Effect And Mechanism Of Bushen Huoxue Recipe Based On Tiangui Theory In The Treatment Of Postmenopausal Knee Osteoarthritis

Objective:To seek the internal relationship between postmenopausal women and Tiangui theory aiming at postmenopausal women,a high incidence group of knee osteoarthritis;To observe the clinical efficacy and advantages of Bushen Huoxue Recipe in treating postmenopausal knee osteoarthritis based on Tiangui theory,and to discuss its mechanism of action,so as to enrich the clinical application and scientific connotation of Tiangui theory.Methods:(1)Through Cochrance systematic review,the literature of randomized controlled trials of oral administration of traditional Chinese medicine decoction compared with oral administration of western medicine in the treatment of knee osteoarthritis was studied,and the adverse reactions,total effective rate,WOMAC score and other aspects were demonstrated.At the same time,the Chinese medicine decoction in the screening results literature was excavated,and the Bushen Huoxue Recipe was analyzed.(2)67 cases of postmenopausal knee osteoarthritis were included.33 cases in the treatment group were given Bushen Huoxue Recipe,and 34 cases in the control group were given Celebrex.After treatment,VAS score,WOMAC score,osteoarthritis TCM syndrome grading quantitative score and improved Kupperman index score were evaluated before and after treatment,and the clinical efficacy of TCM was evaluated.(3)Observe the changes of serological E2,MMP-13,CTX-?,IL-1?,TNF-? in the two groups of patients before and after treatment,and evaluate the articular cartilage,subchondral bone marrow edema,subarticular cyst,meniscus and articular cavity effusion in the knee joint MRI before and after treatment by WORMS scoring system.Analyze their correlation between serological changes and imaging changes.(4)The postmenopausal knee osteoarthritis model was established by ovariectomy and injection of MIA into the right knee cavity,and was divided into Control group,Model group,BSHXR-L group,BSHXR-M group,and BSHXR-H group.The protective effect of Bushen Huoxue Recipe on articular cartilage injury in rats was determined by the methods of maximum running speed,naked eye observation score,safranine O-solid green staining,ELISA and immunohistochemistry.At the same time,Western-blot was used to detect the expression of apoptosis and autophagy-related proteins,qPCR was used to detect the relative expression of Akt,mTOR and downstream autophagy genes,and the correlation between miR-107 and Akt and mTOR was analyzed.(5)The cartilage injury model in vitro was established and divided into control group,IL-1?group,BSHXR group,BSHXR+miR-107 inhibitor group and BSHXR+miR-107 inhibitor NC group.The expression of COL-?,MMP-13,Aggrecan,TNF-? and IL-6 in chondrocyte supernatant of each group was detected by ELISA,the expression of autophagosomes in each group was observed by transmission electron microscope,and the expression of Akt,mTOR and downstream autophagy protein was detected by Western-Blot.At the same time,TargetScan 7.2 software was used to predict the possible target gene of miR-107 and the double fluorescein reporter gene detection system was used to verify it.Results:(1)Systematic review of literature revealed that compared with western medicine group,the total effective rate was higher and the adverse reaction rate was lower in the traditional Chinese medicine group.WOMAC score and Lequesne-Mery arthritis index were significantly improved,but there was no significant difference in walking ability,KSS score,VAS score and Lysholms knee function score.In the traditional Chinese medicine group,deficiency-tonifying drugs,blood-activating and blood-stasis-removing drugs,and wind-dampness-dispelling drugs are the most common,which belong to the liver and kidney meridians,have warm and mild properties,and are mainly sweet and bitter in taste.A total of 54 groups of commonly used drug combinations,33 association rules and 16 core drugs were found,and the main components of the kidney-tonifying and blood-activating recipe were included,and the frequency of use of each drug was?10 times.(2)After treatment,the VAS score,the WOMAC score and the total score of TCM syndromes in the treatment group were significantly lower than those before treatment(P<0.01),especially for patients with liver and kidney deficiency type.It has obvious advantages in the symptoms of joint soreness and weakness,joint cold pain,warm preference and aversion to cold,dizziness and tinnitus.At the same time,Bushen Huoxue Recipe can reduce the improved Kupperman index score and significantly improve many discomfort after menopause.The total effective rate of medical effect evaluation is higher than that of the control group,and the difference is statistically significant(P<0.05).(3)Bushen Huoxue Recipe can increase the expression of E2 in serum and inhibit the expression of MMP-13,CTX-?,IL-1? and TNF-?.MRI observation showed that articular cartilage injury was the most common in early and middle menopausal knee osteoarthritis.The first lesion areas were patellofemoral joint and medial tibiofemoral joint.At the same time,the incidence of degeneration injury of medial meniscus was higher than that of lateral meniscus and the degree of lesion was severe.Cartilage injury score was strongly negatively correlated with E2,positively correlated with MMP-13,CTX-? and IL-1?,and weakly positively correlated with TNF-?.There was a good positive correlation with K-L grade and meniscus degeneration injury,but a weak positive correlation with subchondral bone marrow edema.Bushen Huoxue Recipe significantly reduced the scores of subchondral bone marrow edema and joint cavity effusion,but had little effect on the scores of articular cartilage injury,subarticular cyst and meniscus degeneration injury.(4)After modeling,BSHXR(L,M,H)groups can increase the maximum running speed of rats,reduce the degree of cartilage injury,and inhibit the expression of IL-1?,IL-6,and TNF-?in joint fluid.Immunohistochemistry showed that the expression of Col-? and Aggrecan gradually increased,and the expression of MMP-13 gradually decreased in a dose-dependent manner.The difference was statistically significant compared with the model group(P<0.05).The results of Western-blot showed that the apoptotic protein Bcl-2 gradually increased in BSHXR(L,M,H)groups,while Bax,clean caspase-3,and clean PARP gradually decreased.The autophagy pathway proteins p-Akt/Akt and p-mTOR/mTOR were inhibited,and the downstream proteins Beclin-1 and LC3 ? were gradually increased,while p62 was gradually decreased in a dose-dependent manner.The difference was statistically significant compared with the model group(P<0.05).QPCR results showed that BSHXR(L,M,H)groups could promote the relative expression of Beclin-1 and LC3 ? mRNA,and inhibit the relative expression of p62,Akt and mTOR mRNA in a dose-dependent manner;At the same time,the relative expression of miR-107 was negatively correlated with Akt and mTOR mRNA.(5)COL-? and Aggrecan in chondrocyte supernatant decreased,while MMP-13,TNF-?and IL-6 increased after OA induction.Transmission electron microscopy showed that the number of autophagosomes in chondrocytes in BSHXR group was significantly higher than that in IL-1? group,and the morphology was large and the autophagy ability was strong When miR-107 inhibitor was transfected,the autophagosomes of chondrocytes were inhibited;When miR-107 inhibitor NC was transfected,the inhibition was not obvious.Western-blot results showed that the autophagy pathway proteins p-Akt/Akt and p-mTOR/mTOR were inhibited in BSHXR group,and the downstream proteins Beclin-1 and LC3 ? were increased,while p62 was decreased.QPCR results showed that BSHXR group could promote the relative expression of miR-107 mRNA in chondrocytes,and inhibit the relative expression of Akt and mTOR mRNA.Targetscan 7.2 predicts that the direct target gene of miR-107 may be TRAF3.The results of Western-blot and qPCR showed that the BSHXR group could inhibit the relative expression of TRAF3 protein and mRNA,and the dual luciferase reporter gene detection system confirmed that miR-107 targeted the TRAF3 gene of chondrocytes.Conclusions:(1)Literature studies proved that the internal administration of traditional Chinese medicine decoction in the treatment of knee osteoarthritis had definite efficacy and good safety.The clinical treatment was mainly based on tonifying the liver and kidney,Combined with drugs for treating symptoms such as promoting blood circulation and removing blood stasis,dispelling wind and dampness,etc.,it could simultaneously treat principal and subordinate symptoms.The scientific nature and effectiveness of Bushen Huoxue Recipe were confirmed.(2)Based on Tiangui theory,Bushen Huoxue Recipe had clinical advantages in the treatment of postmenopausal knee osteoarthritis,especially for postmenopausal women with general symptoms of better efficacy.(3)MRI images of early and middle postmenopausal knee osteoarthritis were the most common injuries of patellofemoral joint and medial tibiofemoral joint.Bushen Huoxue Recipe inhibited inflammation,improved subchondral bone marrow edema and joint cavity effusion,and plays an estrogen-like effect to a certain extent,and there was no significant correlation between the two changes.(4)Bushen Huoxue Recipe inhibited the expression of Akt/mTOR signal pathway by upregulating miR-107 targeting TRAF3,and then enhance the autophagy reaction of chondrocytes to protect the cartilage.