探讨活血化瘀方剂对肺间质纤维化TGF-β/Smad2/3信号转导通路糖基化修饰的影响

Explore the effect of the prescription of Huoxuehuayu Chinese herbs on glycosylation modifi cation of TGF-β/Smad2/3 signal transduction pathway in pulmonary fi brosis

目的:明确活血化瘀方剂是否影响TGF-β/Smad2/3信号通路中TGF-β受体糖基化修饰,用现代医学手段阐明活血化瘀方剂治疗肺间质纤维化的机制。方法:胚胎成纤维细胞WI-38细胞随机分为3组,①正常组为单独的DMEM培养;②TGF组加入浓度为10ng/mL的TGF-β1孵育48h;③TGFH组先用10%含活血化瘀方剂血清孵育24h,然后加入10ng/mL TGF-β1继续孵育48h。应用荧光定量PCR、免疫荧光、免疫印记技术检测各组WI-38细胞的FUT8、细胞的岩藻糖基化水平,TGF-β受体ALK5及TGF-βRⅡ的表达,应用ELISIA技术检测细胞培养液中Ⅰ、Ⅲ、Ⅳ型胶原纤维(COLⅠ、Ⅲ、Ⅳ)和纤维连接蛋白(FN)的变化。结果:活血化瘀方剂能明显地抑制WI-38细胞的FUT8的表达和细胞的岩藻糖基化水平,活血化瘀方剂对TGF-β受体ALK5及TGF-βRⅡ的表达无明显影响,但可以减少TGF-β1对WI-38细胞p-Smad2/3的影响,使其表达降低。COLⅠ在TGF组中表达约为正常组的1.5倍,COLⅢ在TGF组中表达约为正常组的2.5倍,COLⅣ在TGF组中表达约为正常组的1倍,FN在TGF组中表达约为正常组的2.5倍,TGFH组COLⅠ、Ⅲ、Ⅳ及FN表达略高于正常组,与TGF组间具有统计学差异。结论:活血化瘀方剂可以抑制TGF-β诱导后肺成纤维细胞ALK5/TGF-βRⅡ-Smad2/3信号通路的糖基化修饰,减少Ⅰ、Ⅲ、Ⅳ型胶原纤维和FN的表达。

Objective： To find whether the prescription of Huoxuehuayu Chinese herbs effects glycosylation of the receptor of TGF-β and the signaling pathway of TGF-β/Smad2/3 and to explain the mechanism of prescription of Huoxuehuayu Chinese herbs for treatment of pulmonary fibrosis by modern medicine means. Methods： The WI-38 cells were divided randomly into 3 groups： ①normal group： cultured in DMEM medium; ②TGF group： cultured in 10 ng/mL TGF-β1 medium for 48 hours; ③TGFH group： cultured in 10% prescription of Huoxuehuayu Chinese herbs with rat serum for 24 hours ,then added 10 ng/ml TGF-β1 into the medium and cultured for another 48 hours. The expression of FUT8, LCA, ALK5 and TGF-βRⅡ in the three groups were detected by real-time PCR, immunofluorescence and western blotting, and the level of collagenⅠ, Ⅲ Ⅳ（COL Ⅰ , Ⅲ, Ⅳ） and fibronectin （FN） of the three groups were detected by ELISA. Results： The prescription of huoxuehuayu Chinese herbs could inhibit the expression of FUT8 and cell fucosylation level in WI-38 cell. Prescription of huoxuehuayu Chinese herbs didn＇t affect the expression of ALK5 and TGF-βR Ⅱ but it reduced the expression of TGF-β1 on p-Smad2/3 in WI-38 cell. The expression of COL I was 1.5 times higher in TGF group as in normal group. The expression of COLⅡ was 2.5 times higher in TGF group as in normal group. The COLⅣ was 1 times higher in TGF group as in normal group. The FN was 2.5 times higher in TGF group as in normal group （P〈0.05）. The expression of COL Ⅰ, COLⅢ, COLⅣ and FN in TGFH group was a little higher than in normal group, but there was no significantly difference between two groups. Conclusion： The prescription of Huoxuehuayu Chinese herbs can inhibit TGF-β-induced lung fibroblast ALK5/TGF-βR Ⅱ -Smad2/3 signaling pathway glycosylation. It can reduce the expression of collagen of Ⅰ, Ⅱ, Ⅳ and FiN.