| Objective1 To explore the clinical effectiveness and safety of acupuncture treatment of knee osteoarthritis(KOA)through the improvement of knee pain and movement function after receiving acupuncture therapy,and to provide evidence-based evidence for acupuncture treatment of KOA.2 The rat model of knee osteoarthritis was prepared by the drug induction method of sodium iodoacetate injection.The KOA rats were respectively treated with acupuncture and minimal acupuncture for 14 days to detect the genes and proteins related to autophagy and inflammation in the knee cartilage tissue of the rats,and to explore whether acupuncture could regulate autophagy and inflammation through AMPK/mTOR pathway and play a therapeutic role.To explore the partial target and mechanism of acupuncture therapy in treating KOA.Methods1.Clinical trialsThe patients recruited in this study were all from the outpatient department of Acupuncture and Rehabilitation of Guangdong Second Hospital of Traditional Chinese Medicine.Patients diagnosed with KOA were collected from January 2022 to February 2023.A total of 60 patients were included for the study,which were included by random number table method and randomly assigned to the acupuncture group(experimental group)and the non-meridian and non-acupoint group(control group)at a ratio of 1:1.Before treatment,patients in both groups were instructed to take uniform daily functional exercise and preventive care measures,and the course of treatment was 2 weeks.VAS score and WOMAC score were recorded at three time points before treatment,7 days and 14 days after treatment,and clinical efficacy evaluation(JOA)was recorded before and after treatment for knee osteoarthritis,and the occurrence of adverse events was recorded.2.Animal experimentForty SD rats were numbered and evenly assigned to acupuncture group,non-meridian and non-acupoint group,blank group and model group with 10 rats in each group according to random number table method.The blank group did not need any modeling or intervention,and the other three groups underwent KOA modeling.After modeling,acupuncture group and non-meridian and non-acupoint group underwent acupuncture and shallow acupuncture,once a day,respectively.The intervention lasted for 14 days.After the intervention,all the rats were sacrificed with excessive anesthesia,and the serum was separated after abdominal aorta blood was extracted.The appearance and morphological changes of knee joints in each group were observed by naked eye first,then samples were taken for HE staining and rufan-solid green staining pathology observation.The pathological results were evaluated by modified Markin score,and some cartilage tissues of knee joints were cut off for detection.Serum levels of IL-1β and TNF-α were determined by ELISA(enzyme-linked immunosorbent assay).The expression levels of IL-1β,TNF-α,NLRP3,MMP13,Beclinl,ULK1,LC3B in rat knee cartilage were detected by RT-qPCR.The protein expression levels of MMP13,COX2,iNOS,p-mTOR,ULK1,Beclinl and p-AMPK were detected by Western blot.Results1.Clinical trialAfter 2 weeks of treatment,VAS score and WOMAC pain score of acupuncture group and non-meridian and non-acupoint group were significantly decreased compared with before treatment(P<0.05).Compared between the two groups,the improvement of VAS score and WOMAC pain score of acupuncture group was better than that of non-meridian and non-acupoint group after 1 and 2 weeks of treatment(P<0.05).The WOMAC stiffness score of acupuncture group and non-meridian and non-acupoint group was significantly decreased compared with that before treatment(P<0.05).After 2 weeks of treatment,the improvement of WOMAC stiffness score in acupuncture group was better than that in non-meridian and non-acupoint group(P<0.05).The scores of WOMAC activity function in the acupuncture group and the non-meridian and non-acupoint group were significantly decreased compared with those before treatment(P<0.05).The improvement of WOMAC activity function score in the acupuncture group was better than that in the non-meridian and non-acupoint group after 1 and 2 weeks of treatment(P<0.05).The total WOMAC score of the acupuncture group and the non-meridian and non-acupoint group was significantly decreased compared with that before treatment(P<0.05).The improvement of the total WOMAC score of the acupuncture group was not significantly different from that of the non-meridian and non-acupoint group at 2 weeks before treatment(P>0.05).Compared with before treatment,JOA scores in acupuncture group and non-meridian and non-acupoint group were significantly improved(P<0.05).After 2 weeks of treatment,JOA scores in acupuncture group were better than those in non-meridian and non-acupoint group(P<0.05).The total effective rate of the acupuncture group was 90.00%,which was higher than that of the non-meridian and non-point group(66.67%),and the comparison between the two groups was statistically significant(P<0.05).2.Animal experimentThe changes of appearance and morphology of knee joint in each group were observed by naked eye,and the degree of degeneration in model group was the most serious.The degree of degeneration in acupuncture group and non-meridian and non-point group was less than that in model group,and the degree of degeneration in acupuncture group was less than that in non-meridian and non-point group.Pathological observation of cartilage samples by HE staining and saffron red-solid green staining showed that the degree of degeneration in the model group was the most serious,the degree of degeneration in the acupuncture group and the non-meridian and non-point group was less than that in the model group,and the degree of degeneration in the acupuncture group was less than that in the non-meridian and non-point group.The modified Mankin score evaluated pathological observation results showed that the Mankin score of model group was significantly higher than that of blank group(P<0.05),the score of acupuncture group and non-meridian and non-point group was significantly lower than that of model group(P<0.05),and the score of acupuncture group was significantly lower than that of non-meridian and non-point group(P<0.05).ELISA results showed that the serum levels of IL-1β and TNF-α in model group were significantly higher than those in blank group(P<0.05),the levels of IL-1β and TNF-α in acupuncture group and non-meridian and non-point group were significantly lower than those in model group(P<0.05),and the levels of IL-1β and TNF-α in acupuncture group were significantly lower than those in non-meridian and non-point group(P<0.05).RT-qPCR results showed that compared with blank group,the expressions of IL-1β,TNF-α,NLRP3 and MMP13 in model group were significantly increased(P<0.05),while the expressions of Beclinl,ULK1 and LC3B were significantly decreased(P<0.05).Compared with model group,the expressions of IL-1β,TNF-α,NLRP3 and MMP13 in acupuncture group and non-acupuncture group were significantly decreased(P<0.05),while the expressions of Beclinl,ULK1 and LC3B were significantly increased(P<0.05).Compared with the non-meridian and non-point group,the expressions of IL-1β,TNF-α,NLRP3 and MMP13 in the acupuncture group were significantly decreased compared with the non-meridian and non-point group(P<0.05),and the expressions of Beclinl and ULK1 in the acupuncture group were significantly increased compared with the non-meridian and non-point group(P<0.05).There was no significant difference in LC3B expression level between acupuncture group and non-acupuncture group(P>0.05).Western blot results showed that compared with blank group,the expressions of MMP13,COX2,iNOS and P-MTor in model group were significantly increased(P<0.05),while the expressions of ULK1,Beclinl and P-AMPK were significantly decreased(P<0.05).Compared with model group,the expressions of MMP13,COX2,iNOS and P-Mtor in acupuncture and non-acupuncture and non-acupoint groups were significantly decreased(P<0.05),while the expressions of ULK1,Beclinl and P-AMPK were significantly increased(P<0.05).Compared with the non-meridian and non-point group,the expressions of MMP13,COX2,iNOS and P-Mtor in the acupuncture group were significantly decreased compared with the non-meridian and non-point group(P<0.05),and the expressions of ULK1,Beclinl and P-AMPK in the acupuncture group were significantly increased compared with the non-meridian and non-point group(P<0.05).Conclution1.Clinical researchIn the study,most of the KOA patients were improved in VAS score,WOMAC score and JO A score to varying degrees after 14 days of acupuncture treatment,indicating that acupuncture therapy can improve knee pain,stiffness and mobility function.In terms of clinical efficacy evaluation,the total effective rate of acupuncture therapy is 90.00%,and has high safety.2.Animal experimentsAcupuncture therapy can significantly reduce the levels of IL-1β and TNF-α in serum of KOA rats,and reduce the expression of IL-1β TNF-α,NLRP3,MMP13,COX2,iNOS and p-mTOR gene or protein in cartilage tissue of KOA rats.Improve Beclinl,ULK1,LC3B,p-AMPK gene or protein expression.AMPK/mTOR signaling pathway can activate the autophagy effect of chondrocytes to inhibit the inflammatory response,thus reducing the cartilage damage and finally achieving the protective effect of cartilage.Acupuncture may activate the autophagy effect through AMPK/mTOR signaling pathway to treat KOA. |
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