山楂来源外泌体样纳米囊泡通过调控SOX9信号通路抑制AngⅡ和TGF-β诱导的心脏成纤维细胞活化

Hawthorn-derived exosome-like nanovesicles inhibit AngⅡ- and TGF-β-induced cardiac fibroblast activation by regulating the SOX9 signaling pathway

摘要:

目的]研究山楂来源外泌体样纳米囊泡(HDELN)抑制血管紧张素Ⅱ(AngⅡ)和转化生长因子β(TGF-β)诱导的大鼠心脏成纤维细胞(RCF)活化的作用机制。 [方法]体外提取并培养原代RCF,使用AngⅡ和TGF-β诱导RCF活化,通过CCK-8法测定细胞存活率,采用Western blot检测蛋白表达水平,采用RT-qPCR检测mRNA表达水平,利用细胞划痕实验检测细胞迁移能力,利用siRNA转染RCF构建SRY盒转录因子9(SOX9)低表达细胞模型。 [结果]CCK-8法检测结果显示,AngⅡ和TGF-β可显著提升RCF的细胞活力(P＜0.05)。RT-qPCR结果显示,AngⅡ和TGF-β可上调RCF中Ⅰ型胶原蛋白α1(COL1A1)、Ⅲ型胶原蛋白α1(COL3A1)、α-平滑肌肌动蛋白(α-SMA)、骨膜蛋白(Postn)、Runt相关转录因子2(RUNX2)的mRNA表达水平(P＜0.05)。Western blot结果显示,AngⅡ和TGF-β可上调RCF中COL1A1、COL3A1和α-SMA的蛋白表达水平(P＜0.05)。细胞划痕实验结果表明,AngⅡ和TGF-β可显著提高RCF的迁移能力(P＜0.05)。差速离心法和蔗糖梯度密度离心法提取HDELN。加入HDELN后,AngⅡ和TGF-β诱导的RCF活化现象被显著逆转(P＜0.05)。Western blot和RT-qPCR结果显示,AngⅡ和TGF-β可上调RCF中SOX9的表达水平,而HDELN则下调其表达水平(P＜0.05)。构建SOX9低表达细胞模型后,实验结果显示,低表达SOX9的RCF活化水平显著低于正常RCF(P＜0.05)。 [结论]HDELN可通过抑制SOX9信号通路,进而抑制由AngⅡ和TGF-β诱导的RCF活化。

Abstract:

Aim To investigate the mechanism by which hawthorn-derived exosome-like nanovesicles (HDELN) on inhibiting the activation of rat cardiac fibroblasts (RCF) induced by angiotensinⅡ(AngⅡ) and transforming growth factor-β (TGF-β). Methods Primary RCF was extracted and cultured in vitro. RCF activation was induced by AngⅡ and TGF-β, and the cell viability was detected by CCK-8 assay. Western blot was used to detect protein expression levels. RT-qPCR was used to detect the mRNA expression levels. Cell scratch assay was used to detect cell migration ability. siRNA was transfected into RCF to construct a cell model with low expression of SRY-box transcription factor 9 (SOX9) low expression cell model. Results CCK-8 results showed that AngⅡ and TGF-β could enhance the cell viability of RCF (P＜0.05). RT-qPCR results showed that AngⅡ and TGF-β upregulated the mRNA expression of typeⅠcollagen α1 (COL1A1), type Ⅲ collagen α1 (COL3A1), α-smooth muscle actin (α-SMA), periostin (Postn), Runt-related transcription factor 2 (RUNX2) in RCF (P＜0.05). Western blot results showed that AngⅡ and TGF-β upregulated the protein expression levels of COL1A1, COL3A1, and α-SMA in RCF (P＜0.05). The results of cell scratch assay showed that AngⅡ and TGF-β markedly promoted the migration ability of RCF (P＜0.05). HDELN was extracted by differential centrifugation and sucrose gradient density centrifugation. The activation of RCF induced by AngⅡ and TGF-β was reversed by the addition of HDELN (P＜0.05). Western blot and RT-qPCR results showed that AngⅡ and TGF-β upregulated the expression levels of SOX9 in RCF, while HDELN downregulated its expression (P＜0.05). After constructing a cell model with low expression of SOX9, the experimental results showed that the activation level of RCF with low expression of SOX9 was significantly lower than that of normal RCF (P＜0.05). Conclusion HDELN can inhibit the activation of RCF induced by AngⅡ and TGF-β via suppressing the SOX9 signaling pathway.