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

Effect of atractylode on osteochondral injury repair and chondrocyte apoptosis in osteoarthritis rats

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7~8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Mankin’s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-α(TNF-α)、白细胞介素-1β(IL-1β)、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-3β(GSK-3β)mRNA表达;蛋白质印迹法(Western blotting)检测大鼠HMGA2、GSK-3β、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-3βmRNA和Bcl-2蛋白表达降低,Mankin’s评分[(8.62±0.69)分]比[(1.71±0.12)分]、TNF-α、IL-1β、IL-6、miR-98-5p、Bax蛋白表达水平、细胞凋亡率[(47.82±4.26)%比(8.41±1.74)%]显著升高(P<0.05);与模型组比较,AT低、中、高剂量组大鼠软骨组织HMGA2、GSK-3βmRNA和Bcl-2蛋白表达升高,Mankin’s评分[(5.05±0.47)分、(4.68±0.36)分、(2.15±0.23)分]比[(8.62±0.69)分]、血清TNF-α、IL-1β、IL-6、miR-98-5p、Bax蛋白表达水平、细胞凋亡率[(32.05±2.84)%、(23.68±2.89)%、(12.15±2.13)%比(47.82±4.26)%]显著降低(P<0.05),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-3β蛋白表达发挥作用。

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 Mankin´s score;the level of serum tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)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-3β(GSK-3β)in rat cartilage were detected by real-time fluorescence quantitative method.The protein expression of high mobility group protein 2(HMGA2),glycogen synthase kinase-3β(GSK-3β),apoptotic protein B-cell lymphoma factor 2(Bcl-2)and Bcl-2 related X protein(Bax)in rat cartilage were detected by Western blot.Re⁃sults Compared with the control group,the levels of HMGA2,GSK-3βmRNA expression and Bcl-2 protein expression were decreased,Mankin´s score[(8.62±0.69)score vs.(1.71±0.12)score],the levels of TNF-α,IL-1β,IL-6,miR-98-5p expression,Bax protein expression,and apoptosis rate[(47.82±4.26)%vs.(8.41±1.74)%]were significantly increased in model group(P<0.05).Compared with the model group,the levels of HMGA2,GSK-3βmRNA expression and Bcl-2 protein expression were increased,Mankin´s score[(5.05±0.47)score,(4.68±0.36)score,(2.15±0.23)score vs.(8.62±0.69)score],and the levels of serum TNF-α,IL-1β,IL-6,miR-98-5p expression,Bax protein expression and apoptosis rate[(32.05±2.84)%,(23.68±2.89)%,(12.15±2.13)%vs.(47.82±4.26)%]were significantly decreased in low,medium and high dose of AT groups(P<0.05),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-3βprotein expression plays a role.