苍术内酯对骨关节炎大鼠软骨...伤修复和软骨细胞凋亡的影响_郭跃生.pdf

安 徽 医 药 Anhui Medical and Pharmaceutical Journal 2023 Aug，27（8）苍术内酯对骨关节炎大鼠软骨损伤修复和软骨细胞凋亡的影响郭跃生，穆岭，张锟，赵大伟，姚太顺作者单位：洛阳正骨医院（河南省骨科医院）足踝一科，河南 洛阳471002基金项目：河南省中医药科学研究专项课题（20-21ZY2249）摘要：目的 探讨苍术内酯（AT）对骨关节炎大鼠软骨损伤修复和软骨细胞凋亡的影响及其可能作用机制。方法 60只78周龄SPF级雄性SD大鼠，48只大鼠采用前后交叉韧带断离术建立骨关节炎（OA）模型，造模成功大鼠采用随机分为模型组、AT低、中、高剂量组，各12只，另设对照组（12只）。AT低、中、高剂量组腹腔注射5、10、20 mg/kg AT，对照组及模型组给予等量生理盐水，连续30 d。观察大鼠软骨组织形态计算Mankins评分；酶联免疫吸附试验（ELISA）检测各组大鼠血清肿瘤坏死因子-（TNF-）、白细胞介素-1（IL-1）、白细胞介素-6（IL-6）水平；苏木精-伊红（HE）染色观察软骨组织病理学变化；TUNEL法检测大鼠软骨组织细胞凋亡情况；实时荧光定量逆转录聚合酶链式反应（qRT-PCR）检测大鼠软骨组织中微RNA-98-5p（miR-98-5p）表达、高迁移率族蛋白2（HMGA2）、糖原合成酶激酶-3（GSK-3）mRNA表达；蛋白质印迹法（Western blotting）检测大鼠HMGA2、GSK-3、凋亡蛋白B细胞淋巴瘤因子2（Bcl-2）、Bcl-2相关蛋白（Bax）蛋白表达情况。结果 模型组大鼠软骨组织HMGA2、GSK-3 mRNA和Bcl-2蛋白表达降低，Mankins评分 （8.620.69）分 比 （1.710.12）分、TNF-、IL-1、IL-6、miR-98-5p、Bax蛋白表达水平、细胞凋亡率 （47.824.26）%比（8.411.74）%显著升高（P0.05）；与模型组比较，AT低、中、高剂量组大鼠软骨组织 HMGA2、GSK-3 mRNA 和 Bcl-2 蛋白表达升高，Mankins 评分 （5.050.47）分、（4.680.36）分、（2.150.23）分 比（8.620.69）分、血清TNF-、IL-1、IL-6、miR-98-5p、Bax蛋白表达水平、细胞凋亡率 （32.052.84）%、（23.682.89）%、（12.152.13）%比（47.824.26）%显著降低（P0.05），且以AT高剂量组效果最佳。结论 AT能够调整OA大鼠血清炎性因子水平，抑制软骨细胞凋亡，可能是通过干扰miR-98-5p促进HMGA2、GSK-3蛋白表达发挥作用。关键词：苍术属；骨关节炎；软骨细胞；微RNA-98-5p；高迁移率族蛋白2；糖原合成酶激酶-3；大鼠，Sprague-DawleyEffect of atractylode on osteochondral injury repair and chondrocyte apoptosis in osteoarthritis ratsGUO Yuesheng,MU Ling,ZHANG Kun,ZHAO Dawei,YAO TaishunAuthor Affiliation:First Department of Foot and Ankle,Henan Luoyang Orthopedic Hospital,Luoyang,Henan 471002,ChinaAbstract：Objective To investigate the effect of atractylode(AT)on osteochondral injury repair and chondrocyte apoptosis in osteoarthritis rats and its possible mechanism.Methods Sixty male SD rats aged 7-8 weeks in SPF,48 rats were selected to establish osteoarthritis(OA)model by anterior and posterior cruciate ligamentectomy.The successful model rats were randomly assigned into model group(12 rats),low dose of AT group(12 rats),medium dose of AT group(12 rats),high dose of AT group(12 rats),and the rest were control group(12 rats).The low,medium and high dose of AT groups were intraperitoneally injected with 5,10 and 20 mg/kg at injection(prepared with normal saline),and the control group and model group were given the same amount of normal saline for 30 days.The cartilage tissue morphology of rats in each group was observed to calculate Mankins score;the level of serum tumor necrosis factor-(TNF-),interleukin-1(IL-1)and interleukin-6(IL-6)were detected by ELISA.The pathological changes of cartilage tissue were observed by HE staining.The apoptosis of rat chondrocytes were detected by TUNEL method.The mRNA expression of micRNA-98-5p,high mobility group protein 2(HMGA2)and glycogen synthase kinase-3(GSK-3)in rat cartilage were detected by real-time fluorescence quantitative method.The protein expression of high mobility group protein 2(HMGA2),glycogen synthase kinase-3(GSK-3),apoptotic protein B-cell lymphoma factor 2(Bcl-2)and Bcl-2 related X protein(Bax)in rat cartilage were detected by Western blot.Results Compared with the control group,the levels of HMGA2,GSK-3 mRNA expression and Bcl-2 protein expression were decreased,Mankins score(8.620.69)score vs.(1.710.12)score,the levels of TNF-,IL-1,IL-6,miR-98-5p expression,Bax protein expression,and apoptosis rate(47.824.26)%vs.(8.411.74)%were significantly increased in model group(P0.05).Compared with the model group,the levels of HMGA2,GSK-3 mRNA expression and Bcl-2 protein expression were increased,Mankins score 药学研究引用本文：郭跃生，穆岭，张锟，等.苍术内酯对骨关节炎大鼠软骨损伤修复和软骨细胞凋亡的影响 J.安徽医药，2023，27（8）：1497-1502.DOI：10.3969/j.issn.1009-6469.2023.08.004.1497网络首发时间：2023-07-06 13:09:34网络首发地址：https:/ 徽 医 药 Anhui Medical and Pharmaceutical Journal 2023 Aug，27（8）(5.050.47)score,(4.680.36)score,(2.150.23)score vs.(8.620.69)score,and the levels of serum TNF-,IL-1,IL-6,miR-98-5p expression,Bax protein expression and apoptosis rate(32.052.84)%,(23.682.89)%,(12.152.13)%vs.(47.824.26)%were significantly decreased in low,medium and high dose of AT groups(P0.05),and the effect of AT high dose group was the best.Conclusion AT can adjust the levels of serum inflammatory factors and inhibit chondrocyte apoptosis in OA rats,possibly by interfering with miR-98-5p and promoting HMGA2 and GSK-3 protein expression plays a role.Key words：Atractylodes;Osteoarthritis;Chondrocytes;Micro RNA-98-5p;High mobility group protein 2;Glycogen synthase kinase-3;Rats,Sprague-Dawley骨关节炎（osteoarthritis，OA）为中老年人常见病症，是一种慢性退行性关节疾病，其发病率随着年龄增长而升高，70 岁以上的老人患病率高达70%1。临床病理表现主要为关节肿痛、关节软骨退化或缺失、关节活动僵硬等，严重影响病人日常生活2。软骨组织靠关节液进行物质交换，一旦受到损伤，靠自身代谢修复组织功能十分困难4。OA发病机制复杂，目前临床仍为保守治疗，迫切需要寻找更加安全有效的药物。苍术内酯（atractylenolide，AT）是分离自中草药白术的一种生物活性成分，具有良好的抗炎止痛、祛风散寒药理作用，中医学临床中常用于治疗风湿和消化系统疾病5。研究表明，AT可以调节炎性因子水平，对细胞增殖和凋亡亦有影响，推测其或对软骨损伤有积极作用6。因此，本研究自2022年14月建立OA大鼠模型，探讨AT对软骨损伤的影响，并分析其可能作用机制，旨在为临床提供参考。1材料与方法1.1材料1.1.1实验动物SPF级SD大鼠，雄性，60只，78周龄，购自北京维通利华实验动物技术有限公司，合格证号：SCXK（京）2016-0011。体质量 210230 g。饲养条件设置为：环境温度为2024，保持良好通风，给予充足饮水和普通饲料，适应性饲养1周。1.1.2药品、主要试剂和仪器AT（纯度98%，上海沪峥生物科技有限公司，批号HZ-22023）；肿瘤坏死因子-（tumour necrosis