Differential Gene Expression Profiles of PASMCs Induced by Quercetin under Hypoxia. Rattus norvegicus

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. Overall design: G1(normoxia1,normoxia2,normoxia3),G2(hypoxia1,hypoxia2,hypoxia3)and G3(hypoxia+quercetin 1,hypoxia+quercetin 2,hypoxia+quercetin 3)

低氧性肺动脉高压（hypoxic pulmonary arterial hypertension, PAH）血管重构过程中的关键发病环节，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，这类改变包括持续性血管收缩、细胞异常增殖以及PASMC的凋亡抵抗。
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血管收缩，但其具体分子调控机制尚未完全阐明。
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），分析低氧暴露及最优浓度槲皮素处理下PASMC的整体转录应答情况。与常氧对照组相比，低氧处理组诱导1694个基因上调、2091个基因下调；槲皮素处理组则出现1790个基因上调、1450个基因下调。
已知槲皮素可调控多个与上述功能相关的差异表达基因：其中，促进增殖、诱导凋亡的基因包括Cycs、Ppp3ca、Prkar2b、Akt3、Ppp3cc、Il1rap、Ntrk1；阻滞细胞周期的基因包括Chek2、Cdkn1c、Gadd45b、Stag2、Anapc7、Orc1、Ccne1、Myc3、Skp1、Espl1、Cdc45、Mcm4；逆转PASMC收缩功能的基因包括Ramp1、Ramp3、Adcy5、Gnas、Prkcd、Itpr3、Adra1d、Calm1、Npr1、Avpr1a、Ednra、Adcy8。
本研究通过实时定量逆转录聚合酶链式反应（Real-time quantitative RT-PCR）验证了微阵列的检测结果。
综上，槲皮素可重塑低氧处理下PASMC的基因表达谱，有助于进一步探究槲皮素干预低氧性PAH的潜在作用机制。
整体实验设计：G1（常氧组1、常氧组2、常氧组3）、G2（低氧组1、低氧组2、低氧组3）以及G3（低氧+槲皮素组1、低氧+槲皮素组2、低氧+槲皮素组3）