| Objective1.To evaluate the efficacy and adverse events of Bushen Qiangjin Capsule for kidney deficiency and blood stasis postmenopausal knee osteoarthritis through clinical study,provide the clinical evidence of the Kidney-tonifying and Blood-activating prescription in postmenopausal knee osteoarthritis treatment.2.To study the effect of Bushen Qiangjin Capsule on cartilage morphology and related genes and proteins of ER ?/miR-140 pathway and cartilage metabolism through animal experiments with ovariectomized knee osteoarthritis rats,explore treatment mechanism of Kidney-tonifying and Blood-activating prescription in postmenopausal knee osteoarthritis.Method1.Clinical StudySixty patients with kidney deficiency and blood stasis postmenopausal knee osteoarthritis were selected from January 2019 to December 2019 in the Orthopaedic Clinic of GuangDong Second Traditional Chinese Medicine Hospital,were randomly divided into test group and control group of 30 patients each.The test group was orally administered with Bushen Qiangjin Capsule in combination with Celecoxib Capsule,and the control group was orally administered with Celecoxib Capsule.On the basis of oral medication,the two groups of patients were instructed for functional exercise,during 4 weeks treatment.Record the VAS score and WOMAC score of the two groups of patients before treatment,2 weeks and 4 weeks after the treatment.Record Lequesne index,proprioception and estradiol(E2)level before treatment and 4 weeks after the treatment.After 4 weeks treatment,evaluate the efficacy of TCM syndromes,record the occurrence of adverse events during the study,and comprehensively evaluate the effectiveness of Bushen Qiangjin Capsule in treating kidney deficiency and blood stasis postmenopausal knee osteoarthritis and the occurrence of adverse events.2.Animal ExperimentSixty SD rats were randomly divided into a blank group,a model group,a Bushen Huoxue group,a letrozole group,and a letrozole/Bushen Huoxue group.Except for the blank group,the rats in each group were subjected to ovarian removal KOA modeling.And the drug intervention was performed.The blank group and the model group were given 0.1ml/10g physiological saline daily,and the doses of the other groups were referenced:letrozole(0.5mg·kg-1)and Bushen Qiangjin Capsule(dose 0.243g crude drug·kg-1),all equivalent conversions according to human recommended doses;administration for 6 weeks,once a day.The rats were sacrificed 6 weeks after the administration,and the serum and knee cartilage tissue of each group of rats were collected for related detection.Pathological sections were made,and the changes in the joint tissue structure of each group of rats were observed with HE staining and saffron-fast green staining,and the modified Mankin score was used;the serum serum E2 and CTX-II levels were detected by ELISA;and Expression levels of 18S,miR-140-5p,ER a expression,Aggrecan,COL2A1,adamts-5 and mmp-13 were detected by RTt-qPCR;Western blot was used to detect ER?,Aggrecan,COL2A1,ADAMTS-5,MMP-13 protein expressions in rat knee cartilage Level.Result1.Clinical StudyIn terms of VAS score,there was no significant difference in VAS scores in the two groups before the treatment(P>0.05).The improvement of VAS score in the test group was better than the control group at 2 and 4 weeks of the treatment(P<0.05).Compared with those before treatment,the VAS score of the both groups were significantly different at 2 and 4 weeks of the treatment(P<0.05).There was no significant difference in WOMAC pain,stiffness,activity function score and total score before the treatment in the two groups(P>0.05).The improvement of WOMAC pain,activity function score and total score in the test group was better than the control group at 2 and 4 weeks of the treatment(P<0.05)and the improvement of WOMAC stiffness score was better than the control group at 4 weeks of the treatment.Compared with those before treatment,the WOMAC pain,stiffness,activity function score and total score of the both groups were significantly different at 2 and 4 weeks of the treatment(P<0.05).There was no significant difference in Lequesne Index in the two groups before the treatment(P>0.05).The improvement of Lequesne Index in the test group was better than the control group at 4 weeks of the treatment(P<0.05).Compared with those before treatment,the Lequesne Index of the both groups were significantly different at 4 weeks of the treatment(P<0.05).In terms of TCM syndrome effect,the effective rate of the test group was 93.3%higher than the control group of 89.5%,the evaluation of the TCM syndrome effect of the test group is better the control group(P<0.05).There was no significant difference in proprioception evaluation in the two groups before the treatment(P>0.05).The test group and the control group are equivalent in improving proprioception at 4 weeks of the treatment(P>0.05).Compared with those before treatment,the proprioception of the both groups were significantly different at 4 weeks of the treatment(P<0.05).The incidence of adverse events was low in the two groups during the treatment,and it was 2/30 in the both groups.2.Animal ExperimentAfter 6 weeks of gavage and feeding,the HE staining and safrane-fast green staining of the cartilage tissue of the rats knee joints in each group showed that the letrozole group had the most degeneration of cartilage tissue,followed by the model group,and the Bushen Huoxue group was lighter than the model group,the letrozole+Bushen Huoxue group was lighter than the letrozole group.The modified Mankin score showed that the model group score was significantly higher than the blank group(P<0.05),and the Bushen Huoxue group was significantly lower than the model group(P<0.05).ELISA results showed that the serum E2 level in the model group was significantly lower than that in the blank group(P<0.05),and the serum E2 level in the Bushen Huoxue group was significantly higher than that in the model group(P<0.05).The serum CTX-? level in the model group was significant higher than the blank group(P<0.05),the serum CTX-? level in the Bushen Huoxue group was significantly lower than that in the model group(P<0.05),the serum CTX-? level in the letrozole+Bushen Huoxue group was significantly lower than that in the letrozole group(P<0.05).RT-qPCR results showed that compared with the blank group,the miR-140-5p,ER ?,Aggrecan and COL2A1 mRNA expressions of the model group was significantly decreased(P<0.05),the ADAMTS-5,MMP-13 mRNA expressions was significantly increased(P<0.05).Compared with the model group,the miR-140-5p,ER ?,Aggrecan and COL2A mRNA expressions of Bushen Huoxue group were significantly increased(P<0.05),the ADAMTS-5 and MMP-13 mRNA expressions were significantly decreased(P<0.05).Compared with the letrozole group,the miR-140-5p,ER ?,Aggrecan and COL2A1 mRNA expressions of the letrozole+Bushen Huoxue group were significantly increased(P<0.05),the ADAMTS-5 and MMP-13 mRNA expressions significantly decreased(P<0.05).Western blot results showed that compared with the blank group,the ER ?,Aggrecan and COL2A1 protein expressions of the model group significantly decreased(P<0.05),the ADAMTS-5 and MMP-13 protein expressions significantly increased(P<0.05).Compared with the model group,the ER ?,Aggrecan and COL2A1 protein expression of Bushen Huoxue group were significantly increased(P<0.05),the ADAMTS-5 and MMP-13 protein expressions were significantly decreased(P<0.05).Compared with the letrozole group,the ER ?,Aggrecan and COL2A1 protein expressions of letrozole+Bushen Huoxue group significantly increased(P<0.05),and the ADAMTS-5 and MMP-13 protein expressions significantly decreased(P<0.05).Conclusion1.Clinical StudyBushen Qiangjin Capsule can reduce the pain(VAS score and WOMAC pain score),stiffness(WOMAC stiffness score),improve the activity function(WOMAC function index)and arthritis severity(Lequesn index).It has good curative effect on TCM syndromes and the incidence of adverse events is low.2.Animal ExperimentBushen Qiangjin Capsule can significantly increase serum E2 level,reduce serum CTX-? level in ovariectomized KOA rats,increase the miR-140-5p,ER ?,Aggrecan,COL2A1 genes or proteins expressions in cartilage tissue,decrease ADAMTS-5,MMP-13 genes or proteins expressions.It may play a role in protecting cartilage by activating the ER?/miR-140 pathway and regulating cartilage metabolism. |
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