Objective: To observe the effect of acupotomy on heat shock protein A family member 5 (HSPA5)/glutathione peroxidase 4 (GPX4) signaling pathway in the chondrocytes of the rabbits with knee osteoarthritis (KOA) and explore the mechanism of acupotomy on chondrocyte ferroptosis in KOA.
Methods: Twenty-seven New Zealand rabbits were randomly divided into a normal group, a model group and an acupotomy group, with 9 rabbits in each group. The left hind limb was fixed by the modified Videman method for 6 weeks to establish KOA model. After modeling, acupotomy was given in the acupotomy group, once a week and for consecutive 3 weeks. Using Lequesne MG score, the local symptoms, physical signs and functions of knee joint were evaluated. With HE staining and saffrane-solid green staining adopted, the morphology of chondrocytes and cartilage tissue was observed. Under transmission electron microscope, the mitochondrial structure of chondrocytes was observed. The iron content of cartilage tissue was detected by iron ion kit. The mitochondrial membrane potential (Δψm) and the reactive oxygen species (ROS) level in cartilage tissue were determined by flow cytometry, and the mitochondrial damage rate was calculated. The mRNA expression of HSPA5, GPX4, type Ⅱ collagen α1 chain (COL2A1), matrix metalloproteinases (MMP) 3 and MMP13 was detected by the real-time quantitative PCR; and the protein expression of HSPA5, GPX4, type Ⅱ collagen (COL-Ⅱ), MMP3 and MMP13 was detected by Western blot. The mean flourscence intensity of HSPA5 and GPX4 in cartilage tissue was determined by immunofluorescence.
Results: Before intervention, compared with the normal group, the Lequesne MG scores were increased in the model group and the acupotomy group (P<0.01). After intervention, the Lequesne MG score in the acupotomy group was decreased when compared with that in the model group. In comparison with that in the normal group, the number of chondrocytes was reduced and the cells were disarranged; the layers of cartilage structure were unclear, the tide lines disordered and blurred; the mitochondria were wrinkled and the mitochondrial crista decreased or even disappeared in the model group. Compared with the model group, the number of chondrocytes was increased, the layers of cartilage structure were clear, the tide lines recovered, the number of mitochondria elevated, with normal structure and more crista in the acupotomy group. The iron content of cartilage tissue was increased (P<0.01), the Δψm of chondrocytes was declined, the mitochondrial damage rate was increased (P<0.01), the average fluorescence intensity of ROS was increased (P<0.01); the mRNA and corresponding protein expression of HSPA5, GPX4 and COL2A1 was decreased (P<0.01), the mRNA and protein expression of MMP3 and MMP13 was increased (P<0.01) and the average fluorescence intensity of HSPA5, GPX4 was decreased (P<0.01) in the model group when compared with those in the normal group. Compared with the model group, the iron content in cartilage tissue was reduced (P<0.01), the Δψm of chondrocytes was increased, the mitochondrial damage rate was decreased (P<0.01), and the average fluorescence intensity of ROS was decreased (P<0.01); the mRNA and corresponding protein expression of HSPA5, GPX4 and COL2A1 was higher (P<0.01), and the mRNA and protein expression of MMP3 and MMP13 was lower, and the average fluorescence intensity of HSPA5, GPX4 was increased (P<0.01) in the acupotomy group.
Conclusion: Acupotomy can alleviate cartilage injury of KOA rabbits, and its mechanism may be related to the regulation of HSPA5/GPX4 signaling pathway to maintain iron homeostasis in articular cartilage, thus inhibiting chondrocyte ferroptosis and relieving extracellular matrix degradation.
目的：观察针刀疗法对膝关节骨关节炎（KOA）兔膝关节软骨细胞热休克蛋白A家族成员5（HSPA5）/谷胱甘肽过氧化物酶4（GPX4）信号通路的影响，探讨针刀干预KOA软骨细胞铁死亡的作用机制。方法：将27只新西兰兔随机分为正常组、模型组和针刀组，每组9只。采用改良Videman法连续固定兔左侧后肢6周建立KOA模型。造模后，针刀组给予针刀干预，每周1次，连续3周。运用奎森功能障碍指数（Lequesne MG评分）评估兔膝关节局部症状、体征及功能；HE染色法与番红-固绿染色法分别观察兔软骨细胞与组织形态；透射电镜观察兔软骨细胞线粒体结构；运用铁离子试剂盒检测兔软骨组织铁含量；流式细胞仪检测兔软骨组织线粒体膜电位（Δψm）和活性氧（ROS）水平，并计算线粒体损伤率；实时荧光定量PCR检测兔软骨组织HSPA5、GPX4、Ⅱ型胶原α1链（COL2A1）、基质金属蛋白酶（MMP）3、MMP13 mRNA表达；Western blot法检测兔软骨组织HSPA5、GPX4、Ⅱ型胶原蛋白（COL-Ⅱ）、MMP3、MMP13蛋白表达；免疫荧光法检测兔软骨组织HSPA5、GPX4平均荧光强度。结果：干预前，与正常组比较，模型组和针刀组兔膝关节Lequesne MG评分升高（P<0.01）；干预后，与模型组比较，针刀组兔膝关节Lequesne MG评分降低（P<0.01）。与正常组比较，模型组兔膝关节软骨细胞数量减少且排列紊乱，软骨结构层次不清，潮线紊乱、模糊不清，线粒体皱缩、线粒体嵴减少甚至消失；与模型组比较，针刀组软骨细胞数量较多，软骨结构层次清楚，潮线恢复，线粒体数量较多，结构正常，有较多的嵴。与正常组比较，模型组软骨组织铁含量增加（P<0.01），细胞呈低Δψm，线粒体损伤率增高（P<0.01），ROS平均荧光强度升高（P<0.01），软骨组织HSPA5、GPX4、COL2A1 mRNA及相应蛋白表达降低（P<0.01），软骨组织MMP3、MMP13 mRNA及蛋白表达升高（P<0.01），软骨组织HSPA5、GPX4平均荧光强度降低（P<0.01）；与模型组比较，针刀组软骨组织铁含量减少（P<0.01），软骨细胞Δψm升高，线粒体损伤率降低（P<0.01），ROS平均荧光强度降低（P<0.01），软骨组织HSPA5、GPX4、COL2A1 mRNA及相应蛋白表达升高（P<0.01），软骨组织MMP3、MMP13 mRNA和蛋白表达降低（P<0.01），软骨组织HSPA5、GPX4平均荧光强度升高（P<0.01）。结论：针刀干预能够缓解KOA模型兔膝关节软骨损伤，其机制可能与调节HSPA5/GPX4信号通路维持关节软骨铁稳态，从而抑制软骨细胞铁死亡，缓解细胞外基质（ECM）降解有关。.
Keywords: HSPA5/GPX4 signaling pathway; acupotomy; chondrocyte ferroptosis; extracellular matrix; knee osteoarthritis.