宁心涤痰汤通过miR-150/Stat1通路调控巨噬细胞极化抑制支架内再狭窄的机制研究

Role of Ningxin Ditan Decoction in In-sent Restenosis by Regulating Macrophage Polarization Through miR-150

目的 观察宁心涤痰汤对miR-150/Stat1通路介导的巨噬细胞极化的影响,从而阐明其抑制支架内血管再狭窄的作用机制。方法 通过脂多糖诱导复制巨噬细胞炎症模型,以宁心涤痰汤含药血清或miR-150模拟剂干预。采用ELISA法检测细胞上清中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、TGF-β和白细胞介素-10(IL-10)的含量,流式细胞术检测巨噬细胞极化表型的变化,Western blotting和RT-PCR法检测miR150/Stat1通路中相关分子的表达水平。在此基础上,提取上述各组细胞分别与内皮祖细胞进行共培养,采用CCK8法和流式细胞术检测巨噬细胞对其活性和凋亡的影响。结果 与模型组相比,宁心涤痰汤能够显著增加巨噬细胞中TGF-β、IL-10的含量和CD206阳性细胞百分比,降低TNF-α、IL-6的含量和CD86阳性细胞百分比,上调miR-150和下调p-Stat1蛋白的表达,差异均具有统计学意义(P <0.01),然而各组间Total-Stat1蛋白的表达水平无明显改变,差异无统计学意义(P> 0.05)。同时,宁心涤痰汤还能够显著促进内皮祖细胞的活性,抑制其凋亡水平,差异均具有统计学意义(P <0.01)。结论 宁心涤痰汤通过抑制miR-150/Stat1信号通路的活化从而促进巨噬细胞的M1向M2型的转化,继而增加内皮祖细胞的活性,达到治疗PCI术后支架内血管再狭窄的作用。

Objective:To observe the effect of Ningxin Ditan Decoction on macrophage polarization mediated by miR-150/Statl pathway,to clarify the mechanism of Ningxin Ditan Decoction in inhibiting in-stent restenosis.Methods:Macrophage inflammation model was established by lipopolysaccharide inducing RAW264.7 and was intervened by Ningxin Ditan Decoction containing serum or miR-150 mimic.The contents of TNF-α,IL-6,TCF-β,and IL-10 in cell supernatant were detected by ELISA.The changes of macrophage polarization phenotype were detected by flow cytometry,and the expression levels of related molecules in miR150/Statl pathway were detected by Western blotting and RT-PCR.Furthermore,macrophage was extracted and co-cultured with endothelial progenitor cells,and the effects of macrophages on activity and apoptosis of endothelial progenitor cells were de-tected by CCK8 and flow cytometry.Results:Compared with the model group,Ningxin Ditan Decoction could sig-nificantly increase the contents of TGF-β,IL-10 and the percentage of CD206 positive cells(P<0.01),while the contents of TNF-α,IL-6 and the percentage of CD86 positive cells were significantly decreased in the Ningxin Di-tan Decoction group(P<0.01).Ningxin Ditan Decoction could also significantly upregulate the expression level of miR-150 and downregulate the expression level of p-Statl protein(P<0.01),with on effect on the expression lev-el of total-Statl protein(P>0.01).At the same time,Ningxin Ditan Decoction could also significantly promote the activity of endothelial progenitor cells and inhibit their apoptosis levels,with significant differences(P<0.01).Conclusion:Ningxin Ditan Decoction can promote the transformation of macrophage M1 into M2 by inhibiting the activation of miR-150/Statl signal pathway,and then increase the activity of endothelial progenitor cells,so as to achieve the effect of treating In-stent restenosis after PCI.