电针膝眼穴对膝骨关节炎模型大鼠RhoA/ROCK信号通路的影响

Effects of Electroacupuncture at Knee-Eye Point on RhoA/ROCK Signaling Pathway in Knee Osteoarthritis Model Rats

摘要: 目的：基于RhoA/ROCK信号通路研究电针抑制膝骨关节炎软骨细胞凋亡、延缓退变的作用机制。方法：选择2月龄SD雄性大鼠50只，随机分为5组，即正常组、模型组、电针膝眼组、电针非穴组和抑制剂组。选用改良Hulth手术建立大鼠膝骨性关节炎模型，正常组不作干预，模型组和抑制剂组于两电针组电针治疗时给予同样的抓取和袜套固定15 min，电针膝眼组予内外膝眼电针15 min，电针非穴组予电针非经非穴15 min以作对照，抑制剂组大鼠每次腹腔注射0.1 ml Y-27632溶液，1天注射1次，每周连续注射5天，休息2天，连续注射12周。两组电针均采用疏密波刺激模式，脉冲频率调节2/10 Hz，强度0.5~1.5 mA，以肌肉局部开始抽搐，大鼠不挣扎为度。每周干预5天，1天干预1次，共干预12周。收集大鼠右侧膝关节软骨组织，通过HE染色观察各组软骨组织形态学变化，Western Blot法检测软骨组织中RhoA/ROCK信号通路上RhoA、ROCK、ERK1/2、p-ERK1/2、Bax及Bcl-2等相关蛋白表达情况。结果：HE染色结果显示：电针膝眼组和抑制剂组的关节软骨组织形态均较模型组或电针非穴组有明显改善，而电针膝眼组和抑制剂组两组间的差异不明显。Western Blot结果显示：与模型组或电针非穴组相比，电针膝眼组和抑制剂组均明显下调了RhoA、ROCK、ERK1/2、bax等蛋白的表达水平，上调Bcl-2的蛋白表达水平，差异均具有显著意义(P < 0.01)，而电针膝眼组和抑制剂组两组间的差异无统计意义(P > 0.05)。结论：电针膝眼穴可有效减轻膝骨关节炎大鼠软骨组织形态的损伤程度，电针膝眼穴通过调控RhoA/ROCK信号通路，下调软骨组织中RhoA、ROCK、ERK1/2、bax等关键蛋白的表达水平，上调Bcl-2的蛋白表达水平，抑制关节软骨细胞凋亡，减轻软骨破坏，维持关节软骨结构的相对完整性，从而达到延缓软骨退变的作用。

Abstract: Objective: Based on the RhoA/ROCK signaling pathway, to study the mechanism of electroacu-puncture in inhibiting chondrocyte apoptosis and delaying degeneration in knee osteoarthritis. Methods: Fifty 2-month-old male SD rats were selected and randomly divided into 5 groups, namely normal group, model group, electroacupuncture knee-eye group, electroacupuncture non-acupoint group and inhibitor group. The modified Hulth operation was used to establish a rat model of knee osteoarthritis. The normal group was not intervened. The model group and the inhibitor group were given the same grasping and sock fixation for 15 min during the electroacupuncture treat-ment in the two electroacupuncture groups. Electroacupuncture knee-eye group was given to the eyes of the inner and outer knees for 15 min, and the electroacupuncture non-acupoint group was given electroacupuncture to non-meridian and non-acupoints for 15 min as a control. The rats in the inhibitor group were intraperitoneally injected with 0.1 ml of Y-27632 solution each time, once a day, continuously every week. 5 days of injection, 2 days of rest, 12 weeks of continuous injection. Both groups of electroacupuncture used the sparse-dense wave stimulation mode, the pulse fre-quency was adjusted to 2/10 Hz, and the intensity was 0.5~1.5 mA. The strength is based on the fact that the muscle starts to twitch locally and the rats do not struggle. Intervention was conducted 5 days a week, once a day, for a total of 12 weeks. The cartilage tissue of the right knee joint of the rats was collected, and the morphological changes of the cartilage tissue in each group were observed by HE staining, and detect the expression of RhoA, ROCK, ERK1/2, p-ERK1/2, Bax, Bcl-2 and other re-lated proteins on rhoa/ROCK signal pathway in the cartilage tissue by Western blot. Results: HE staining results showed that the morphology of articular cartilage in the electroacupuncture knee-eye group and the inhibitor group was significantly improved compared with the model group or the electro-acupuncture non-acupoint group, while the difference between the electroacupunc-ture knee-eye group and the inhibitor group was no significant. Western Blot results showed: Com-pared with the model group or the electroacupuncture non-acupoint group, the electroacupuncture knee eye group and inhibitor group significantly down-regulated the expression levels of RhoA, ROCK, ERK1/2, bax and other proteins, and up-regulated the expression levels of Bcl-2. There were significant differences in protein expression levels (P < 0.01), while there was no significant differ-ence between the electroacupuncture knee-eye group and the inhibitor group (P > 0.05). Conclusion: Electro-acupuncture at knee-eye point can effectively reduce the damage degree of cartilage tissue morphology in rats with knee osteoarthritis. Electro-acupuncture at knee-eye point can regulate RhoA/ROCK signaling pathway and down-regulate RhoA, ROCK, ERK1/2, bax, etc. in cartilage tissue. The expression level of key proteins increases the protein expression level of Bcl-2, inhibits the apoptosis of articular cartilage cells, reduces cartilage damage, and maintains the relative integrity of articular cartilage structure, thereby delaying cartilage degeneration.