黄腐酚抑制膝骨关节炎大鼠软骨组织细胞自噬作用

Inhibitory effect of xanthohumol on autophagy of chondrocytes in rats with knee osteoarthritis

目的探讨黄腐酚对膝骨关节炎大鼠软骨损伤和软骨细胞自噬水平的影响及其可能的作用机制。方法选取48只大鼠用前后交叉韧带断离术建立膝骨关节炎(knee osteoarthritis,KOA)模型,将造模成功大鼠随机分为模型组、黄腐酚低剂量组、黄腐酚高剂量组和西药组,各12只,另设对照组12只。黄腐酚低剂量、高剂量组分别灌胃50、100 mg·kg^(-1)黄腐酚混悬液(生理盐水配制),西药组灌胃0.17 g·kg^(-1)硫酸氨基葡萄糖胶囊,对照组及模型组给予等量生理盐水,连续干预28 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(enzyme linked immunosorbent assy,ELISA)检测各组大鼠血清白细胞介素-6(interleukin-6,IL-6)、白细胞介素-1β(interleukin-1β,IL-1β)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)水平;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;反转录聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)检测大鼠软骨组织磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)、蛋白激酶B(protein kinase B,Akt)和哺乳动物雷帕霉菌靶蛋白(mammalian rapamydia target protein,mTOR)mRNA的表达情况;Western blotting检测大鼠软骨组织PI3K、磷酸化蛋白激酶B(phosphorylated protein kinase B,p-Akt)、磷酸化哺乳动物雷帕霉菌靶蛋白(phosphorylated mammalian rapamydia target protein,p-mTOR)、哺乳动物ATG同源蛋白(mammalian ATG homologous protein,Beclin 1)和微管相关蛋白1轻链3(protein 1 light chain 3,LC3)蛋白的表达情况。结果与模型组比较,3个给药组大鼠软骨组织学Mankin’s评分、IL-6、IL-1β、TNF-α、软骨细胞凋亡率和PI3K、Akt、mTOR mRNA的表达水平及PI3K、p-Akt、p-mTOR蛋白的表达水平降低,Beclin 1和LC3-Ⅱ蛋白的表达水平升高(P<0.05)。与黄腐酚低剂量组比较,高剂量组和西药组大鼠软骨组织学Mankin’s评分、IL-6、IL-1β、TNF-α、软骨细胞凋亡率和PI3K、Akt、mTOR mRNA的表达水平及PI3K、p-Akt、p-mTOR蛋白的表达水平降低,Beclin 1和LC3-Ⅱ蛋白的表达水平升高(P<0.05),后2组比较差异无统计学意义。结论黄腐酚能减轻KOA大鼠的炎症反应、提高软骨细胞自噬水平、抑制细胞凋亡,可能是通过抑制PI3K/Akt/mTOR信号通路发挥作用的。

Objective To investigate the effects of xanthohumol on cartilage injury and chondrocyte autophagy in knee osteoarthritis rats and to explore its possible mechanism.Methods 48 rats were used to establish the knee osteoarthritis(KOA)model by anterior and posterior cruciate ligament disconnection.The successful modeled rats were randomly divided into model group,low and high dose of xanthohumol groups and western medicine group,with 12 rats in each group and 12 rats in the control group.Low and high dose of xanthohumol groups were given 50 and 100 mg·kg^(-1) xanthohumol suspension(prepared with normal saline)by gavage,the western medicine group was given 0.17 g·kg^(-1) Glucosamine Sulfate Capsules by gavage,and the control group and model group were given the same amount of normal saline for 28 days.The morphology of rat cartilage was observed and Mankin’s score was calculated;the level of serum interleukin-6(IL-6),interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)were detected by ELISA;the pathological changes of cartilage tissue were observed by HE staining;the proliferation and apoptosis of rat chondrocytes were detected by TUNEL method;the mRNA expression of phosphatidylinositol 3-kinase(PI3K),protein kinase B(Akt)and mammalian rapamydia target protein(mTOR)in rat cartilage were detected by reverse transcription-polymerase chain reaction(RT-PCR);the protein expression of PI3K,phosphorylated protein kinase B(p-Akt),phosphorylated mammalian rapamydia target protein(p-mTOR),mammalian ATG homologous protein(Beclin 1),microtubule associated protein 1 light chain 3(LC3)were detected by Western blotting.Results Compared with the model group,the Mankin’s score of cartilage histology,IL-6,IL-1β,TNF-α,apoptosis rate of chondrocytes,the mRNA expression of PI3K,Akt and mTOR,the protein expression of PI3K,p-Akt and the p-mTOR were decreased,and the protein expression levels of Beclin 1 and LC3-Ⅱin low,high dose xanthohumol groups and western medicine group were increased(P<0.05).Compared with low dose xanthohumol group,the Mankin’s score of cartilage histology,IL-6,IL-1β,TNF-α,apoptosis rate of chondrocytes,the mRNA expression of PI3K,Akt and mTOR,the protein expression of PI3K,p-Akt and p-mTOR were decreased,and the protein expression levels of Beclin 1 and LC3-Ⅱin high dose xanthohumol group and western medicine group were increased(P<0.05).There was no significant difference in the above indexes between high dose xanthohumol group and western medicine group(P>0.05).Conclusion Xanthohumol can increase the level of autophagy,reduce inflammatory response and inhibit chondrocyte apoptosis in KOA rats,which may be related to the inhibiting PI3K/Akt/mTOR signal pathway.