中国寄生虫学与寄生虫病杂志 ›› 2011, Vol. 29 ›› Issue (2): 3-93-98.

• 论著 • 上一篇    下一篇

芍药苷通过IL-13/STAT6信号转导通路抑制成纤维细胞产生胶原

杜明占1,沈继龙2,吴强1,胡向阳1,储德勇2 *   

  1. 安徽医科大学1 病理学教研室;2 病原生物学教研室,安徽病原生物学省级实验室,人兽共患病安徽省重点实验室,合肥 230032
  • 出版日期:2011-04-30 发布日期:2012-09-27

Inhibitory Effect of Paeoniflorin on the Collagen Production by Fibroblasts through IL-13/STAT6 Signaling Pathway

DU Ming-zhan1,SHEN Ji-long2,WU Qiang1,HU Xiang-yang1,CHU De-yong2 *   

  1. 1 Department of Pathology:2 Department of Microbiology and Parasitology,Anhui Key Laboratory of Microbiology and Parasitology,Anhui Key Laboratory of Zoonoses,Anhui Medical University,Hefei 230032,China
  • Online:2011-04-30 Published:2012-09-27

摘要: 目的  观察芍药苷通过白细胞介素13(IL-13)信号转导通路调控3T3成纤维细胞的细胞激活、增殖和胶原产生。 方法  分别用不同浓度的芍药苷(200、 400、 600、 800和1 000 mg/L)或重组白介素13(rIL-13, 6.25、 12.5、 50、100和200 μg/L),在体外刺激经无血清培养基培养12 h的3T3细胞,同时设空白对照组。培养24 h后,用细胞计数试剂盒-8(CCK-8)检测rIL-13和芍药苷对细胞增殖的影响,并根据其结果选择1个适宜的rIL-13刺激浓度。用该浓度rIL-13(100 μg/L)刺激无血清培养基培养12 h的3T3细胞12 h后,再分别加入不同浓度的芍药苷(200、 400、 600、 800和1 000 mg/L),继续培养24 h,同时设空白对照组。用CCK-8法检测细胞增殖情况,碱裂解法测定培养上清羟脯氨酸含量。蛋白质印迹(Western blotting)分析α-平滑肌肌动蛋白(α-SMA)、 白介素13受体α1 (IL-13Rα1)和信号转导和转录激活因子6(STAT6)的表达情况。RT-PCR检测细胞Ⅰ型胶原(Col-Ⅰ)、 Ⅲ型胶原(Col-Ⅲ)、 IL-13Rα1和STAT6的mRNA水平。 结果  芍药苷能浓度依赖性地抑制3T3细胞增殖(r=-0.980, P<0.01),且各浓度组间差异有统计学意义(F=198.599, P<0.01)。rIL-13能明显促进3T3细胞增殖(r=0.538, P<0.05)。芍药苷(200、 400、 600、 800和1 000 mg/L)能浓度依赖性地抑制rIL-13刺激的3T3细胞增殖(1.780±0.177、 1.636±0.073、 0.965±0.066、 0.623±0.037和0.337±0.022, r=-0.971, P<0.01),且各浓度组间差异有统计学意义(F=198.537, P<0.01)。芍药苷还能浓度依赖性地抑制rIL-13刺激的3T3细胞分泌羟脯氨酸(3.030±0.094、2.976±0.047、2.814±0.047、2.652±0.124和2.408±0.124,r=-0.916,P<0.01),且各浓度组间差异有统计学意义(F=13.642, P<0.01)。Western blotting分析结果显示,芍药苷能抑制rIL-13刺激的3T3细胞α-SMA、IL-13Rα1和STAT6蛋白的表达。RT-PCR结果显示,芍药苷能抑制rIL-13刺激的3T3细胞Col-Ⅰ、Col-Ⅲ、IL-13Rα1和STAT6 mRNA的转录水平。 结论  芍药苷通过IL-13/STAT6信号转导通路抑制成纤维细胞增殖、激活和胶原的产生,可能是芍药苷抗日本血吸虫病肝纤维化的机制之一。

关键词: 芍药苷;成纤维细胞;IL-13;IL-13R&alpha, 1;Ⅰ型胶原;Ⅲ型胶原

Abstract: Objective   To observe the effects of paeoniflorin on 3T3 fibroblast activation, proliferation and collagen production through IL-13/STAT6 signaling pathway.   Methods   3T3 cell strain was cultured with serum-free medium for 12 h, then stimulated by paeoniflorin (200, 400, 600, 800, and 1 000 mg/L) or rIL-13 (6.25, 12.5, 50, 100, and 200 μg/L) for another 24 h. At the same time the blank control group for paeoniflorin or rIL-13 was observed. 3T3 cell proliferation was assayed by Cell Counting Kit-8 (CCK-8), and an appropriate concentration (100 μg/L) of rIL-13 was chosen according to the result of cell proliferation. Subsequently, 3T3 cell cultured with serum-free medium for 12 h was stimulated by 100 μg/L rIL-13 for 12 h, and then was treated with different concentrations of paeoniflorin (200, 400, 600, 800, and 1 000 mg/L) for another 24 h. Untreated 3T3 cell served as blank control. Cell proliferation was measured by CCK-8. Hydroxyproline content in cell supernatant was determined by alkaline lysis method. IL-13Rα1, α-SMA and STAT6 protein expression were detected by Western blotting. Col-Ⅰ, Col-Ⅲ, IL-13Rα1 and STAT6 mRNA expression were analyzed by RT-PCR.   Results   Pae-oniflorin inhibited 3T3 cell proliferation in a concentration-dependent manner (r=-0.980, P<0.01), and there was a statistically significant difference among all groups (F=198.599, P<0.01). rIL-13 caused a remarkably concentration-dependent increase in proliferation of 3T3 cells (r=0.538, P<0.05). Paeoniflorin (200, 400, 600, 800, and 1 000 mg/L) inhibited proliferation of 3T3 cell stimulated by rIL-13 in a concentration-dependent manner (1.780±0.177, 1.636±0.073, 0.965±0.066, 0.623±0.037, 0.337±0.022, r=-0.971, P<0.01), and among all groups there existed a significant difference (F=198.537, P<0.01). Moreover, paeoni-florin also suppressed secretion of hydroxyproline from 3T3 cell stimulated by rIL-13 in a concentration-dependent manner (3.030±0.094, 2.976±0.047, 2.814±0.047, 2.652±0.124, 2.408±0.124, r=-0.916, P<0.01) with a statistical significance among all groups (F=13.642, P<0.01). Further investigations showed that paeoniflorin decreased both protein expression of α-SMA, IL-13Rα1, and STAT6, and mRNA expression of Col-Ⅰ, Col-Ⅲ, IL-13Rα1, and STAT6 in 3T3 cell stimulated by rIL-13.  Conclusion   Paeoniflorin inhibits activation, proliferation of fibroblasts and production of collagen from fibroblasts through IL-13/STAT6 signaling pathway, which might be one of mechanisms of anti-hepatic fibrosis of paeoniflorin in schistosomiasis japonica.

Key words: Paeoniflorin;Fibroblast;Interleukin-13;Interleukin-13R&alpha, 1;CollagenⅠ;CollagenⅢ