王枣子总黄酮对佐剂性关节炎大鼠的治疗作用及机制研究

目的研究王枣子中总黄酮(TFIA)对佐剂性关节炎(AA)的治疗作用及机制。方法采用注射完全弗氏佐剂制备AA模型大鼠后随机分为4组：AA组、AA+TFIA 50 mg/kg组、AA+TFIA 100 mg/kg组、AA+TFIA 150 mg/kg组，每组10只。另设空白对照组大鼠，不造模（n=10）。造模后4 d，TFIA各剂量组大鼠采用TFIA灌胃治疗，分别按上述3个剂量灌胃，每天1次，共24 d，空白对照组和AA组灌胃生理盐水。治疗第12天、16天、20天、24天采用大鼠关节炎评分法评价TFIA对AA大鼠的治疗作用。治疗第24天处死各组大鼠，分离大鼠滑膜组织，原代培养各组大鼠成纤维样滑膜细胞(FLS)，ELISA检测各组FLS中白细胞介素（IL-1）表达，MTT检测各组FLS增殖活力，real time qPCR检测各组FLS miR-152和经典Wnt信号通路关键基因〔β-catenin、细胞周期蛋白基因（ccnd1）〕表达。向AA大鼠FLS转染miR-152 mimics和对照NC mimics，向TFIA 100 mg/kg剂量组大鼠FLS转染miR-152 inhibitors和对照NC inhibitors，36 h后检测miR-152、β-catenin、ccnd1、IL-1表达和FLS的增殖。结果 TFIA各剂量灌胃治疗均能抑制AA大鼠关节炎评分，抑制β-catenin、ccnd1、IL-1表达和FLS增殖活力（P<0.05)，各剂量组间比较差异无统计学意义（P>0.05)，与空白对照组相比，各剂量组均未恢复至正常水平（P<0.05)。与空白对照组相比，AA组FLS中miR-152表达降低（P<0.05)，向AA组FLS转染miR-152 mimics后，与对照组相比，过表达的miR-152抑制β-catenin、ccnd1、IL-1表达，抑制FLS增殖（P<0.05)。向TFIA 100 mg/kg剂量组FLS转染miR-152 inhibitors，与对照组相比，抑制了miR-152的表达，促进了β-catenin、ccnd1、IL-1表达，促进了FLS增殖（P<0.05)。结论 TFIA对AA大鼠有一定治疗作用，此作用可能通过上调miR-152的表达，影响经典Wnt信号通路实现。

英文摘要:

Objective The therapeutic effect and mechanism of total flavonoids in Isodon amethystoides (Ben-th) Cy Wu et Hsuan (TFIA) on adjuvant arthritis (AA) were investigated. Methods AA model rats were set and complete Freund’s adjuvant injection, randomly divided into 4 groups: AA group, AA+TFIA 50 mg/kg group, AA+TFIA 100 mg/kg group, AA+TFIA 150 mg/kg group, and each group has 10 rats. Blank control group was set without modeling (n=10). Four days post-modeling rats in each TFIA groups were treated once a day with TFIA at 50 mg/kg, 100 mg/kg and 150 mg/kg for 24 d, and rats in blank control and AA groups were given saline as control. At the 12th day, 16th day, 20th day and 24th day of treatment, the effect of TFIA on AA rats was evaluated by rat arthritis score. Then the rats were sacrificed on the 24th day of treatment, and the synovial tissue of rats was isolated and the fibroblast-like synoviocytes (FLS) were primary cultured. The expressions of IL-1 in FLS was detected by ELISA, the FLS proliferation activity was detected by MTT assay, and the expression of miR-152, β-catenin and cyclin D1 gene (ccnd1) were detected by real time qPCR. MiR-152 mimics and NC mimics (control) were transfected into FLS in AA rats, and miR-152 inhibitors and NC inhibitors (control) were transfected into FLS in AA+TFIA 100 mg/kg group rats. The expressions of miR-152, β-catenin,ccnd1,IL-1 and FLS proliferation were detected 36 h post-transfection. Results TFIA significantly inhibited the arthritis socre of rats and the expressions of β-catenin,ccnd1,IL-1 and the proliferation of FLS in AA rats (P<0.05). There was no significant difference between the dose groups, all of which were significant when compared with the blank control group (P<0.05). Compared with the control group, the expression of miR-152 in AA group was significantly decreased (P<0.05). After transfection of miR-152 mimics into AA FLS, overexpression of miR-152 significantly inhibited the expressions of β-catenin,ccnd1,IL-1 and the proliferation of FLS (P<0.05). After transfection of miR-152 inhibitors into FLS from AA+TFIA 100 mg/kg group, inhibition of miR-152 significantly promoted the expressions of β-catenin,ccnd1,IL-1 and the proliferation of FLS. Conclusion TFIA has a certain therapeutic effect on AA rats via the up-regulation of miR-152 expression, possibly affecting the classical Wnt signaling pathway.

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