-related factor 2(NRF2),matrix metalloproteinase 13(MMP13)and SOD2 mRNA expression were detected by using real-time qPCR(RT-qPCR).The protein expressions of NRF2,a disintegrin and metalloproteinase with thrombospondin motif 5(ADAMTS5),type Ⅱ collagen(Coll2),MMP13 and SOD2 were detected by Western blot.The nuclear metastasis of NRF2 was detected by immunofluorescence.Meanwhile,ML385(NRF2 specific inhibitor)was selected to inhibit the express of NRF2 in chondrocytes.ML385 was used with or without H2O2 in the inhibitor group(ML385 group),ML385+H2O2 group,and Bushen Qiangjin Capsule+ML385+H2O2 group(BSQJ+ML385+H2O2 group),while Control group were cultured in normal medium.After the intervention,the expression of NRF2 protein in each group was detected by Western blot,the production of ROS in chondrocytes was detected by DCFH-DA fluorescent probe,and the cell survival rate was detected by using CCK8 assay.Results1.Clinical researchBushen Qiangjin Capsule significantly reduce the activity VAS score and WOMAC score,and decrease difficulty in ascending and descending stairs and KOA TCM syndrome score for KOA patients.The effective rate of TCM syndrome in the Experimental group is significantly higher than that in the Control group.Furthermore,Bushen Qiangjin Capsule significantly increase the activity of total SOD in joint fluid of patients with KOA and reduce the content of MDA.2.Animal experimentBushen Qiangjin Capsule significantly reduce Lequsene MG score and Mankin score of articular cartilage in KOA rats,and promote the expression of NRF2 in articular chondrocytes.Moreover,Bushen Qiangjin Capsule significantly increase the activity of total SOD and CAT in the serum of KOA rats,and reduce the content of MDA in the serum.3.Cell experimentBushen Qiangjin Capsule increase the mRNA expression of NRF2 and SOD2 in chondrocytes under oxidative stress and reduce the expression of MMP-13 mRNA;Up regulate the expression of NRF2 and promote its nuclear transcription,increase the protein expression of coll2 and SOD2,inhibit the protein expression of ADAMTS5 and MMP13.reduce the content of ROS in chondrocytes under oxidative stress,and reduce the apoptosis of chondrocytes caused by oxidative stress.Conclusions①Bushen Qiangjin Capsule can effectively improve the joint pain,function and TCM syndrome score of patients with KOA,especially in alleviating the difficulty of ascending and descending stairs,reducing the clinical symptoms of joint fear of cold,and improving joint soreness,weakness,and fatigue;②Bushen Qiangjin Capsule can up regulate the expression of NRF2 in articular cartilage,reduce oxidative stress,delay the degeneration of articular cartilage in KOA rats and improve the symptoms of KOA.③Bushen Qiangjin Capsule can up regulate the expression of Nrf2 in chondrocytes and promote Nrf2 nuclear transcription activation,so as to reduce the oxidative damage of chondrocytes and protect chondrocytes.