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        4-苯基丁酸鈉對(duì)缺氧復(fù)氧誘導(dǎo)心肌細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激的影響*

        2016-05-07 02:33:48徐磊張雙徐繼前譚春燕陳海燕賀集賢何元軍
        西部醫(yī)學(xué) 2016年3期
        關(guān)鍵詞:復(fù)氧苯基內(nèi)質(zhì)網(wǎng)

        徐磊 張雙 徐繼前 譚春燕 陳海燕 賀集賢 何元軍

        4-苯基丁酸鈉對(duì)缺氧復(fù)氧誘導(dǎo)心肌細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激的影響*

        徐磊1,2張雙2徐繼前2譚春燕2陳海燕2賀集賢1何元軍1

        (1.廣元市中心醫(yī)院心內(nèi)科, 四川 廣元 628000;2,川北醫(yī)學(xué)院附屬醫(yī)院心內(nèi)科, 四川 南充 637000)

        目的 探討4-苯基丁酸鈉(4-phenyl butyric acid,4-PBA)對(duì)缺氧復(fù)氧誘導(dǎo)心肌細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激的影響。方法 建立原代培養(yǎng)的心肌細(xì)胞缺氧復(fù)氧模型,隨機(jī)將細(xì)胞分為對(duì)照組(Control組)、4-苯基丁酸鈉預(yù)處理組(4-PBA組)、缺氧復(fù)氧組(H/R組)和4-苯基丁酸鈉+缺氧復(fù)氧組(4-PBA+H/R組),應(yīng)用CCK-8試劑盒檢測細(xì)胞活力,caspase-3活性試劑盒測定caspase-3活性及Tunel染色測定凋亡細(xì)胞比率,western blot檢測內(nèi)質(zhì)網(wǎng)應(yīng)激標(biāo)志性蛋白C/EBP同源蛋白(C/EBP homologous protein, CHOP)表達(dá)。結(jié)果 與對(duì)照組[(100±5.31)%]比較,缺氧復(fù)氧組細(xì)胞活力[(77.45±5.67)%]顯著降低(P<0.01);而與H/R組比較,4-PBA+H/R組[(87.64±4.62)%]細(xì)胞活力明顯升高(P<0.05);H/R組caspase活性(1.86±0.14)較對(duì)照組(0.82±0.14)顯著增加(P<0.01),4-PBA預(yù)處理組(1.09±0.11)較H/R組相比明顯降低(P<0.05);H/R組Tunel陽性細(xì)胞比率[(37.55±3.51)%]較對(duì)照組[(6.78±1.75)%]相比顯著升高(P<0.01),而4-PBA+H/R組[(20.81±2.72)%]較H/R組相比顯著降低(P<0.01);H/R組CHOP表達(dá)(1.99±0.21)較對(duì)照組(0.98±0.14)顯著上調(diào)(P<0.01),而4-PBA預(yù)處理組CHOP表達(dá)(1.36±0.18)與H/R組相比明顯降低(P<0.05)。結(jié)論 缺氧復(fù)氧能夠誘導(dǎo)心肌細(xì)胞凋亡及內(nèi)質(zhì)網(wǎng)應(yīng)激,而4-PBA能夠減輕內(nèi)質(zhì)網(wǎng)應(yīng)激及心肌細(xì)胞缺氧復(fù)氧損傷。

        4-苯基丁酸鈉; 缺氧復(fù)氧; 內(nèi)質(zhì)網(wǎng)應(yīng)激; 細(xì)胞凋亡

        內(nèi)質(zhì)網(wǎng)應(yīng)激(Endoplasmic reticulum stress,ERS)是細(xì)胞應(yīng)對(duì)刺激的一種適應(yīng)性反應(yīng),但是過度內(nèi)質(zhì)網(wǎng)應(yīng)激又可以通過激活相關(guān)的信號(hào)通路引起細(xì)胞損傷甚至細(xì)胞凋亡[1,2]。研究表明,內(nèi)質(zhì)網(wǎng)應(yīng)激參與心肌缺血再灌注(ischemia/reperfusion ,I/R)損傷[3],抑制內(nèi)質(zhì)網(wǎng)應(yīng)激可減輕心肌I/R損傷[4],然而內(nèi)質(zhì)網(wǎng)應(yīng)激抑制劑4-苯基丁酸鈉(4-PBA)能否有效減輕心肌缺氧復(fù)氧(hypoxia/reoxygenation,H/R)損傷還有待進(jìn)一步研究。本研究擬通過使用內(nèi)質(zhì)網(wǎng)應(yīng)激抑制劑4-PBA預(yù)處理心肌細(xì)胞并觀察其對(duì)H/R心肌細(xì)胞作用,探索其能否應(yīng)用于心血管疾病的防治。

        1 材料與方法

        1.1 原代小鼠心肌細(xì)胞培養(yǎng) 根據(jù)以前的方法[5],選取出生1~3天小鼠乳鼠,斷頭法處死后采用胰酶、膠原酶混合消化的方法提取心室肌細(xì)胞,采用差速貼壁方法分離成纖維細(xì)胞而純化心肌細(xì)胞,純化細(xì)胞經(jīng)培養(yǎng)48~72小時(shí)后用于實(shí)驗(yàn)。

        1.2 分組及缺氧復(fù)氧模型建立 原代培養(yǎng)小鼠心肌細(xì)胞分為4組:對(duì)照組(Control組)、4-苯基丁酸鈉預(yù)處理組(4-PBA組,在實(shí)驗(yàn)前使用含有4-PBA的培養(yǎng)基在正常條件下培養(yǎng)4小時(shí),然后換用正常培養(yǎng)基)、缺氧復(fù)氧組(H/R組,通過將正常培養(yǎng)基更換為不含血清及葡萄糖的培養(yǎng)基,并用含5%CO2的N2充分飽和30min達(dá)到缺氧目的,然后在含5%CO2的N2填充小室中培養(yǎng)4小時(shí)模擬缺氧培養(yǎng),其后換用正常培養(yǎng)基在正常培養(yǎng)箱中培養(yǎng)4小時(shí)模擬復(fù)氧[6])和4-苯基丁酸鈉+缺氧復(fù)氧組(4-PBA+H/R組,即在實(shí)驗(yàn)前用含4-PBA培養(yǎng)基預(yù)處理4小時(shí),然后進(jìn)行缺氧復(fù)氧處理)。

        1.3 心肌細(xì)胞凋亡水平及細(xì)胞活力測定 根據(jù)先前方法[6,7],心肌細(xì)胞晚期凋亡水平通過Tunel染色法按照羅氏公司提供的染色操作流程染色,使用熒光顯微鏡鏡下觀察拍片并計(jì)算Tunel陽性核細(xì)胞比率評(píng)估;另外,按照caspase-3試劑盒操作說明,測定各組提取蛋白中caspase-3活性來評(píng)估心肌細(xì)胞早期凋亡水平;按照日本同仁公司CCK-8試劑盒操作流程檢測各組心肌細(xì)胞活力。

        1.4 內(nèi)質(zhì)網(wǎng)應(yīng)激CHOP蛋白檢測 缺氧復(fù)氧處理后,提取各組細(xì)胞蛋白,通過western blot檢測心肌細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)凋亡標(biāo)志性蛋白CHOP蛋白表達(dá)水平。

        1.5 統(tǒng)計(jì)學(xué)分析 數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)誤表示,采用 Graph-Pad Prism 5. 0軟件統(tǒng)計(jì)分析并作圖, 兩組間差異比較用 Newman Keuls分析,以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

        2 結(jié)果

        2.1 4-PBA對(duì)缺氧復(fù)氧引起心肌細(xì)胞活力下降的影響 各組心肌細(xì)胞活力測定結(jié)果見圖1,與control組相比,4-PBA組細(xì)胞活力并沒有明顯差異(P>0.05),而H/R組細(xì)胞活力明顯下降(P<0.01);與H/R組相比,4-PBA預(yù)處理組細(xì)胞活力有顯著增加(P<0.05)。

        圖1 缺氧氧復(fù)對(duì)心肌細(xì)胞活力影響

        Fig 1 Effect of H/R on cardiomyocytes viability

        注:與對(duì)照組比較,①P<0.05;與H/R組比較,②P<0.05

        2.2 4-PBA對(duì)缺氧復(fù)氧引起的心肌細(xì)胞凋亡的影響 為評(píng)估心肌細(xì)胞凋亡水平,我們通過capase-3活性測定早期凋亡及Tunel染色方法測定晚期心肌細(xì)胞。結(jié)果顯示,與control組相比,H/R組心肌細(xì)胞capase-3活性和Tunel陽性核細(xì)胞比例顯著增加(均P<0.01);與H/R組相比,4-PBA+H/R組caspase-3活性和Tunel陽性核細(xì)胞比率均明顯降低(均P<0.05),見圖2。H/R組與control組心肌細(xì)胞凋亡率相比有顯著增加(P<0.01),4-PBA+H/R組與H/R組相比有明顯減少(P<0.01),見圖3。

        2.3 4-PBA減輕缺氧復(fù)氧誘導(dǎo)的內(nèi)質(zhì)網(wǎng)應(yīng)激 為明確4-PBA對(duì)心肌細(xì)胞缺氧復(fù)氧誘導(dǎo)的內(nèi)質(zhì)網(wǎng)應(yīng)激的影響,我們檢測各組CHOP蛋白表達(dá)水平。結(jié)果發(fā)現(xiàn),與control組相比,H/R組CHOP表達(dá)水平明顯升高(P<0.01);與H/R組相比,4-PBA+H/R組CHOP表達(dá)有明顯下降(P<0.05),見圖4。

        圖2 各組測定caspase-3活性結(jié)果

        Fig 2 Caspase-3 activity in different groups

        注:與control組相比,①P<0.05;與H/R組相比,②P<0.05

        圖4 各組CHOP蛋白表達(dá)

        Fig 4 The expression of CHOP in different groups

        注:與control組相比,①P<0.01;與H/R組相比,②P<0.05

        3 討論

        心肌缺血再灌注損傷中,心肌細(xì)胞超微結(jié)構(gòu)、細(xì)胞功能以及基本生物學(xué)活性都會(huì)發(fā)生改變,其分子機(jī)制目前包括胞質(zhì)內(nèi)鈣超載、氧化應(yīng)激、線粒體通透性轉(zhuǎn)換孔開放、胞質(zhì)促凋亡因子釋放等等[6,8]。ERS是細(xì)胞應(yīng)對(duì)各種刺激的一種適應(yīng)性反應(yīng),然而這種反應(yīng)對(duì)于細(xì)胞本身卻是一把“雙刃劍”,在輕度的損傷性刺激下細(xì)胞可通過促進(jìn)未折疊蛋白折疊為成熟蛋白以及相關(guān)細(xì)胞轉(zhuǎn)錄因子作用減輕細(xì)胞內(nèi)質(zhì)網(wǎng)穩(wěn)態(tài)的失衡,從而減輕細(xì)胞損傷[9];但是另一方面,過度的長時(shí)間的ERS卻可以通過激活內(nèi)質(zhì)網(wǎng)特征性的JNK、CHOP、caspase-12信號(hào)通路引起細(xì)胞凋亡損傷[10,11]。越來越多的研究表明,ERS的促凋亡作用參與許多病理生理狀態(tài),其在心肌I/R損傷、心肌梗塞、心力衰竭、高血壓等疾病的發(fā)病中具有重要作用,而抑制ERS能夠顯著減輕心肌細(xì)胞損傷,改善心臟功能[12~14]。

        4-PBA是一種小分子化合物,目前主要是作為ERS抑制劑被用于研究癌癥、糖尿病、阿爾茨海默病、帕金森癥及神經(jīng)系統(tǒng)退行性病變等與ERS相關(guān)的疾病。研究表明,4-PBA能夠通過減輕ERS,改善糖尿病小鼠的肝臟、骨骼肌、脂肪組織的血糖濃度,正因?yàn)?-PBA有極大可能成為糖尿病治療的藥物所以受到廣泛關(guān)注和研究[15,16]。Ayala P等[17]在異丙腎上腺素構(gòu)建的小鼠心室重構(gòu)模型中發(fā)現(xiàn),4-PBA預(yù)處理能夠減輕ERS而減少心肌細(xì)胞損傷、延緩心肌纖維化的進(jìn)展和對(duì)抗心室重構(gòu)。而Luo T等[18]在另外一項(xiàng)研究中發(fā)現(xiàn),4-PBA灌胃處理能夠減輕主動(dòng)脈縮窄大鼠的心室重量以及心肌間質(zhì)中的膠原沉積,這與其選擇性減輕肥厚心肌ERS具有重要關(guān)系,4-PBA從理論上可以作為對(duì)抗壓力依賴性心室重構(gòu)的藥物研究。

        目前對(duì)于4-PBA是否能夠通過減輕ERS而減輕心肌細(xì)胞I/R損傷尚未見報(bào)道。因此,我們擬通過4-PBA預(yù)處理原代培養(yǎng)心肌細(xì)胞構(gòu)建缺氧復(fù)氧損傷模型來模擬I/R損傷,觀察其對(duì)H/R誘導(dǎo)心肌細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激的影響,以驗(yàn)證其應(yīng)用心血管疾病研究的可能性。結(jié)果發(fā)現(xiàn),心肌細(xì)胞H/R后心肌細(xì)胞活力明顯下降,心肌細(xì)胞凋亡水平顯著升高,ERS相關(guān)的促凋亡蛋白CHOP表達(dá)顯著增加,4-PBA預(yù)處理則能夠明顯減輕這種效應(yīng)。另外,這說明了ERS相關(guān)的細(xì)胞凋亡途徑明確參與的心肌細(xì)胞H/R損傷,4-PBA能夠通過抑制ERS來減輕H/R引起的心肌細(xì)胞損傷,并且是通過抑制ERS相關(guān)的CHOP促凋亡信號(hào)通路實(shí)現(xiàn)的。

        4 結(jié)論

        H/R能夠引起原代培養(yǎng)心肌細(xì)胞ERS,并激活ERS相關(guān)的CHOP促凋亡信號(hào)通路導(dǎo)致心肌細(xì)胞凋亡;4-PBA能通過減輕ERS及降低ERS相關(guān)的促凋亡蛋白CHOP表達(dá)減少心肌細(xì)胞凋亡;4-PBA是一種有用的抗心肌細(xì)胞H/R損傷藥物,但其是否能夠作為臨床藥物應(yīng)用于心肌I/R損傷的防治還有待更進(jìn)一步研究。

        [1]Logue SE, Cleary P, Saveljeva S,etal. New directions in ER stress-induced cell death[J]. Apoptosis, 2013, 18(5): 537-546.

        [2]Liu X, Wang M, Chen H,etal. Hypothermia protects the brain from transient global ischemia/reperfusion by attenuating endoplasmic reticulum response-induced apoptosis through CHOP[J]. PLoS One, 2013, 8(1): e53431.

        [3]Grall S, Prunier-Mirebeau D, Tamareille S,etal. Endoplasmic reticulum stress pathway involvement in local and remote myocardial ischemic conditioning[J]. Shock, 2013, 39(5): 433-439.

        [4]Guo J, Bian Y, Bai R,etal. Globular adiponectin attenuates myocardial ischemia/reperfusion injury by upregulating endoplasmic reticulum Ca(2)(+)-ATPase activity and inhibiting endoplasmic reticulum stress[J]. J Cardiovasc Pharmacol, 2013, 62(2): 143-153.

        [5]徐磊. Lycopene通過調(diào)節(jié)內(nèi)質(zhì)網(wǎng)應(yīng)激減輕心肌缺氧復(fù)氧損傷[D].川北醫(yī)學(xué)院研究生論文集,2014:3-31.

        [6]Yue R, Hu H, Yiu KH,etal. Lycopene protects against hypoxia/reoxygenation-induced apoptosis by preventing mitochondrial dysfunction in primary neonatal mouse cardiomyocytes[J]. PLoS One, 2012, 7(11): e50778.

        [7]Hu H, Li X, Li Y,etal. Calpain-1 induces apoptosis in pulmonary microvascular endothelial cells under septic conditions[J]. Microvasc Res, 2009, 78(1): 33-39.

        [8]Luo T, Yue R, Hu H,etal. PD150606 protects against ischemia/reperfusion injury by preventing mu-calpain-induced mitochondrial apoptosis[J]. Arch Biochem Biophys, 2015,586:1-9.

        [9]Chen JC, Wu ML, Huang KC,etal. HMG-CoA reductase inhibitors activate the unfolded protein response and induce cytoprotective GRP78 expression[J]. Cardiovasc Res, 2008, 80(1): 138-150.

        [10] Lu H, Lu L, Xu ZC,etal. Tauroursodeoxycholic acid and 4-phenyl butyric acid alleviate endoplasmic reticulum stress and improve prognosis of donation after cardiac death liver transplantation in rats[J]. Hepatobiliary Pancreat Dis Int, 2014, 13(6): 586-593.

        [11] Yuan Y, Guo Q, Ye Z,etal. Ischemic postconditioning protects brain from ischemia/reperfusion injury by attenuating endoplasmic reticulum stress-induced apoptosis through PI3K-Akt pathway[J]. Brain Res, 2011, 13(67):85-93.

        [12] Doroudgar S, Glembotski CC. New concepts of endoplasmic reticulum function in the heart: programmed to conserve[J]. J Mol Cell Cardiol, 2013, 55:85-91.

        [13] Wang J, Wang Y, Shan S,etal. Salusins protect myocardium against ischemic injury by alleviating endoplasmic reticulum stress[J]. Sci China Life Sci, 2012, 55(4): 358-366.

        [14] Groenendyk J, Sreenivasaiah PK, Kim do H,etal. Biology of endoplasmic reticulum stress in the heart[J]. Circ Res, 2010, 107(10): 1185-1197.

        [15] Shang L, Hua H, Foo K,etal. beta-cell dysfunction due to increased ER stress in a stem cell model of Wolfram syndrome[J]. Diabetes, 2014, 63(3): 923-933.

        [16] Ozcan U, Yilmaz E, Ozcan L,etal. Chemical chaperones reduce ER stress and restore glucose homeostasis in a mouse model of type 2 diabetes[J]. Science, 2006, 313(5790): 1137-1140.

        [17] Ayala P, Montenegro J, Vivar R,etal. Attenuation of endoplasmic reticulum stress using the chemical chaperone 4-phenylbutyric acid prevents cardiac fibrosis induced by isoproterenol[J]. Exp Mol Pathol, 2012, 92(1): 97-104.

        [18] Luo T, Chen B, Wang X. 4-PBA prevents pressure overload-induced myocardial hypertrophy and interstitial fibrosis by attenuating endoplasmic reticulum stress[J]. Chem Biol Interact, 2015, 242:99-106.

        Effect of 4-PBA on hypoxia/reoxygenation induced endoplasmic reticulum stress in cardiomycytes

        XU Lei1,2,ZHANG Shuang2,XU Jiqian2,etal

        (1.DepartmentofCardiology,CentralHospitalofGuangyuan,Guangyuan628000,Sichuan,China;2.DepartmentofCardiology,TheAffiliatedHospitalofNorthSichuanMedicalCollege,Nanchong637000,Sichuan,China)

        Objective This study was designed to explore the effect of 4-PBA on hypoxia/reoxygenation induced endoplasmic reticulum stress (ERS) in cardiomyocytes H/R injury. Methods Primary cultured cardiomyocyctes were exposure to H/R to stimulating I/R injury and randomly divided to control group (Control), 4-PBA group (4-PBA), H/R group, 4-PBA +H/R group (4-PBA +H/R). CCK-8 assessed the cell viability of cardiomyocyctes, Caspase-3 activity was measured and Tunel staining was performed to estimate the apoptotic cardiomyocytes. Western blot was used to assess the protein expression of CHOP. Results Compared to the control group [(100±5.31)%[], cell viability in H/R group[(77.45±5.67)%] was significantly decreased (P<0.01). 4-PBA pretreatment [(87.64±4.62)%] ameliorated, compared to the H/R group(P<0.05). Caspase-3 activity in H/R group(1.86±0.14) was obviously increased compared to that in the control group(0.82±0.14)(P<0.01). 4-PBA pretreatment decreased viability, compared to the H/R group(P<0.05). Apoptotic index in H/R group[(37.55±3.51)%] was increased compared to that in the control group [(6.78±1.75)% ](P<0.01). 4-PBA pretreatment[(20.81±2.72)%] decreased the apoptotic index, compared to the H/R group(P<0.01). Expression of CHOP in H/R group(1.99±0.21) was upregulated compared to the control group(0.98±0.14)(P<0.01) and the upregulating of CHOP in H/R group was attenuated as compared to the 4-PBA+H/R group(1.36±0.18)(P<0.05). Conclusion H/R exposure treatment could induce cardiomyocytes apoptosis and ERS, and 4-PBA pretreatment could effectively protect cardimyocytes against H/R injury via attenuating ERS and apoptotic cell death.

        4-phenyl butyric acid; Hypoxia/reoxygenation; Endoplasmic reticulum stress; Aopoptosis

        國家自然科學(xué)基金(81070101);四川省教育廳課題(15ZB0200)

        R-33

        A

        10.3969/j.issn.1672-3511.2016.03.010

        2015-12-01; 修回日期: 2016-01-17; 編輯: 母存培)

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