电针通过HIF-1α和SOX-9维持兔膝骨关节炎软骨稳态及抗炎机制研究

Mechanism of Electroacupuncture Maintaining Cartilage Homeostasis and Antiinflammation Through HIF-1αand SOX-9 in Rabbit Knee Osteoarthritis

目的观察电针对兔膝骨关节炎(knee osteoarthritis,KOA)模型软骨缺氧诱导因子1α(hypoxia inducible factor-1α,HIF-1α)、性别决定区Y框蛋白9(SRY-box transcription factor 9,SOX-9)、基质金属蛋白酶1(matrix metalloproteinase-1,MMP-1)、基质金属蛋白酶13(matrix metalloproteinase-13,MMP-13)、Ⅱ型胶原蛋白和炎症因子表达的影响,探讨电针干预改善KOA软骨稳态以及抗炎的可能作用机制。方法采用随机数字表法将实验兔分为对照组、模型组和电针组,每组10只。对模型组和电针组实验兔的右膝关节采用木瓜蛋白酶制造兔KOA模型。制模完成后,电针组给予电针犊鼻及内膝眼穴干预,每次30 min,每天1次,共14 d。模型组每天在固定器上固定15 min。对照组右膝关节注射等量0.9%氯化钠溶液。使用苏木精-伊红染色(hematoxylin-eosin staining,HE染色)检测软骨组织的病理情况,原位末端转移酶标记技术(terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay,TUNEL)检测软骨细胞的凋亡情况,免疫组化检测软骨中Ⅱ型胶原蛋白的表达情况,酶联免疫吸附实验(enzyme linked immunosorbent assay,ELISA)检测软骨中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和白细胞介素-1β(interleukin-1β,IL-1β)的水平,蛋白质免疫印迹(Western Blot,WB)检测HIF-1α、SOX-9、MMP-1和MMP-13的蛋白表达水平,实时荧光定量PCR(real-time quantitative PCR,qRT-PCR)检测HIF-1α、SOX-9、MMP-1和MMP-13的mRNA水平。结果HE染色结果显示,与对照组相比,模型组软骨细胞数量明显减少,细胞核皱缩,基质染色呈浅色,潮线不完整,而电针组较模型组显著改善;改良Mankin's评分结果显示,模型组较对照组显著升高,电针组较模型组显著降低;TUNEL结果显示,电针组软骨细胞凋亡率显著低于模型组;免疫组化结果显示,与模型组相比,电针组的Ⅱ型胶原蛋白表达明显升高;ELISA结果显示与模型组相比,电针组的TNF-α、IL-1β表达明显降低;WB和qRT-PCR结果显示,与对照组相比,模型组的HIF-1α和SOX-9蛋白表达水平和mRNA水平显著降低(P<0.05),与模型组相比,电针组显著升高(P<0.05)。结论电针可能通过上调HIF-1α和SOX-9的表达,减少MMP-1、MMP-13、TNF-α、IL-1β的表达,增加Ⅱ型胶原蛋白的形成,从而发挥缓解软骨损伤、减少炎症反应的作用。

Objective This study aims to observe the effects of electroacupuncture on the homeostasis of articular cartilage and the expression of HIF-1α,SOX-9,MMP-1,MMP-13,typeⅡcollagen,and inflammatory factors in a rabbit model of KOA.AND to investigate the possible mechanism of electroacupuncture intervention in improving cartilage homeostasis and anti-inflammation of KOA.Methods The experimental rabbits were randomly divided into control,model,and electroacupuncture groups,with 10 rabbits in each group.The rabbit KOA model was established by injecting papain into the right knee joint of the model and electroacupuncture groups.After model establishment,the electroacupuncture group received electroacupuncture at the Dubi(ST35)and Neixiyan(EX-LE5)acupoint for 30 minutes once daily for 14 days.The model group was immobilized on a fixator for 15 minutes daily.The control group received normal saline in the right knee joint.Histopathological changes of cartilage tissue were assessed by HE staining,apoptosis of chondrocytes was detected by TUNEL staining,expression of typeⅡcollagen in cartilage was examined by immunohistochemistry,levels of TNF-αand IL-1βin cartilage were measured by ELISA,protein expression levels of HIF-1α,SOX-9,MMP-1,and MMP-13 were determined by Western blot,and mRNA levels of HIF-1α,SOX-9,MMP-1,and MMP-13 were analyzed by qRT-PCR.Results HE staining showed that the number of chondrocytes in the model group significantly decreased,with condensed nuclei and lightly stained matrix,whereas the electroacupuncture group exhibited improved compared with the model group.The modified Mankin's score showed that the model group was significantly higher than the control group,and the electroacupuncture group was significantly lower than the model group.TUNEL results showed that the apoptosis rate of chondrocytes in electroacupuncture group was significantly lower than that in model group.Immunohistochemistry showed a significant increase in typeⅡcollagen expression in the electroacupuncture group compared to the model group.ELISA results showed that the expressions of TNF-αand IL-1βin the electroacupuncture group were significantly decreased compared with those in the model group.Western blot and qRT-PCR results demonstrated that the protein and mRNA levels of HIF-1αand SOX-9 in the electroacupuncture group were significantly decreased compared to the control group(P<0.05),but significantly increased compared to the model group(P<0.05).Conclusion Electroacupuncture may alleviate cartilage damage and reduce inflammation by upregulating the expression of HIF-1αand SOX-9,downregulating the expression of MMP-1,MMP-13,TNF-α,IL-1βand promoting the formation of typeⅡcollagen.