【摘要】：一、槲皮素對大鼠低氧肺動(dòng)脈高壓防治作用的研究目的：體內(nèi)實(shí)驗(yàn)探討槲皮素對低氧肺動(dòng)脈高壓的作用效果,為肺動(dòng)脈高壓治療尋求新的方式及治療靶點(diǎn)。方法：隨機(jī)將30只雄性Sprague-Dawley大鼠分為常氧組、低氧組、低氧+槲皮素組,每組10只。持續(xù)4周后,在生理多導(dǎo)儀監(jiān)視下,通過右心漂浮導(dǎo)管測定大鼠體循環(huán)壓、右心室收縮壓、右心室比重[右心室／(左心室+室間隔)],免疫組化檢測各組大鼠肺小動(dòng)脈中PCNA、Ki67以及TUNEL的表達(dá)變化。結(jié)果：槲皮素可顯著緩解低氧肺動(dòng)脈高壓(P0.05),逆轉(zhuǎn)右心室肥厚以及肺血管重塑(P0.05),并導(dǎo)致肺小動(dòng)脈PASMCs增殖減少,凋亡增加。結(jié)論：槲皮素可有效促進(jìn)大鼠PASMCs增殖減少、凋亡增加,緩解低氧肺血管重塑,逆轉(zhuǎn)低氧肺動(dòng)脈高壓。二、槲皮素通過調(diào)控TrkA/AKT信號通路緩解低氧肺動(dòng)脈高壓目的：探討槲皮素抑制低氧肺動(dòng)脈平滑肌細(xì)胞(PASMC)增殖和誘導(dǎo)細(xì)胞凋亡從而緩解缺氧肺動(dòng)脈高壓的潛在的分子機(jī)制,為槲皮素治療低氧肺動(dòng)脈高壓進(jìn)一步提供理論基礎(chǔ)。方法：分離、培養(yǎng)大鼠PASMC,在Oxycycler model C21低氧細(xì)胞培養(yǎng)箱中建立細(xì)胞低氧模型。MTT法檢測槲皮素的IC50,流失細(xì)胞法檢測槲皮素濃度梯度下細(xì)胞周期及凋亡細(xì)胞數(shù),Western-blot檢測周期蛋白及凋亡蛋白的變化；Tanswell實(shí)驗(yàn)檢測槲皮素對低氧PASMCs遷移的影響;全組基因芯片篩選低氧PASMCs增殖、凋亡及遷移的相關(guān)調(diào)控基因變化。結(jié)果：槲皮素可顯著緩解PH(P0.05),逆轉(zhuǎn)右心室肥厚以及肺血管重塑(P0.05),并導(dǎo)致肺小動(dòng)脈PASMCs增殖減少,凋亡增加：體外實(shí)驗(yàn)表明：槲皮素能有效抑制低氧PASMCs增殖,使其周期停滯在G1/G0期、促進(jìn)其凋亡和遷移;此外,結(jié)果顯示槲皮素能增加低氧PASMCs cyclin D1的表達(dá),同時(shí)減少cyclin B1和Cdc2的表達(dá);Western-blot結(jié)果顯示槲皮素能逆轉(zhuǎn)低氧所導(dǎo)致的BAX/Bcl-2的比率,同時(shí)減少M(fèi)MP2、MMP9、CXCR4、integrin β1和integrin a 5的表達(dá)；全組基因芯片分析表明槲皮素調(diào)控低氧PASMCs增殖、凋亡、細(xì)胞周期停滯、以及遷移與酪氨酸受體激酶A (TrkA)相關(guān)；低氧激活TrkA/AKT信號通路,槲皮素可有效抑制該通路的激活,更為重要的是在加入TrkA受體激活劑NGF-β后,槲皮素對低氧PASMCs抑制增殖、細(xì)胞周期阻滯,促進(jìn)其凋亡和抑制遷移的作用明顯減弱。結(jié)論：槲皮素可能是通過抑制TrkA/AKT信號通路發(fā)揮抑制低氧PASMCs增殖、細(xì)胞周期阻滯,促進(jìn)其凋亡和抑制其遷移從而緩解肺血管重塑,達(dá)到治療肺動(dòng)脈高壓的目的。三、槲皮素通過FOX01依賴的信號通路激活自噬,抑制自噬后槲皮素促進(jìn)低氧肺動(dòng)脈平滑肌細(xì)胞凋亡增加目的：進(jìn)一步深入探討槲皮素緩解低氧肺動(dòng)脈高壓(PH)的分子機(jī)制,明確細(xì)胞自噬行為有無參與其中。方法：分離、培養(yǎng)大鼠PASMC,在Oxycycler model C21低氧細(xì)胞培養(yǎng)箱中建立細(xì)胞低氧模型。流式細(xì)胞術(shù)、Western Blot和TUNEL細(xì)胞免疫熒光評估槲皮素促凋亡;電鏡、LC3細(xì)胞免疫熒光觀察細(xì)胞處理前后自噬形態(tài)學(xué)變化;構(gòu)建攜帶mRFP-GFP-LC3雙熒光的慢病毒觀察槲皮素對自噬流的影響；利用siRNA技術(shù)沉默F(xiàn)OXO1, SESN3,使用3MA抑制自噬,檢測槲皮素對自噬以及自噬對低氧PASMC增殖的影響。最后建立Sprague-Dawley大鼠PH模型,在給予槲皮素的基礎(chǔ)上抑制自噬,觀察有無疊加效應(yīng)。結(jié)果：槲皮素有效促進(jìn)低氧PASMC凋亡并促進(jìn)凋亡相關(guān)蛋白的表達(dá)。同時(shí),槲皮素增加了低氧所激發(fā)的自噬以及上調(diào)自噬相關(guān)調(diào)控蛋白的表達(dá),并增加自噬流的形成;槲皮素增加低氧FOXO1轉(zhuǎn)錄以及活性;抑制FOXO1表達(dá)后可有效增加mTOR以及其下游靶蛋白P70-S6K.4E-BPI的磷酸化,也明顯削弱槲皮素增加低氧所激發(fā)的自噬;此外,我們觀察到FOXO1通過上調(diào)SESN3表達(dá)來實(shí)現(xiàn)提高mTOR活性從而激發(fā)自噬的深入機(jī)制,抑制SESN3亦可有效抑制槲皮素所增強(qiáng)的自噬。最后,我們發(fā)現(xiàn)明顯抑制自噬可增強(qiáng)槲皮素所介導(dǎo)的低氧PASMC凋亡,而且在給予槲皮素的基礎(chǔ)上加上自噬抑制劑可增加槲皮素治療PH的效果。結(jié)論：槲皮素可有效促進(jìn)低氧PASMC凋亡從而緩解PAH,同時(shí)加劇了低氧所激發(fā)的自噬,抑制自噬后槲皮素對低氧肺動(dòng)脈高壓的防治效果明顯增加,在槲皮素治療低氧肺動(dòng)脈高壓的同時(shí)加用自噬抑制劑會(huì)獲取更令人滿意的效果。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R544.1

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