槲皮素对低氧肺动脉高压的防治作用及其机制研究

发布时间：2018-08-30 12:31

【摘要】：一、槲皮素对大鼠低氧肺动脉高压防治作用的研究目的：体内实验探讨槲皮素对低氧肺动脉高压的作用效果,为肺动脉高压治疗寻求新的方式及治疗靶点。方法：随机将30只雄性Sprague-Dawley大鼠分为常氧组、低氧组、低氧+槲皮素组,每组10只。持续4周后,在生理多导仪监视下,通过右心漂浮导管测定大鼠体循环压、右心室收缩压、右心室比重[右心室／(左心室+室间隔)],免疫组化检测各组大鼠肺小动脉中PCNA、Ki67以及TUNEL的表达变化。结果：槲皮素可显著缓解低氧肺动脉高压(P0.05),逆转右心室肥厚以及肺血管重塑(P0.05),并导致肺小动脉PASMCs增殖减少,凋亡增加。结论：槲皮素可有效促进大鼠PASMCs增殖减少、凋亡增加,缓解低氧肺血管重塑,逆转低氧肺动脉高压。二、槲皮素通过调控TrkA/AKT信号通路缓解低氧肺动脉高压目的：探讨槲皮素抑制低氧肺动脉平滑肌细胞(PASMC)增殖和诱导细胞凋亡从而缓解缺氧肺动脉高压的潜在的分子机制,为槲皮素治疗低氧肺动脉高压进一步提供理论基础。方法：分离、培养大鼠PASMC,在Oxycycler model C21低氧细胞培养箱中建立细胞低氧模型。MTT法检测槲皮素的IC50,流失细胞法检测槲皮素浓度梯度下细胞周期及凋亡细胞数,Western-blot检测周期蛋白及凋亡蛋白的变化；Tanswell实验检测槲皮素对低氧PASMCs迁移的影响;全组基因芯片筛选低氧PASMCs增殖、凋亡及迁移的相关调控基因变化。结果：槲皮素可显著缓解PH(P0.05),逆转右心室肥厚以及肺血管重塑(P0.05),并导致肺小动脉PASMCs增殖减少,凋亡增加：体外实验表明：槲皮素能有效抑制低氧PASMCs增殖,使其周期停滞在G1/G0期、促进其凋亡和迁移;此外,结果显示槲皮素能增加低氧PASMCs cyclin D1的表达,同时减少cyclin B1和Cdc2的表达;Western-blot结果显示槲皮素能逆转低氧所导致的BAX/Bcl-2的比率,同时减少MMP2、MMP9、CXCR4、integrin β1和integrin a 5的表达；全组基因芯片分析表明槲皮素调控低氧PASMCs增殖、凋亡、细胞周期停滞、以及迁移与酪氨酸受体激酶A (TrkA)相关；低氧激活TrkA/AKT信号通路,槲皮素可有效抑制该通路的激活,更为重要的是在加入TrkA受体激活剂NGF-β后,槲皮素对低氧PASMCs抑制增殖、细胞周期阻滞,促进其凋亡和抑制迁移的作用明显减弱。结论：槲皮素可能是通过抑制TrkA/AKT信号通路发挥抑制低氧PASMCs增殖、细胞周期阻滞,促进其凋亡和抑制其迁移从而缓解肺血管重塑,达到治疗肺动脉高压的目的。三、槲皮素通过FOX01依赖的信号通路激活自噬,抑制自噬后槲皮素促进低氧肺动脉平滑肌细胞凋亡增加目的：进一步深入探讨槲皮素缓解低氧肺动脉高压(PH)的分子机制,明确细胞自噬行为有无参与其中。方法：分离、培养大鼠PASMC,在Oxycycler model C21低氧细胞培养箱中建立细胞低氧模型。流式细胞术、Western Blot和TUNEL细胞免疫荧光评估槲皮素促凋亡;电镜、LC3细胞免疫荧光观察细胞处理前后自噬形态学变化;构建携带mRFP-GFP-LC3双荧光的慢病毒观察槲皮素对自噬流的影响；利用siRNA技术沉默FOXO1, SESN3,使用3MA抑制自噬,检测槲皮素对自噬以及自噬对低氧PASMC增殖的影响。最后建立Sprague-Dawley大鼠PH模型,在给予槲皮素的基础上抑制自噬,观察有无叠加效应。结果：槲皮素有效促进低氧PASMC凋亡并促进凋亡相关蛋白的表达。同时,槲皮素增加了低氧所激发的自噬以及上调自噬相关调控蛋白的表达,并增加自噬流的形成;槲皮素增加低氧FOXO1转录以及活性;抑制FOXO1表达后可有效增加mTOR以及其下游靶蛋白P70-S6K.4E-BPI的磷酸化,也明显削弱槲皮素增加低氧所激发的自噬;此外,我们观察到FOXO1通过上调SESN3表达来实现提高mTOR活性从而激发自噬的深入机制,抑制SESN3亦可有效抑制槲皮素所增强的自噬。最后,我们发现明显抑制自噬可增强槲皮素所介导的低氧PASMC凋亡,而且在给予槲皮素的基础上加上自噬抑制剂可增加槲皮素治疗PH的效果。结论：槲皮素可有效促进低氧PASMC凋亡从而缓解PAH,同时加剧了低氧所激发的自噬,抑制自噬后槲皮素对低氧肺动脉高压的防治效果明显增加,在槲皮素治疗低氧肺动脉高压的同时加用自噬抑制剂会获取更令人满意的效果。
[Abstract]:Objective: To investigate the effect of quercetin on hypoxic pulmonary hypertension in rats in vivo in order to find a new method and target for the treatment of pulmonary hypertension.Methods: 30 Sprague-Dawley male rats were randomly divided into normoxia group, hypoxia group, hypoxia + quercetin group, each group. Ten rats were monitored by physiological polygraph for 4 weeks. The systemic circulatory pressure, right ventricular systolic pressure and right ventricular specific gravity were measured by right ventricle floating catheter. The expressions of PCNA, Ki67 and TUNEL in pulmonary arterioles were detected by immunohistochemistry. CONCLUSION: Quercetin can effectively promote the proliferation of PASMCs, decrease apoptosis, relieve hypoxic pulmonary vascular remodeling and reverse hypoxic pulmonary hypertension in rats. 2. Quercetin relieves hypoxia by regulating TrkA / AKT signaling pathway. AIM: To investigate the molecular mechanism of quercetin inhibiting the proliferation and inducing apoptosis of hypoxic pulmonary artery smooth muscle cells (PASMC) and alleviating hypoxic pulmonary hypertension (PHPH). Methods: Rat PASMC was isolated and cultured in Oxycycler model C21. Cell hypoxia model was established in cell incubator. IC50 of quercetin was detected by MTT, cell cycle and apoptotic cell number were detected by loss cell method, cyclin and apoptotic protein were detected by Western blot, the migration of hypoxic PASMCs was detected by Tanswell assay, hypoxic PASMCs were screened by gene chip. Results: Quercetin could significantly alleviate PH (P 0.05), reverse right ventricular hypertrophy and pulmonary vascular remodeling (P 0.05), reduce the proliferation of PASMCs in pulmonary arterioles, and increase the apoptosis. In vitro experiments showed that quercetin could effectively inhibit the proliferation of hypoxic PASMCs, arrest its cycle in G1/G0 phase, and promote it. In addition, quercetin increased the expression of cyclin D1 and decreased the expression of cyclin B1 and CD2 in hypoxic PASMCs; Western-blot showed that quercetin reversed the ratio of BAX/Bcl-2 induced by hypoxia, and decreased the expression of MMP2, MMP9, CXCR4, integrin beta 1 and integrin a 5; whole set of gene chip analysis tables Quercetin regulates the proliferation, apoptosis, cell cycle arrest and migration of hypoxic PASMCs related to tyrosine receptor kinase A (TrkA); hypoxia activates the TrkA/AKT signaling pathway, quercetin can effectively inhibit the activation of this pathway, more importantly, after the addition of TrkA receptor activator NGF-beta, quercetin inhibits the proliferation and cell cycle of hypoxic PASMCs. Conclusion: Quercetin may inhibit the proliferation and cell cycle of hypoxic PASMCs by inhibiting the TrkA / AKT signaling pathway, promote their apoptosis and inhibit their migration, thereby alleviating pulmonary vascular remodeling and achieving the purpose of treating pulmonary hypertension through FOX01 dependence. AIM: To investigate the molecular mechanism of quercetin in alleviating hypoxic pulmonary hypertension (PH) and ascertain whether autophagy is involved. METHODS: Rat PASMC was isolated and cultured in Oxycycler model C21 hypoxic cell culture. Cell hypoxia model was established in incubator. Quercetin-induced apoptosis was assessed by flow cytometry, Western Blot and TUNEL immunofluorescence; autophagy morphological changes were observed by electron microscopy and LC3 immunofluorescence before and after treatment; lentiviruses carrying mRFP-GFP-LC3 double fluorescence were constructed to observe the effect of quercetin on autophagy; FOXO1 was silenced by siRNA technique. Finally, the Sprague-Dawley rat PH model was established. Quercetin inhibited autophagy on the basis of quercetin, and the superposition effect was observed. Results: Quercetin effectively promoted the apoptosis of hypoxic PASMC and the expression of apoptosis-related proteins. Quercetin increased hypoxia-induced autophagy and up-regulated autophagy-related protein expression, and increased autophagy flow formation; quercetin increased hypoxia FOXO1 transcription and activity; inhibited FOXO1 expression can effectively increase the phosphorylation of mTOR and its downstream target protein P70-S6K.4E-BPI, but also significantly weakened quercetin increased hypoxia-induced autophagy. In addition, we observed that FOXO1 enhanced the activity of mTOR by up-regulating the expression of SESN3, which stimulated the in-depth mechanism of autophagy. Inhibition of SESN3 also effectively inhibited quercetin-enhanced autophagy. Finally, we found that inhibition of autophagy significantly enhanced quercetin-mediated apoptosis of PASMC, and added quercetin to the quercetin-induced hypoxia. Conclusion: Quercetin can effectively promote apoptosis of hypoxic PASMC and alleviate PAH. At the same time, quercetin can aggravate hypoxic-stimulated autophagy. The effect of quercetin on hypoxic pulmonary hypertension after inhibiting autophagy is obviously increased. Quercetin can also increase autophagy inhibition in the treatment of hypoxic pulmonary hypertension. The agent will achieve more satisfactory results.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R544.1

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