花椒水提物通过抑制滑膜细胞增殖及诱导细胞凋亡治疗类风湿性关节炎  

Zanthoxylum bungeanum Aqueous Extract Treated Rheumatoid Arthritis by Inhibiting Synovial Cell Proliferation and Inducing Cell Apoptosis

作　　者：刘子琪 吴雨柔 张礼林 孙雪 袁星[1] 吴纯洁[1] LIU Ziqi;WU Yurou;ZHANG Lilin;SUN Xue;YUAN Xing;WU Chunjie(School of Pharmacy,The Second Affiliated Hospital,Innovative Institute of Chinese Medicine and Pharmacy,Chengdu University of Traditional Chinese Medicine,Chengdu 611137,China)

机构地区：[1]成都中医药大学药学院,第二附属医院,中药创新研究所,成都611137

出　　处：《中国实验方剂学杂志》2023年第20期30-38,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：四川省中医药管理局2020年度花椒药用价值研发专项(2020HJZX001)。

摘　　要：目的:探讨花椒水提物(ZBAE)对类风湿性关节炎的治疗作用。方法:将60只SD大鼠分为正常组、模型组[完全弗氏佐剂(CFA),10 mg·kg^(-1)]、甲氨蝶呤组(0.25 mg·kg^(-1))和ZBAE低、中、高剂量组(90、180、360 mg·kg^(-1))。甲氨蝶呤组腹腔注射给药2周,每周给药3次,ZBAE组连续灌胃给药14 d。给药期间,每3 d测量大鼠体质量和足跖肿胀度,同时进行关节炎评分。给药结束后,采用计算机断层(CT)扫描,苏木素-伊红(HE)染色及番红固绿染色观察ZBAE对滑膜增生及骨保护的作用。采用细胞增殖活性检测-8(CCK-8)试剂盒检测ZBAE对细胞增殖活力的影响;根据CCK-8实验结果确定最佳给药浓度与时间,设置空白组、ZBAE低、中、高质量浓度组(0.08、0.10、0.12 g·L^(-1))。流式细胞仪分析花椒作用24 h后细胞的周期分布和细胞凋亡率;采用体外划痕实验检测类风湿关节炎成纤维样滑膜细胞(RA-FLSs)迁移能力;蛋白免疫印迹法(Western blot)检测RA-FLSs中磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(Akt)、周期素依赖性激酶2(CDK2)、周期蛋白A(Cyclin A)及磷酸化(p)-PI3K、p-Akt蛋白的表达。结果:与正常组比较,模型组大鼠足肿胀度及关节炎评分明显升高(P<0.05),踝关节部位骨质严重受损,有明显的滑膜增生现象;与模型组比较,ZBAE各组的足肿胀程度明显降低(P<0.05),抑制了滑膜增生和软骨破坏现象。体外研究表明,与空白组比较,ZBAE能抑制RA-FLSs细胞的迁移(P<0.05),促进细胞凋亡(P<0.05),作用于RA-FLSs细胞的S期抑制细胞增殖。Western blot结果显示,与空白组比较,ZBAE高剂量组可明显降低p-PI3K、p-Akt、CDK2和Cyclin A蛋白表达(P<0.05)。结论:花椒水提物可能通过抑制滑膜增生、促进滑膜凋亡及抑制滑膜迁移对类风湿性关节炎起到治疗作用。Objective:To investigate the therapeutic effect of the water extract of Zanthoxylum bungeanum aqueous extract(ZBAE)on rheumatoid arthritis.Method:The sixty SD rats were divided into normal group,model group[complete Freund's adjuvant(CFA),10 mg·kg^(-1)],methotrexate(MTX)group(0.25 mg·kg^(-1)),low-,medium-,and high-dose ZBAE groups(90,180,360 mg·kg^(-1)).The rats in MTX group were given intraperitoneal injection for two weeks,three times a week,and the rats in ZBAE group were administrated for 14 days. The swelling of the ankle joint and body weight were observed, and arthritis scores were also performed. Computed tomography (CT), hematoxylin-eosin (HE) staining and safranine-O and fast green staining were used to observe the effect of ZBAE on synovial hyperplasia and bone protection. Cell counting kit-8(CCK-8)method was used to detect the proliferation of the RA-FLSs cells treated with ZBAE. According to the results of CCK-8 experiment, the optimal concentration and time of administration were determined, blank group, low -, medium -, and high-dose ZBAE groups (0.08,0.10,0.12 g·L^(-1)) were set up. The cell cycle distribution and apoptosis rate were analyzed by flow cytometry,the migration ability of RA-FLSs cells was examined by scratch test. Western blot was used to detect the effect of ZBAE on phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), cyclin-dependent kinase 2 (CDK2), Cyclin A and phosphorylated PI3K, Akt (p-PI3K,p-Akt) protein expression in RA-FLSs cells. Result: Compared with the normal group,joint swelling index and arthritis score were increased in the model group (P<0.05),the bone of the ankle was seriously damaged, and there was obvious synovial hyperplasia. Compared with the model group, the ZBAE group could significantly reduce the joint swelling index (P<0.05), inhibit synovial hyperplasia and cartilage destruction. In vitro study showed that compared with the blank group, ZBAE could inhibit the migration of RAFLSs (P<0.05), promoted cell apoptosis (P<0.05), and acted on RA-FL

关 键 词：花椒水提物 滑膜成纤维样细胞 类风湿关节炎 佐剂性关节炎 

分 类 号：R2-0[医药卫生—中医学] R33R593.22R289