粉防己碱对类风湿模型作用的体内与体外试验

Effect of tetrandrine on rheumatoid model in vivo and in vitro

摘要:

[目的]探讨粉防己碱对蛋白聚糖诱导的类风湿性关节炎的体内与体外作用，探讨其可能的作用机制。[方法]体内试验，42 只 BALB/C 小鼠随机分为正常组、模型组、粉防己碱低量组、中量组、高量组和对照组。除正常组外，其他动物给予蛋白聚糖诱导的类风湿性关节炎（proteoglycan-induced arthritis, PGIA）小鼠模型。低量组每天给予粉防己碱 7.5 mg/kg，中量组每天 15 mg/kg，高量组每天 30 mg/kg 灌胃，正常组和模型组均给予等体积的生理盐水，对照组每天给予塞来昔布 60.7 mg/kg， 连续治疗 28 d。体外试验，MH7A 细胞同上分组给予粉防己碱处理。[结果] 体内实验：用药第 28 d，与模型组相比，低、 中、高量组和对照组在后爪厚度 [(5.1±0.1) mm vs (3.6±0.2) mm vs (3.0±0.2) mm, (2.4±0.2) mm, (3.6±0.1) mm, P＜0.001]、关节炎评分 [7.0 (7.0, 7.0) vs 5.0 (4.0, 5.0) vs 4.0 (4.0, 4.0), 2.0 (1.0, 2.0), 2.0 (1.0, 2.0), P＜0.001]、血清中 TNF-α [(320.6±7.2) pg/mL vs (264.5± 13.1) pg/mL vs (217.3±40.0) pg/mL vs (159.8±17.2) pg/mL vs (160.7±11.3) pg/mL, P＜0.001]、IL-1β [(365.1±32.2) pg/mL vs (217.7± 18.8) pg/mL vs (161.1±18.3) pg/mL vs (110.6±4.8) pg/mL vs (110.4±3.8) pg/mL, P＜0.001]、IL-6 含量 [(435.5±33.3) pg/mL vs (261.7± 30.6) pg/mL vs (189.0±8.1) pg/mL vs (152.9±13.3) pg/mL vs (154.7±11.1) pg/mL, P＜0.001]、组织学评分和 μCT 检查评分均有显著改善 （P＜0.05）。体外实验：与模型组相比，低、中、高量组在 EdU 阳性细胞数量、p-Akt 和 p-P65 的蛋白表达水平、M2 巨噬细胞浸润和激活状态的肥大细胞浸润均显著改善（P＜0.05）。[结论]粉防己碱可抑制类风湿模型的炎症进展和滑膜增生，其机制可能与调控 AKT 和 NF-κB 信号通路有关。

Abstract:

[Objective] To investigate the effects of tetrandrine (TET) on proteoglycan-induced rheumatoid arthritis (RA) in vivo and in vitro, search the possible mechanism of action. [Methods] In vivo experiment, 42 BALB/C mice were randomly divided into the normal control group, model group, as well as tetrandrine low dose (LD) group, medium dose (MD) group, high dose (HD) group and control group. Except the normal group, all animals in other groups underwent proteoglycan-induced arthritis (PGIA) treatment to create RA mouse model. The LD group was given TET 7.5 mg/kg · d-1 , the MD group was 15 mg/kg · d-1 , the HD group was 30 mg/kg · d-1 , while the normal group and the model group were given equal volume of normal saline, and the control group was given celecoxib 60.7 mg/kg · d-1 for 28 days. In vitro experiments, MH7A cells were treated with TET in similar manner as in vivo test. [Results] In vivo experiment, compared with the model group, the LD group, MD group, HD groups and the control group had significant improvements in terms of the paw thickness [(5.1± 0.1) mm vs (3.6±0.2) mm vs (3.0±0.2) mm vs (2.4±0.2) mm vs (3.6±0.1) mm, P＜0.001], arthritis score [7.0 (7.0, 7.0) vs 5.0 (4.0, 5.0) vs 4.0 (4.0, 4.0) vs 2.0 (1.0, 2.0) vs 2.0 (1.0, 2.0), P＜0.001], serum TNF-alpha [(320.6±7.2) pg/mL vs (264.5±13.1) pg/mL vs (217.3±40.0) pg/mL vs (159.8±17.2) pg/mL vs (160.7±11.3) pg/mL, P＜0.001], IL-1 beta [(365.1±32.2) pg/mL vs (217.7±18.8) pg/mL vs (161.1±18.3) pg/mL vs (110.6 ±4.8) pg/mL, (110.4±3.8) pg/mL, P＜0.001], IL-6 [(435.5±33.3) pg/mL vs (261.7±30.6) pg/mL vs (189.0±8.1) pg/mL vs (152.9±13.3) pg/mL vs (154.7±11.1) pg/mL, P＜0.001], the histological score (P＜0.05) and μCT score (P＜0.05). In vitro experiment, compared with the model group, the LD group, MD group and HD groups had significant improvements in terms of number of EdU positive cells (P＜0.05), PAkt and P-P65 protein expression levels (P＜0.05), infiltration of M2 macrophages and infiltration of activated mast cells (P＜0.05). [Conclusion] Tetrandrine can inhibit the progression of inflammation and synovial hyperplasia in rheumatoid models, and the mechanism may be related to the regulation of AKT and NF-κB signaling pathways.