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谢芳,刘永利,李树冬,戴斌玉,刘涛,陈绍军,邵先舫.活膝汤对膝骨关节炎模型大鼠软骨细胞焦亡的作用机制[J].湖南中医药大学学报英文版,2023,43(2):225-231.[Click to copy
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| 活膝汤对膝骨关节炎模型大鼠软骨细胞焦亡的作用机制 |
| 谢芳,刘永利,李树冬,戴斌玉,刘涛,陈绍军,邵先舫 |
| (湖南省中医药研究院附属医院, 湖南 长沙 410006;海口市中医医院, 海南 海口 570216;湖南中医药大学附属常德医院, 湖南 常德 415000) |
| 摘要: |
| 目的 观察活膝汤对膝骨关节炎模型大鼠膝关节软骨组织形态结构及趋化因子配体2（CC chemokine ligand 2, CCL2）、趋化因子受体2（CC chemokine receptor 2, CCR2）、半胱氨酸天冬氨酸蛋白酶-1（Caspase-1）、磷酸化p65（phosphorylated p65, p-p65）表达水平的影响。方法 32只SPF级大鼠随机分为正常组（9只）与造模组（23只）。造模组采用改良Hulth造模法制备大鼠膝骨关节炎模型。造模后各组随机分别取2只大鼠验证造模效果。造模组剩余的21只大鼠随机分成3组：模型组、活膝汤组（12.81 mL·kg-1·d-1）、阳性药物组（20 mg·kg-1·d-1），每组7只。正常组与模型组予以等体积蒸馏水灌胃，活膝汤组予以活膝汤灌胃，阳性药物组予以塞来昔布溶液灌胃，每日1次，灌胃4周后留取大鼠膝关节软骨组织标本。HE染色和番红O-固绿染色观察大鼠膝关节软骨细胞病理形态学改变；RT-PCR检测CCL2、CCR2、Caspase-1 mRNA表达；Western blot检测CCL2、CCR2、Caspase-1、p-p65蛋白表达；免疫荧光双重染色定性检测大鼠软骨细胞焦亡情况。结果 与正常组比较，模型组大鼠软骨退变情况明显加剧，软骨焦亡水平上升，CCL2、CCR2、Caspase-1 mRNA表达水平明显增高（P<0.01），CCL2、CCR2、Caspase-1、p-p65蛋白表达水平明显增高（P<0.01）。与模型组比较，活膝汤组及阳性药物组大鼠软骨退变情况明显改善，软骨细胞焦亡水平下降。CCL2、CCR2、Caspase-1 mRNA表达水平明显降低（P<0.01），CCL2、CCR2、Caspase-1、p-p65蛋白表达水平明显降低（P<0.05，P<0.01）。与活膝汤组对比，阳性药物组Caspase-1 mRNA及蛋白表达水平显著增高（P<0.05，P<0.01）。结论 活膝汤可能通过降低CCL2、CCR2、Caspase-1、p-p65的表达水平，抑制大鼠软骨细胞焦亡，改善大鼠关节软骨的退变情况。 |
| 关键词: 活膝汤 膝骨关节炎 软骨 细胞焦亡 趋化因子配体2 趋化因子受体2 |
| DOI：10.3969/j.issn.1674-070X.2023.02.008 |
| Received:September 01, 2022 |
| 基金项目:湖南省中医药科研计划重点项目（2021048）；湖南省研究生科研创新资助项目（CX20200800）；湖南省常德市科技创新发展项目（2020S045）；全国第五批名老中医药专家学术继承导师；湖南省名中医邵先舫教授工作室建设项目。 |
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| The mechanism of Huoxi Decoction for chondrocyte pyroptosis in rats with knee osteoarthritis |
| XIE Fang,LIU Yongli,LI Shudong,DAI Binyu,LIU Tao,CHEN Shaojun,SHAO Xianfang |
| (The Hospital of Hunan Institute of Chinese Medicine, Changsha, Hunan 410006, China;Haikou Hospital of Chinese Medicine, Haikou, Hainan 570216, China;Changde Hospital of Hunan University of Chinese Medicine, Changde, Hunan 415000, China) |
| Abstract: |
| Objective To observe the effects of Huoxi Decoction on the morphological structure of knee joint cartilage and the expression levels of CC chemokine ligand 2 (CCL2), CC chemokine receptor 2 (CCR2), Caspase-1 and phosphorylated p65 (p-p65) in knee joint osteoarthritis model rats. Methods A total of 32 SPF rats were randomly divided into normal group (n=9) and model group (n=23). The model group of rat knee osteoarthritis was established by the modified Hulth method. After modeling, 2 rats were randomly selected from each group to verify the modeling effects. The remaining 21 rats in model group were randomly divided into three groups:model group, Huoxi Decoction group (12.81 mL/kg/d) and positive drug group (20 mg/kg/d), with 7 rats in each group. The normal group and model group were given the same volume of distilled water by gavage once a day, the Huoxi Decoction group was administered the Huoxi Decoction by gavage once a day, the positive drug group received the celecoxib solution by gavage once a day. After 4 weeks of gavage, the cartilage tissue samples of the rat knee joint were taken. HE staining and safranin O-fast green staining were taken to observe the pathological changes of chondrocytes in rat knee joint; the expression of CCL2, CCR2 and Caspase-1 mRNA was detected by RT-PCR; the expression of CCL2, CCR2, Caspase-1 and p-p65 was detected by Western blot; the pyroptosis of rat chondrocytes was detected by Tunel-IF (Caspase-1) fluorescence double staining. Results Compared with the normal group, the cartilage degeneration of model group was significantly aggravated, the level of cartilage pyrosis was raised, the expression levels of CCL2 mRNA, CCR2 mRNA and Caspase-1 mRNA were significantly higher (P<0.01), and the expression levels of CCL2, CCR2, Caspase-1 and p-p65 protein were significantly higher (P<0.01). Compared with model group, the cartilage degeneration of Huoxi Decoction group and positive drug group was significantly improved, and the level of chondrocyte apoptosis was reduced. The expression levels of CCL2 mRNA, CCR2 mRNA and Caspase-1 mRNA were significantly lower (P<0.01), and the expression levels of CCL2, CCR2, Caspase-1 and p-p65 protein were significantly lower (P<0.05, P<0.01). The expression levels of Caspase-1 mRNA and protein in positive drug group were significantly higher than those in Huoxi Decoction group (P<0.05, P<0.01). Conclusion Huoxi Decoction can inhibit the pyroptosis of rat chondrocytes and improve the degeneration of rat articular cartilage by reducing the expression levels of CCL2, CCR2, Caspase-1, and p-p65. |
| Key words: Huoxi Decoction knee osteoarthritis chondrocyte pyroptosis chemokine ligand 2 chemokine receptor 2 |
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