Differential Gene Expression Profiles of PASMCs Induced by Quercetin under Hypoxia

As the critical step of pathogenesis during hypoxic pulmonary arterial hypertension (PAH) vascular remodeling is closely associated with pulmonary arterial smooth muscle cell (PASMC) alterations induced by hypoxia that include persistent vasoconstriction, abnormal proliferation and PASMC resistance to apoptosis. Quercetin is a flavonoid compound extracted from green plants that inhibits proliferation, induces apoptosis, arrests the cell cycle, and rescues the constriction of PASMCs, but the underlying mechanisms remain poorly understood. In this study, we used a commercial Agilent Whole Rat Genome Oligo Microarray to determine the overall transcriptional response of PASMCs in response to exposure to hypoxia and the optimal concentration of quercetin. Hypoxia induced the upregulation of 1694 genes and the downregulation of 2091 genes compared with the normoxia group. Quercetin treatment resulted in 1790 upregulated genes and 1450 downregulated genes. Quercetin is known to cause differential expression of several of these genes that are known to promote proliferation, induce apoptosis (Cycs, Ppp3ca, Prkar2b, Akt3, Ppp3cc, Il1rap, Ntrk1), arrest the cell cycle (Chek2, Cdkn1c, Gadd45b, Stag2, Anapc7, Orc1, Ccne1, Myc3, Skp1, Espl1, Cdc45, Mcm4), and rescue PASMC constriction (Ramp1, Ramp3, Adcy5, Gnas, Prkcd, Itpr3, Adra1d, Calm1, Npr1, Avpr1a, Ednra, Adcy8). Real-time quantitative RT-PCR was performed to verify the microarray results. In conclusion, quercetin altered the expression profile of many genes regulated by hypoxia in PASMCs, which helps to further explore the mechanism of the effects of quercetin treatment on hypoxic PAH.低氧性肺动脉高压（hypoxic pulmonary arterial hypertension, PAH）血管重构的关键发病环节，与低氧诱导的肺动脉平滑肌细胞（pulmonary arterial smooth muscle cell, PASMC）异常改变密切相关，这类改变包括血管持续性收缩、细胞异常增殖以及PASMC对细胞凋亡的抵抗。槲皮素是一类从绿色植物中提取的黄酮类化合物，可抑制PASMC增殖、诱导其凋亡、阻滞细胞周期并逆转其异常收缩状态，但其具体作用机制仍不甚明确。本研究采用商业化安捷伦全大鼠基因组寡核苷酸微阵列（Agilent Whole Rat Genome Oligo Microarray），检测PASMC在低氧暴露及不同浓度槲皮素处理下的整体转录应答情况，并筛选槲皮素的最优作用浓度。与常氧组相比，低氧处理可诱导1694个基因上调、2091个基因下调；而槲皮素处理则导致1790个基因上调、1450个基因下调。已知槲皮素可调控多个参与上述病理过程的差异表达基因：其中，促进增殖、诱导凋亡的相关基因包括Cycs、Ppp3ca、Prkar2b、Akt3、Ppp3cc、Il1rap、Ntrk1；阻滞细胞周期的相关基因包括Chek2、Cdkn1c、Gadd45b、Stag2、Anapc7、Orc1、Ccne1、Myc3、Skp1、Espl1、Cdc45、Mcm4；逆转PASMC异常收缩状态的相关基因包括Ramp1、Ramp3、Adcy5、Gnas、Prkcd、Itpr3、Adra1d、Calm1、Npr1、Avpr1a、Ednra、Adcy8。本研究通过实时定量反转录聚合酶链反应（real-time quantitative RT-PCR）验证了微阵列检测结果。综上，槲皮素可调控低氧处理后PASMC的大量差异表达基因，该结果有助于进一步探索槲皮素干预低氧性PAH的分子机制。