白藜蘆醇及鋅對(duì)心肌缺血再灌注損傷的線粒體保護(hù)機(jī)制

1、白藜蘆醇及鋅對(duì)心肌缺血/再灌注損傷的線粒體保護(hù)機(jī)制【中文摘要】 第一部分白藜蘆醇的心肌保護(hù)作用及其機(jī)制的研究缺血性心肌損傷是當(dāng)今人類疾病導(dǎo)致死亡的主要原因之一， 冠狀動(dòng)脈血流中斷和再灌注致心肌壞死和心肌收縮功能喪失,稱為心肌缺血/再灌注損傷。再灌注對(duì)心肌產(chǎn)生一系列影響,包括心肌超微結(jié)構(gòu)的變化以及心肌能量代謝的變化。目前,缺血/再灌注損傷發(fā)生的機(jī)制尚未完全闡明。研究表明,自由基、鈣超載、心肌纖維能量代謝障礙、中性粒細(xì)胞、血管內(nèi)皮細(xì)胞、細(xì)胞粘附因子與細(xì)胞凋亡等均參與缺血/再灌注損傷。其中,自由基和鈣超載互為因果,在缺血/再灌注損傷中具有重要意義。近年來對(duì)心肌缺血再灌注損傷的研究取得了很大進(jìn)展,很多

2、動(dòng)物實(shí)驗(yàn)證明許多藥物能夠減輕心肌缺血再灌注損傷。其中白藜蘆醇(Resveratrol,化學(xué)名為3,4,5-三羥基二苯乙烯)的心肌保護(hù)作用受到廣泛關(guān)注。這是因?yàn)榘邹继J醇長(zhǎng)久以來被認(rèn)為是“法國悖論”的關(guān)鍵因素,即有喝紅酒習(xí)慣的法國人雖然吃了很多高熱量的飲食,但心血管疾病發(fā)病率卻很低。白藜蘆醇是一種在紅酒和葡萄中存在的多酚類化學(xué)物質(zhì),具有強(qiáng)大的抗氧化、抗腫瘤、抗衰老、抗炎癥等生物學(xué)特性。目前研究認(rèn)為,白藜蘆醇通過誘導(dǎo)藥物性預(yù)處理機(jī)制減輕缺血/再灌注損傷,但其詳細(xì)的細(xì)胞.更多信號(hào)轉(zhuǎn)導(dǎo)和分子機(jī)制尚不清楚。最近研究表明,線粒體膜通透性轉(zhuǎn)移孔(mitochondrial permeabilitytransi

3、tion pore,mPTP)在心肌缺血/再灌注損傷中起關(guān)鍵性作用。缺血時(shí),mPTP保持關(guān)閉狀態(tài),而再灌注時(shí)mPTP開放。而且研究發(fā)現(xiàn),mPTP開放發(fā)生在再灌注早期而不是在缺血期。因此,在早期再灌注時(shí)如能有效地抑制mPTP的開放,是保護(hù)再灌注心肌的關(guān)鍵。糖原合成酶激酶3(glycogen synthase kinase-3,GSK-3)的作用最初認(rèn)為是調(diào)節(jié)糖原合成,并參與細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)、蛋白質(zhì)合成、細(xì)胞增殖與分化、凋亡等過程。最近研究證實(shí),GSK-3在心肌保護(hù)中也起非常重要的作用,其機(jī)制主要是通過其Ser9位點(diǎn)的磷酸化,使其活性被抑制,進(jìn)一步阻止mPTP的開放而保護(hù)再灌注損傷的心肌。鑒于以上研究

4、,白藜蘆醇在心肌缺血/再灌注損傷中的保護(hù)作用可能是通過GSK-3的失活并抑制mPTP的開放而引起。因此,本研究對(duì)GSK-3和mPTP在白藜蘆醇誘導(dǎo)的心肌保護(hù)中的作用進(jìn)行了初步探討。本實(shí)驗(yàn)首先制備大鼠離體心臟缺血再灌注損傷模型(Langendorff裝置),用TTC染色法測(cè)定心肌梗塞面積。結(jié)果顯示,離體大鼠心臟再灌注時(shí)給予白藜蘆醇(10mol/L),可以明顯減少心肌梗塞面積,明顯增加再灌注時(shí)冠狀動(dòng)脈流量(coronary flow,CF)和左心室發(fā)展壓(left ventricular-developed pressure,LVDP)。白藜蘆醇的這種抗梗塞作用可被mPTP開放激動(dòng)劑蒼術(shù)苷(atr

5、actyloside)所逆轉(zhuǎn),提示白藜蘆醇通過抑制mPTP的開放而保護(hù)再灌注心臟。另外,線粒體腫脹試驗(yàn)顯示,白藜蘆醇能夠抑制Ca2+誘導(dǎo)的線粒體腫脹,再次證明白藜蘆醇能夠抑制心肌細(xì)胞mPTP的開放。其次,用Western blotting的方法檢測(cè)離體大鼠心臟GSK-3的磷酸化(Ser9位點(diǎn))。結(jié)果顯示,白藜蘆醇明顯增加再灌注時(shí)GSK-3的磷酸化(Ser9位點(diǎn)),說明白藜蘆醇能夠使GSK-3失活。白藜蘆醇抑制GSK-3活性的作用被鳥苷酸環(huán)化酶抑制劑ODQ所阻斷,并且白藜蘆醇使再灌注心臟血管擴(kuò)張刺激磷蛋白(vasodilator-stimulated phosphoporotein,VASP)的

6、磷酸化增加,說明白藜蘆醇誘導(dǎo)的GSK-3磷酸化作用與鳥苷酸環(huán)化酶-環(huán)磷酸鳥苷-蛋白激酶G(GC-cGMP-PKG)信號(hào)轉(zhuǎn)導(dǎo)通路有關(guān)。由于GSK-3失活可以抑制mPTP的開放,從而保護(hù)再灌注心肌,因此本實(shí)驗(yàn)進(jìn)一步檢測(cè)了線粒體GSK-3的水平。結(jié)果顯示,再灌注時(shí)給予白藜蘆醇明顯增加線粒體GSK-3水平。由于在生理?xiàng)l件下GSK-3主要存在于細(xì)胞漿,因此白藜蘆醇使GSK-3從細(xì)胞漿轉(zhuǎn)移到線粒體,從而使GSK-3有機(jī)會(huì)與mPTP結(jié)合。在分離的大鼠心肌細(xì)胞中,白藜蘆醇使線粒體GSK-3增加的作用同樣被鳥苷酸環(huán)化酶抑制劑ODQ所阻斷,進(jìn)一步證實(shí)cGMP-PKG信號(hào)轉(zhuǎn)導(dǎo)通路介導(dǎo)白藜蘆醇誘導(dǎo)的GSK-3由胞漿向

7、線粒體的轉(zhuǎn)移。另外,利用免疫共沉淀方法探討轉(zhuǎn)移到線粒體的GSK-3與mPTP成分相互作用時(shí)發(fā)現(xiàn),GSK-3與mPTP的組成成分親和素D(Cyclophilin D,CypD)相結(jié)合,而不和外膜上的電壓依賴性陰離子通道(the Voltage-Dependent Anion Channel,VDAC)或內(nèi)膜上的腺嘌呤核苷酸轉(zhuǎn)位酶(the Adenine-Nucleotide Translocase,ANT)相互作用,表明白藜蘆醇通過誘導(dǎo)GSK-3與線粒體膜成分親和素D結(jié)合而抑制mPTP開放,從而保護(hù)心臟。小結(jié)1.白藜蘆醇通過使GSK-3失活而抑制mPTP開放,保護(hù)再灌注心肌。2.白藜蘆醇通過cG

8、MP-PKG信號(hào)轉(zhuǎn)導(dǎo)通路使GSK-3失活并誘導(dǎo)其從細(xì)胞漿轉(zhuǎn)移至線粒體。3.轉(zhuǎn)移至線粒體的GSK-3最終與線粒體膜成分親和素D結(jié)合而抑制mPTP開放,從而保護(hù)心臟。第二部分鋅的心肌保護(hù)作用及其機(jī)制的研究鋅存在于所有細(xì)胞中,除了構(gòu)成細(xì)胞結(jié)構(gòu)蛋白、酶、及轉(zhuǎn)錄因子外,其本身參與多種生理功能。它通過調(diào)節(jié)細(xì)胞信號(hào)識(shí)別、第二信使代謝、蛋白激酶的磷酸化等在細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)中起重要作用。最近研究報(bào)道,鋅能夠激活PI3K/Akt信號(hào)途徑,抑制GSK-3活性,而PI3K/Akt及GSK-3在心肌缺血/再灌注損傷的保護(hù)中起重要作用。GSK-3失活而阻止mPTP開放,是許多藥物性預(yù)處理保護(hù)心臟的共同機(jī)制。因此,本實(shí)驗(yàn)利用H

9、9C2大鼠心肌細(xì)胞株,探討外源性鋅能否通過抑制GSK-3活性而阻止mPTP的開放,從而保護(hù)缺血再灌注心肌細(xì)胞。本實(shí)驗(yàn)首先用ZnCl2(10mol/L)及鋅載體Pyrithion處理H9C2細(xì)胞20分鐘,然后利用Western blotting的方法分析GSK-3磷酸化情況。結(jié)果顯示,鋅能使GSK-3(Ser9)磷酸化明顯增加,提示外源性鋅可抑制H9C2細(xì)胞中GSK-3的活性。其次,為了分析鋅抑制GSK-3的信號(hào)介導(dǎo)機(jī)制,本實(shí)驗(yàn)用PI3K、mTOR以及PKC抑制劑LY-294002、Rapamycin及Chelerythrine處理了細(xì)胞。結(jié)果顯示,鋅誘導(dǎo)的GSK-3磷酸化增加的作用被LY-29

10、4002而不是Rapamycin及Chelerythrine所抑制,鋅能夠明顯增加Akt而不是mTOR的磷酸化水平,提示PI3K/Akt而不是mTOR或PKC參與鋅的作用。進(jìn)一步的研究顯示,鋅能夠明顯增加線粒體GSK-3磷酸化水平,表明線粒體參與鋅的作用。鋅誘導(dǎo)的線粒體GSK-3磷酸化增強(qiáng)的作用沒有被線粒體ATP敏感性鉀離子通道(mKATP)抑制劑5-HD所阻斷,表明mKATP不參與鋅誘導(dǎo)的線粒體GSK-3磷酸化過程。在H9C2細(xì)胞缺血/再灌注模型中,鋅能夠明顯減輕再灌注時(shí)細(xì)胞死亡,表明鋅能防止再灌注損傷。為了進(jìn)一步探討GSK-3是否在其中發(fā)揮作用,利用持續(xù)激活型GSK-3質(zhì)粒DNA(GSK-

11、3-S9A-HA,不受GSK-3上游激酶下調(diào)而持續(xù)激活)轉(zhuǎn)染了細(xì)胞。結(jié)果顯示,在轉(zhuǎn)染了這種質(zhì)粒的細(xì)胞中,鋅不能減輕再灌注損傷。用持續(xù)抑制型GSK-3(GSK-3-KM-HA)轉(zhuǎn)染細(xì)胞后,細(xì)胞死亡率也明顯降低,提示GSK-3失活在鋅引起的心肌保護(hù)中起重要作用。另外,共聚焦顯微鏡觀察顯示,鋅明顯抑制H2O2誘導(dǎo)的TMRE熒光減少,而在GSK-3-S9A-HA轉(zhuǎn)染細(xì)胞中鋅的這種作用消失,表明鋅通過抑制GSK-3活性而阻止mPTP的開放。小結(jié):(1)外源性鋅可抑制H9C2心肌細(xì)胞中GSK-3的活性,PI3K/Akt信號(hào)途徑可能介導(dǎo)鋅的這種作用。(2)鋅通過使GSK-3失活而阻止mPTP開放,從而減輕再

12、灌注損傷。 【英文摘要】 Part 1 Resveratrol-induced cardioprotection and the mechanism underlying the protectionAcute myocardial infarction (AMI) is the leading cause of death worldwide. The occlusion of coronary artery followed by revasculation induces myocardial necrosis and the myocardial contractile dysfunc

13、tion. In spite of substantial investigation, no specific therapy was yet been established to effectively save myocardium from AMI. First isolated from the roots of white hellebore in 1940, resveratrol (3,4, 5-trihydroxystilbene) is a polyphenolic product found abundantly in grapes and red wines. Stu

14、dies have shown that resveratrol exerts numerous biological effects including antioxidant , anti-ageing , and anti-inflammation. Resveratrol is a key explanation for the French Paradox - the low incidence of heart disease among the French people, who eat a relatively high-fat diet. Resveratrol pretr

15、eatment can protect the heart from ischemia/reperfusion injury by inducing pharmacological preconditioning. T.he mitochondrial permeability transition pore (mPTP) opening has been proposed to play an important role in myocardial ischemia/reperfusion injury. The mPTP remains closed during ischemia bu

16、t opens at the onset of reperfusion, and modulation of the mPTP opening at early reperfusion can protect the heart from reperfusion injury. Identified as a regulator of glycogen metabolism, glycogen synthase kinase 3(GSK-3) is now a well established component contributing to cell signaling, protein

17、synthesis, cell proliferation, cell differentiation, cell adhesion, and apoptosis. Studies have shown that GSK-3plays a role in ischemic preconditioning and cardioprotection at reperfusion. In addition, a recent study has shown that GSK-3plays a central role in pharmacological preconditioning induce

18、d modulation of the mPTP opening. Therefore, it is intriguing to determine whether GSK-3is involved in the action of resveratrol, if resveratrol protects the heart at reperfusion by targeting the mPTP. First, we examined the effect of resveratrol on myocardial infarct size when given at reperfusion

19、and investigated the mechanism underlying the effect. Isolated rat hearts were subjected to 30 min ischemia followed by 2h of reperfusion, and myocardial samples were collected from the risk zone for Western blot analysis. Mitochondrial swelling was spectrophotometrically measured as a decrease in a

20、bsorbance at 520 nm (A520). Resveratrol reduced infarct size, an effect that was reversed by the mPTP opener atractyloside, indicating that resveratrol may protect the heart by modulating the mPTP opening. This finding was further supported by the observation that resveratrol prevented cardiac mitoc

21、hondrial swelling.Second, Resveratrol enhanced GSK-3phosphorylation at Ser9 upon reperfusion, an effect that was blocked by the guanylyl cyclase inhibitor ODQ, suggesting the cGMP-PKG signaling pathway is involved in the action of resveratrol. Resveratrol also increased phosphorylation of Vasodilato

22、r-stimulated phosphoprotein (VASP), implying that resveratrol does activate PKG. Resveratrol translocated GSK-3from cytosol to mitochondria via the cGMP-PKG pathway.Further studies showed that mitochondrial GSK-3was co-immunoprecipitated with cyclophilin D but not with VDAC (voltage dependent anion

23、channel) or ANT (adenine nucleotide translocator).These data suggest that resveratrol prevents myocardial reperfusion injury presumably by targeting the mPTP through translocation of GSK-3from cytosol to mitochondria. Translocated GSK-3may ultimately interact with cyclophilin D to modulate the mPTP

24、opening. Part 2 Zinc-induced cardioprotection and the mechanism underlying the protectionFound in most tissues, zinc is fundamental to the structure and function of many proteins, enzymes, and transcriptional factors. Zinc has also been demonstrated to play an important role in cellular signaling by

25、 modulating signal recognition, second messenger metabolism, protein kinase, and Phosphatase activities. Recent studies have shown that exogenous zinc can regulate activities of several important intracellular signaling elements such as phosphatidylinositol 3-kinase (PI3K), Akt/PKB, p70S6 kinase, ma

26、mmalian target of rapamycin (mTOR), extracellular signal-regulated kinase (ERK), and glycogen synthase kinase-3(GSK-3). Since these signaling kinases have been demonstrated to be involved in the mechanism underlying cardioprotection against reperfusion injury, the treatment of cardiac cells with zin

27、c may lead to cardioprotection against reperfusion injury. The purpose of this study was to determine whether exogenous zinc prevents cardiac reperfusion injury by targeting the mitochondrial permeability transition pore (mPTP) via glycogen synthase kinase 3(GSK.-3).The treatment of cardiac H9C2 cel

28、ls with ZnCl2 (10M) in the presence of zinc ionophore pyrithione for 20 min significantly enhanced GSK-3phosphorylation at Ser9, indicating that exogenous zinc can inactivate GSK-3in H9C2 cells. The effect of zinc on GSK-3activity was blocked by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY-

29、294002 but not by the mammalian target of rapamycin (mTOR) inhibitor rapamycin or the PKC inhibitor chelerythrine, implying that PI3K but not mTOR or PKC accounts for the action of zinc. In support of this interpretation, zinc induced a significant increase in Akt but not mTOR phosphorylation.Furthe

30、r experiments found that zinc also increased mitochondrial GSK-3phosphorylation. This may indicate an involvement of the mitochondria in the action of zinc. The effect of zinc on mitochondrial GSK-3phosphorylation was not altered by the mitochondrial ATP-sensitive KATP channel blocker 5 hydroxydecanoic acid. Zinc applied at reperfusion reduced cell death in cells subjected to simulated ischemia/reperfusion, indicating that zinc c