理气活血滴丸含药血清对ISO诱导乳鼠心脏成纤维细胞转分化的影响及其机制研究

Study on the effects and mechanism of Liqi Huoxue Drop Pill-containing serum on ISO-induced transdif⁃ferentiation of suckling rat cardiac fibroblasts

目的:研究理气活血滴丸含药血清对异丙肾上腺素(isoprenaline,ISO)诱导的乳鼠心脏成纤维细胞(CFs)转分化的影响及其机制。方法:离体培养SD乳鼠CFs并经CCK-8法筛选理气活血滴丸含药血清的最适工作浓度。将细胞分为正常对照组(NC组)、空白大鼠血清组(BRS组)、ISO诱导模型组(NC+ISO组)、空白大鼠血清+ISO诱导模型组(BRS+ISO组)、理气活血滴丸含药血清+ISO诱导模型组(LQHXDP+ISO组)。生化法和ELISA法分别检测羟脯氨酸(Hyp)和TGF-β1水平,流式细胞术和Western blot分别检测细胞周期分布和CollagenⅠ、CollagenⅢ、α-SMA、IL-6、p-STAT3、STAT3蛋白表达情况。结果:经Vimentin免疫荧光鉴定所得细胞为CFs,10%的理气活血滴丸以175.0 mg·kg^(-1)剂量灌胃大鼠制备的含药血清为最佳工作浓度;与NC和BRS组相比,经ISO诱导的CFs增殖活力显著增加,细胞培养上清液中Hyp、TGF-β1含量显著增加,S期细胞显著增多,CollagenⅠ、CollagenⅢ、α-SMA、IL-6、p-STAT3蛋白表达显著升高;与BRS+ISO组相比,LQHXDP+ISO组CFs细胞增殖活力下降,Hyp、TGF-β1含量显著降低,S期细胞显著减少,CollagenⅠ、CollagenⅢ、α-SMA、IL-6、pSTAT3蛋白表达显著降低。结论:理气活血滴丸含药血清可明显抑制ISO诱导CFs的转分化,该作用与其抑制ISO诱导CFs的增殖活力,减少Hyp、TGF-β1的分泌,减少胶原蛋白生成和抑制IL-6/STAT3信号通路有关。

OBJECTIVE To investigate the effect of Liqi Huoxue Drop Pill-containing serum on isoprenaline(ISO)induced transdifferentiation of suckling rat cardiac fibroblasts(CFs)and its mechanism.METHODS In vitro culture of SD suckling mouse cardiac fibroblasts,and the CCK-8 method was used to screen the optimal working concentration of Liqi Huoxue Drop Pilll-containing serum.The cells were divided into normal control group(NC group),blank rat serum group(BRS group),ISO induced model group(NC+ISO group),blank rat serum+ISO induced model group(BRS+ISO group),Liqi Huoxue Drop Pill-containing serum+ISO induced model group(LQHXDP+ISO group).The levels of hydroxyproline(Hyp)and TGF-β1 were detected by biochemical assay and ELISA,respectively,and the cell cycle distribution and protein expression of CollagenⅠ,CollagenⅢ,α-SMA,IL-6,p-STAT3 and STAT3 were detected by flow cytometry and Western blot,respectively.RESULTS The cells identified by Vimentin immunofluorescence as CFs,and the drug-containing serum prepared by 10%Liqi Huoxue Drop Pill at a dose of 175.0 mg·kg^(-1) was the optimal working concentration.Compared with the NC and BRS groups,the proliferation viability of ISO-induced CFs was significantly increased,the contents of Hyp and TGF-β1 in the cell culture supernatant were significantly increased,the number of S phase cells was significantly increased,and the expression of CollagenⅠ,CollagenⅢ,α-SMA,IL-6 and p-STAT3 proteins was significantly increased.Compared with the BRS+ISO group,the proliferation activity of CFs in the LQHXDP+ISO group decreased,the content of Hyp and TGF-β1 was significantly reduced,the S phase cells were significantly reduced,and the expression of CollagenⅠ,CollagenⅢ,α-SMA,IL-6 and p-STAT3 pro⁃teins were significantly reduced.CONCLUSION Liqi Huoxue Drop Pill-containing serum can significantly inhibit the transdiffer⁃entiation of ISO-induced CFs,which is related to its inhibition of ISO-induced proliferation and activity of CFs,reduce the secre⁃tion of Hyp and TGF-β1,reduce collagen production and inhibit IL-6/STAT3 signaling pathway.