【摘要】 目的：探究附子治疗心肌肥大体外细胞模型的分子机制。方法：通过慢病毒载体pLVX-TetOne-Puro-HDAC2转染H9C2心肌细胞建立稳定表达外源性HDAC2基因的体外心肌肥大模型。采用qRT-PCR检测转染后的H9C2细胞中HDAC2mRNA水平，WB检测ANP以及BNP蛋白表达水平；选用附子水溶性生物碱以及水煎液对建立的体外心肌肥大模型进行给药处理，最后采用转录组学解析附子对其治疗的分子机制。结果：与对照组相比，重组慢病毒组中细胞的HDAC2 mRNA水平相对表达量显著升高（P<0.05）。转染后H9C2细胞表面积显著增加（P<0.05）,ANP和BNP蛋白的表达水平也显著提高（P<0.05）。表明HDAC2上调表达可能影响H9C2细胞表面积的增加和心肌肥大标志物的重新表达，心肌肥大体外模型构建成功。转录组分析显示附子主要通过激活ABC transporters和PI3K-Akt信号通路，抑制mTOR、JAKSTAT、MAPK、TNF、Calcium、Wnt、Ras以及p53等信号通路基因的表达从而缓解心肌肥大症状。结论：附子治疗心肌肥大的分子机制可能主要通过激活ATP转运体基因表达调控线粒体功能障碍，抑制钙离子信号、细胞生长、细胞凋亡和炎症反应等通路基因。更多还原

【Abstract】 Objective: To explore the molecular mechanism of Fuzi treating cardiac hypertrophy through an in vitro cell model. Method: An in vitro model of cardiac hypertrophy with stable expression of exogenous HDAC2 gene was established by transfecting H9C2 cardiomyocytes with the lentiviral vector pLVX-TetOne-Puro-HDAC2. The HDAC2 mRNA levels in the transfected H9C2 cells were detected by qRT-PCR, and the expression levels of ANP and BNP proteins were detected by Western blot. The in vitro model of cardiac hypertrophy was treated with the water-soluble alkaloids and aqueous decoction of Fuzi, and the molecular mechanism was analyzed by transcriptomics. Result: The relative expression of HDAC2 mRNA was significantly increased in the cells in the recombinant lentivirus group compared with the control group(P<0.05). The surface area of H9C2 cells was significantly increased after transfection(P<0.05), and the expression levels of ANP and BNP proteins were also significantly increased(P<0.05). These indicated that the upregulated expression of HDAC2 might affect the increase of surface area of H9C2cells and the re-expression of the markers of cardiac hypertrophy, suggesting that the in vitro model of cardiac hypertrophy was successfully established. The transcriptomic analysis showed that Fuzi alleviated the symptoms of cardiac hypertrophy mainly by activating ABC transporters and the PI3K-Akt signaling pathway, inhibiting the expression of mTOR, JAK-STAT, MAPK,TNF, Calcium, Wnt, Ras and p53 signaling pathway genes. Conclusion: The molecular mechanism of Fuzi in the treatment of cardiac hypertrophy may be mainly through activating ATP transporter gene expression to regulate mitochondrial dysfunction, and inhibiting the genes of calcium signal, cell growth, apoptosis and inflammatory response pathways.更多还原