目的:探究电针联合推拿对膝骨关节炎大鼠软骨损伤的保护作用及分子作用机制。方法:选取50只SD大鼠纳入研究,随机分为对照组、模型组、电针组、推拿组、电针+推拿组,每组10只大鼠。空白组不做造模处理,其余各组大鼠均采用关节腔内注射木瓜蛋白酶的方法构建右侧膝骨关节炎模型,电针及推拿干预2周；采用改良Le-quesne MG量表评估大鼠行为学表现；分别与干预前后统计大鼠压痛值、热痛阈值；8周后处死大鼠,取腹主动脉血,ELISA法检测各组大鼠血清疼痛介质(K+)、5-羟色胺(5-HT)、多巴胺(DA)含量；取大鼠右侧膝关节进行HE染色Mankin评分、TUNEL软骨细胞凋亡分析,甲苯胺蓝染色观察软骨基质多聚糖变化；Western-blot检测各组大鼠膝关节组织内软骨基质因子II型胶原(Collagen II)、C端肽(CTX II)、II型胶原C前肽(CPII)、凋亡相关蛋白Bax、Bcl-2、Cleaved-caspase-3、YAP、p-YAP浓度表达。结果:KOA模型建立后,大鼠Lequesne MG评分提升(P<0.05)；与模型组相比,电针组、推拿组大鼠Lequesne MG评分降低(P<0.05)；二者联合可进一步降低Lequesne MG评分(P<0.05)；与对照组组相比,模型组疼痛阈值(TWL、MWT)下调,血清疼痛因子(K+、DA、5-HT)水平上调(P<0.05)；与模型组组相比,电针组、推拿组疼痛阈值(TWL、MWT)上调,血清疼痛因子(K+、DA、5-HT)水平下调(P<0.05)；二者联合体现出最高疼痛阈值(TWL、MWT)及最低血清疼痛因子(K+、DA、5-HT)水平。模型组大鼠关节软骨裂隙明显,软骨基质染色度、着色面积均下降,Markin评分、细胞凋亡能力提升(P<0.05)；与模型组组相比,电针组、推拿组大鼠软骨层排列度、软骨基质染色度、着色面积均提升,Markin评分、细胞凋亡率均降低(P<0.05)；联合可以进一步降低Markin评分及软骨细胞凋亡程度(P<0.05)；与对照组相比,模型组Collagen II、CPII、Bcl-2、p-YAP蛋白表达降低,CTX II、Bax、Cleaved-caspase-3蛋白表达提升(P<0.05)；与模型组相比,电针组、推拿组大鼠软骨组织内Collagen II、CPII、Bcl-2、p-YAP蛋白表达提升,CTX II、Bax、Cleaved-caspase-3蛋白表达降低(P<0.05)；二者联合可以进一步提升Collagen II、CPII、Bcl-2、p-YAP蛋白表达,抑制CTX II、Bax、Cleaved-caspase-3蛋白表达(P<0.05)。结论:电针联合推拿干预可以明显改善骨性关节炎大鼠软骨损伤病理状态,减轻膝关节疼痛度,抑制软骨细胞凋亡,起到软骨损伤保护作用,其分子机制可能与激活Hippo-YAP信号通路有关,值得临床进一步研究。

Objective: To explore the protective effect of electroacupuncture combined with massage on cartilage damage in knee Osteoarthritis rats and its molecular mechanism. Methods: Fifty SD rats were selected for inclusion in the study and randomly divided into a control group, a model group, an electroacupuncture group, a massage group, and an electroacupuncture+massage group, with 10 rats in each group. The rats in the blank group were not treated with modeling, and the rats in the other groups were injected with Papain into the articular cavity to build the right knee Osteoarthritis model, and were intervened with electroacupuncture and massage for 2 weeks; Using the improved Le quesne MG scale to evaluate the behavioral performance of rats; Calculate the pressure pain value and heat pain threshold of rats before and after intervention; Eight weeks later, the rats were euthanized and abdominal aortic blood was collected. The serum levels of pain mediators (K+), 5-hydroxytryptamine (5-HT), and dopamine (DA) were measured using ELISA in each group of rats; The right knee joint of rats was taken for HE staining Mankin score, TUNEL chondrocyte apoptosis analysis, toluidine blue staining to observe the changes of cartilage matrix polysaccharide; Western blot was used to detect the concentration and expression of cartilage matrix factor type II collagen (Collagen II), C-terminal peptide (CTX II), type II collagen C-precursor peptide (CPII), apoptosis related proteins Bax, Bcl-2, Cleared caspase-3, YAP, p-YAP in the knee joint tissues of rats in each group. Result: After the establishment of the KOA model, the Lequesne MG score of rats increased (P<0.05); Compared with the model group, the Lequesne MG scores of rats in the electroacupuncture group and massage group decreased (P<0.05); The combination of the two can further reduce the Lequesne MG score (P<0.05); Compared with the control group, the pain threshold (TWL, MWT) of the model group was down regulated, and the levels of serum pain factors (K+, DA, 5-HT) were up regulated (P<0.05); Compared with the model group, the pain thresholds (TWL, MWT) of the electroacupuncture group and the massage group were upregulated, while the levels of serum pain factors (K+, DA, 5-HT) were downregulated (P<0.05); The combination of the two reflects the highest pain threshold (TWL, MWT) and the lowest serum pain factor (K+, DA, 5-HT) levels. The model group rats had significant joint cartilage fissures, decreased cartilage matrix staining and staining area, and improved Markin score and cell apoptosis ability (P<0.05); Compared with the model group, the arrangement degree of the cartilage layer, staining degree of the cartilage matrix, and staining area of the electroacupuncture group and massage group were all improved, while the Markin score and cell apoptosis rate were all reduced (P<0.05); The combination can further reduce the Markin score and the degree of chondrocyte apoptosis (P<0.05); Compared with the control group, the expression of Collagen II, CPII, Bcl-2, and p-YAP proteins in the model group decreased, while the expression of CTX II, Bax, and Cleared caspase-3 proteins increased (P<0.05).Conclusion: The combination of electroacupuncture and massage intervention can significantly improve the pathological state of cartilage injury in rats with osteoarthritis, reduce knee joint pain, inhibit chondrocyte apoptosis, and play a protective role in cartilage injury. Its molecular mechanism may be related to the activation of Hippo YAP signaling pathway, which is worth further research in clinical practice.

北京中医药大学科技处项目（编号：2019-JYB-JS-118）