【摘要】目的 探究在心肌梗死(MI)后使用卡格列凈(CANA)對(duì)心臟炎癥反應(yīng)及細(xì)胞凋亡的作用。方法 選用15只8~10周齡C57/BL6小鼠(SPF級(jí)),用抽簽法隨機(jī)抽取5只小鼠為Sham組(假手術(shù)+生理鹽水灌胃),剩余10只小鼠通過(guò)結(jié)扎左冠狀動(dòng)脈前降支的方法制作MI模型,制作成功后的10只MI小鼠隨機(jī)分為2組:MI組(生理鹽水灌胃)、CANA+MI組(CANA灌胃),每組5只。CANA+MI組小鼠以20 mg/(kg·d-1)卡格列凈灌胃4周,Sham組及MI組則使用同等體積生理鹽水灌胃4周。4周后,使用小鼠超聲機(jī)測(cè)量左室收縮末期內(nèi)徑及左室射血分?jǐn)?shù)。蘇木精染色、Tunel染色用于明確心臟結(jié)構(gòu)及MI后細(xì)胞凋亡狀況。IF免疫熒光染色用于檢測(cè)MI后心臟炎癥反應(yīng)程度。Western blotting用于檢測(cè)炎癥及凋亡相關(guān)蛋白表達(dá)水平。結(jié)果 與MI組相比,CANA+MI組左室射血分?jǐn)?shù)升高、梗死面積減小。Tunel染色顯示,CANA+MI組小鼠MI邊緣區(qū)凋亡細(xì)胞數(shù)量較MI組顯著減少,相應(yīng)促凋亡蛋白Bax表達(dá)降低,抑凋亡蛋白Bcl-2表達(dá)升高。CANA+MI組小鼠CD3+T細(xì)胞、F480+巨噬細(xì)胞和LY6G+中性粒細(xì)胞數(shù)量較MI組明顯減少,相關(guān)蛋白p-P65、p-IκBα表達(dá)量明顯降低。結(jié)論 CANA抑制了MI后心臟炎癥反應(yīng),減輕了細(xì)胞凋亡。
【關(guān)鍵詞】卡格列凈;炎癥反應(yīng);心肌梗死;細(xì)胞凋亡
【DOI】10.16806/j.cnki.issn.1004-3934.2024.04.018
Canagliflozin Alleviates Apoptosis and Inflammation After
Myocardial Infarction and Improves Prognosis
LI Ming,GAO Jixian,WU Bing,LIU Xiaoxiong,XIA Hao
(Department of Cardiology,Renmin Hospital of Wuhan University,Cardiovascular Research Institute,Wuhan University,Hubei Key Laboratory of Cardiology,Wuhan 430060,Hubei,China)【Abstract】Objective To investigate the effect of canagliflozin(CANA)on cardiac inflammatory response and apoptosis after myocardial infarction (MI).Methods This study selected 15 C57/BL6 mice (SPF grade) aged 8~10 weeks,and randomly selected 5 mice as the Sham group (sham surgery+saline gavage) using a lottery method.The remaining 10 mice were used to create an MI model by ligating the anterior descending branch of the left coronary artery.After successful production,the 10 MI mice were randomly divided into two groups:the MI group (saline gavage) and the CANA+MI group (Canagliflozin gavage),with 5 mice in each group.The mice in the CANA+MI group were gavaged with 20 mg/(kg·d-1) of Canagliflozin for 4 weeks,while the Sham and MI groups were gavaged with the same volume of physiological saline for 4 weeks.After 4 weeks,left ventricular end systolic diameter and left ventricular ejection fraction were measured using a mouse ultrasound machine.Hematoxylin(HE staining) and Tunel staining are used to clarify the cardiac structure and cell apoptosis status after MI.IF immunofluorescence staining is used to detect the degree of cardiac inflammatory response after MI.Western blotting is used to detect the expression levels of inflammation and apoptosis related proteins.Results Compared with the MI group,left ventricular ejection fraction increased and infarct size decreased in CANA+MI group.Tunel staining showed that the number of apoptotic cells in the MI margin area of the CANA+MI group mice was significantly reduced compared to the MI group,with a corresponding decrease in the expression of pro apoptotic protein Bax and an increase in the expression of anti apoptotic protein Bcl-2.The number of CD3+ T cells,F(xiàn)480+ macrophages,and LY6G+ neutrophils in the CANA+MI group mice was significantly reduced compared to the MI group,with associated proteins p-P65,p-IκBα.The expression level was significantly reduced.Conclusion Canagliflozin inhibits cardiac inflammatory response after MI and reduces cell apoptosis.
【Keywords】Canagliflozin;Inflammatory response;Myocardial infarction;Apoptosis
心血管疾病仍然是世界上人類(lèi)死亡的主要原因之一,而心肌梗死(myocardial infarction,MI)是心血管疾病中最嚴(yán)重的表現(xiàn),在發(fā)達(dá)國(guó)家中每年因急性MI造成的死亡人數(shù)超過(guò)1/3[1]。盡管越來(lái)越多的生活干預(yù)及治療方法使65歲以上老年人急性MI的死亡率有所降低,但65歲以下的男性及女性心血管事件發(fā)生率并沒(méi)有類(lèi)似的下降[2],因此MI對(duì)人類(lèi)的健康仍然有著重大威脅,尋找新的治療方案及作用靶點(diǎn)勢(shì)在必行。
鈉-葡萄糖共轉(zhuǎn)運(yùn)蛋白2抑制劑(sodium-glucose cotransporter 2 inhibitor,SGLT2i)最初被發(fā)現(xiàn)是一種治療2型糖尿病的藥物,通過(guò)促進(jìn)尿液中的糖及鈉的排泄,來(lái)發(fā)揮降糖作用。后來(lái)具有重要意義的臨床試驗(yàn)EMPA[3]和CANVAS [4]證實(shí)了SGLT2i在2型糖尿病高?;颊咧芯哂酗@著心血管益處。最近的研究[5]證明長(zhǎng)期口服卡格列凈(canagliflozin,CANA)發(fā)揮心血管保護(hù)作用并不受體內(nèi)血糖的影響。CANA發(fā)揮作用可能與多種因素相關(guān),例如抗炎、抗氧化應(yīng)激、心肌重構(gòu)和離子代謝等。因此,SGLT2i發(fā)揮心血管益處的具體機(jī)制仍不明確,本研究試圖探索CANA影響非糖尿病MI小鼠的炎癥反應(yīng)、心肌細(xì)胞凋亡及心臟重構(gòu)的相關(guān)機(jī)制。
1 材料和方法
1.1 材料
1.1.1 實(shí)驗(yàn)動(dòng)物
8~10周齡雄性C57/BL6小鼠(SPF級(jí))購(gòu)自北京維通利華實(shí)驗(yàn)動(dòng)物技術(shù)有限公司(許可證號(hào):SCXX2021-0006),所有動(dòng)物均飼養(yǎng)于SPF級(jí)環(huán)境中,予以12 h光照及無(wú)光照環(huán)境養(yǎng)育,環(huán)境濕度50%~80%,溫度21~23 ℃,予以普通食物飼養(yǎng)。本實(shí)驗(yàn)采用的動(dòng)物實(shí)驗(yàn)方案經(jīng)武漢大學(xué)人民醫(yī)院動(dòng)物保護(hù)與使用委員會(huì)批準(zhǔn)[倫理編號(hào):WDRM動(dòng)(福)20211203D號(hào)]。
1.1.2 試劑與儀器
GAPDH抗體(AC001)、p-P65抗體(AP1294)、p-IκBα抗體(AP0707)、Bcl-2抗體(A19693)、HRP山羊抗兔IgG二抗、TRIzol(RK30129)均購(gòu)自于武漢愛(ài)博泰克生物(Abclonal)??笲ax一抗(GB114122)、RIPA裂解液(G2002)、5×蛋白上樣緩沖液、SDS-PAGE凝膠快速制備試劑盒、Servicebio RT First Stand cDNA Synthesis Kit、Servicebio 2*SYBR Green qPCR Master Mix試劑盒(Low ROX)均購(gòu)自于武漢塞維爾生物科技有限公司(Servicebio)。
1.2 方法
1.2.1 動(dòng)物MI模型及給藥
小鼠通過(guò)腹腔注射3%戊巴比妥鈉(90 mg/kg)進(jìn)行麻醉,剔除左側(cè)胸部體毛且使用酒精對(duì)相應(yīng)部位進(jìn)行消毒,將其仰臥位固定于手術(shù)臺(tái)上,然后使用小動(dòng)物呼吸機(jī)(Kent Scientific VFA-23-BV型)保證通氣。在胸骨左緣第3、4肋間切開(kāi),逐步暴露心臟,使用7-0絲線結(jié)扎左前降支,結(jié)扎后可以發(fā)現(xiàn)結(jié)扎部位心肌變白或者顏色變淺,接著逐步縫合,抽出殘存空氣后關(guān)閉胸部。術(shù)后將小鼠放于加熱板上,完全蘇醒前將小鼠放回獨(dú)立通氣的籠內(nèi)。Sham組小鼠進(jìn)行相同操作,但不進(jìn)行結(jié)扎。在術(shù)后第3天開(kāi)始,CANA+MI組小鼠每天行CANA灌胃處理[20 mg/(kg·d-1)],持續(xù)4周[6]。Sham組及MI組每天以等量生理鹽水進(jìn)行灌胃,持續(xù)4周。
1.2.2 小鼠心臟超聲檢查
4周后小鼠通過(guò)吸入2%異氟醚進(jìn)行麻醉,通過(guò)小動(dòng)物高分辨超聲成像采集心臟的長(zhǎng)軸及短軸圖像,測(cè)量出左室收縮末期內(nèi)徑,計(jì)算左室射血分?jǐn)?shù)。
1.2.3 免疫熒光染色
取出術(shù)后4周小鼠心臟,先用生理鹽水取出殘存血液,再使用10%氯化鉀溶液浸泡使心臟停搏于舒張期,用4’,6-二脒基-2-苯基吲哚(D4054,Bioscience,中國(guó))對(duì)細(xì)胞核進(jìn)行標(biāo)記。最后在熒光顯微鏡下進(jìn)行圖像采集,用Image J軟件進(jìn)行定量分析。
1.2.4 蘇木精染色
用準(zhǔn)備好的石蠟切片進(jìn)行二甲苯透明、梯度酒精脫水、蘇木精染色、鹽酸酒精分化、清洗后伊紅染色、梯度酒精脫水、二甲苯透明等步驟后,進(jìn)行封片,最后于顯微鏡下觀察。使用Image J軟件進(jìn)行計(jì)算左心室梗死面積。
1.2.5 Tunel染色
將MI后4周小鼠心臟取出,使用Tunel染色對(duì)MI邊緣區(qū)凋亡心肌進(jìn)行檢測(cè),應(yīng)用ApopTag Plus熒光素原位凋亡檢測(cè)試劑盒(S7111,Millipore)進(jìn)行染色,隨后使用熒光顯微鏡下進(jìn)行圖像采集,用Image J軟件進(jìn)行定量分析。
1.2.6 Western blotting檢測(cè)
首先從心臟心室組織(MI小鼠則為MI邊緣區(qū))中提取總蛋白。將準(zhǔn)備好的心臟組織放入蛋白酶及磷酸酶抑制劑混合物中,經(jīng)過(guò)研磨、離心后,取上清液加入蛋白上樣緩沖液后金屬浴100 ℃10 min,提取好的蛋白使用BCA蛋白測(cè)定試劑盒(Servicebio)測(cè)定蛋白濃度。蛋白通過(guò)10% SDS-PAGE(Servicebio)進(jìn)行電泳分離,并轉(zhuǎn)移到聚偏氟乙烯膜上,進(jìn)行封閉及三羥甲基氨基甲烷緩沖液清洗,然后放入對(duì)應(yīng)一抗在4 ℃冰箱中孵育過(guò)夜。隨后,將聚偏氟乙烯膜與相應(yīng)二抗在室溫下孵育90 min。最后在Bio-Rad檢測(cè)系統(tǒng)上使用增強(qiáng)化學(xué)發(fā)光將圖像可視化。根據(jù)目的條帶與加載控制的灰度值比采用Image J軟件評(píng)估蛋白的相對(duì)表達(dá)量。
1.3 統(tǒng)計(jì)學(xué)處理
應(yīng)用GraphPad Prism 9統(tǒng)計(jì)軟件進(jìn)行數(shù)據(jù)分析。計(jì)量資料采用均數(shù)±標(biāo)準(zhǔn)差表示,兩組間比較采用Students’t檢驗(yàn),多組間比較采用單因素方差分析。Plt;0.05為差異有統(tǒng)計(jì)學(xué)意義。
2 結(jié)果
2.1 CANA減輕MI后MI面積和心功能障礙
MI后4周,HE染色結(jié)果顯示,與MI組相比,CANA+MI組小鼠MI面積明顯減少(圖1A、1B),同時(shí)進(jìn)行的小鼠心臟超聲結(jié)果顯示(圖1C、1D及1E),與MI組對(duì)比,CANA+MI組小鼠的左室射血分?jǐn)?shù)顯著提高。綜上所述,這些數(shù)據(jù)表明,CANA可以減少M(fèi)I后的梗死面積,改善心功能,有利于預(yù)后。
2.2 CANA減輕MI后心肌細(xì)胞凋亡
細(xì)胞凋亡是MI后梗死面積大小與患者死亡的主要原因。因此筆者通過(guò)Tunel染色進(jìn)一步研究CANA是否減少M(fèi)I邊緣區(qū)細(xì)胞凋亡。如圖2A、2B所示,Sham組小鼠Tunel陽(yáng)性細(xì)胞極少,且各組間差異沒(méi)有統(tǒng)計(jì)學(xué)意義,MI組小鼠MI邊緣區(qū)凋亡細(xì)胞數(shù)量明顯增加。而相比于MI組小鼠,CANA+MI組小鼠的凋亡細(xì)胞數(shù)量明顯減少。為了收集更多的證據(jù)證實(shí)CANA減輕MI后心肌細(xì)胞凋亡,筆者檢測(cè)了相關(guān)因子蛋白水平。Western blotting結(jié)果顯示,相比于MI組,CANA+MI組小鼠促進(jìn)抗凋亡蛋白Bcl-2的表達(dá),抑制促凋亡蛋白Bax的表達(dá)(圖2C、2D)。這表明CANA可以減輕MI后心肌細(xì)胞凋亡。
2.3 CANA減輕MI后炎癥反應(yīng)
由于MI的發(fā)展過(guò)程中,持續(xù)的炎癥反應(yīng)會(huì)引起大量免疫細(xì)胞及炎性細(xì)胞浸潤(rùn),加重心肌細(xì)胞凋亡及心功能損傷,因此探究CANA對(duì)MI后炎癥反應(yīng)的影響。如圖3A、3B所示,與MI組小鼠相比,CANA+MI組小鼠減輕了MI邊緣區(qū)的炎癥反應(yīng),抑制了炎癥細(xì)胞聚集,CD3+T細(xì)胞、F480+巨噬細(xì)胞及LY6G+中性粒細(xì)胞明顯減少。為了收集更多CANA干預(yù)MI炎癥反應(yīng)的證據(jù),筆者對(duì)炎癥的經(jīng)典通路核因子κB(nuclear factor-κB,NF-κB)進(jìn)行了Western blotting分析,圖3C、3D表明,與MI組相比,CANA+MI組小鼠的p-P65及p-IκBα水平明顯降低。上述結(jié)果表明,CANA可以通過(guò)調(diào)控NF-κB通路來(lái)減輕MI后心臟炎癥反應(yīng)。
3 討論
在MI發(fā)生以后,梗死心臟的修復(fù)依賴于一個(gè)高度協(xié)調(diào)的反應(yīng),這包括炎癥細(xì)胞和間充質(zhì)細(xì)胞的連續(xù)募集和清除[7]。梗死愈合可以分為三個(gè)不同但又相互重疊的階段:炎癥期、增殖期和成熟期[8]。在炎癥期中,壞死的細(xì)胞釋放危險(xiǎn)信號(hào)激活受體系統(tǒng),如Toll樣受體、晚期糖基化終產(chǎn)物受體、白細(xì)胞介素(interleukin,IL)等,這些受體又通過(guò)不同的方式激活下游促炎級(jí)聯(lián)反應(yīng),引起強(qiáng)烈的炎癥反應(yīng)。大量的免疫細(xì)胞及炎性細(xì)胞(巨噬細(xì)胞)浸潤(rùn)于梗死心肌中,炎性巨噬細(xì)胞有助于消化和清除受損的細(xì)胞及細(xì)胞外基質(zhì),但同時(shí)分泌炎癥因子,如腫瘤壞死因子-α、IL-1β、IL-6和IL-12,這些炎癥因子在長(zhǎng)時(shí)間持續(xù)的炎癥反應(yīng)中也加重心肌細(xì)胞凋亡及心功能損傷[9-11]。激活的多種受體系統(tǒng)中,NF-κB轉(zhuǎn)錄因子的激活涉及多種細(xì)胞類(lèi)型,在調(diào)節(jié)炎癥反應(yīng)、細(xì)胞黏附、生存和生長(zhǎng)控制多個(gè)方面起著重要作用。有大量的研究報(bào)告與上述對(duì)應(yīng),NF-κB缺失在再灌注梗死模型中可以減少梗死面積、改善心功能、降低促炎細(xì)胞因子水平[12]。近年來(lái),關(guān)于SGLT2i通過(guò)抑制NF-κB通路減輕炎癥反應(yīng)的研究大量報(bào)道[13-15]。在本次研究中,筆者探究了MI發(fā)生以后,CANA通過(guò)抑制NF-κB通路,也就是抑制p-IκBα的磷酸化,從而降低P65活化,最終減輕了MI后炎癥反應(yīng),改善了預(yù)后。
炎癥反應(yīng)發(fā)生以后,MI區(qū)的梗死細(xì)胞及細(xì)胞質(zhì)基質(zhì)碎片被清除,但長(zhǎng)時(shí)間持續(xù)的炎癥反應(yīng)使促炎介質(zhì)的表達(dá)增強(qiáng),反而可能會(huì)激活促凋亡通路,誘導(dǎo)更多的心肌細(xì)胞損失,這也是MI后心肌細(xì)胞損失的主要原因。細(xì)胞凋亡的激活也決定了MI后的心臟功能以及MI后心力衰竭的發(fā)生。據(jù)相關(guān)文獻(xiàn)[14,16-17]報(bào)道,在葡萄糖脂毒性誘導(dǎo)的心肌模型、阿毒素誘導(dǎo)的心肌缺血再灌注模型中,CANA等SGLT2i均減輕了心肌細(xì)胞凋亡。在其他領(lǐng)域中,同樣有SGLT2i減輕細(xì)胞凋亡,保護(hù)機(jī)體功能的研究報(bào)道[18-19]。細(xì)胞凋亡的原因是促凋亡及抗凋亡相關(guān)蛋白之間的平衡失衡。結(jié)果表明,CANA通過(guò)抑制促凋亡蛋白Bax、促進(jìn)抗凋亡蛋白Bcl-2的表達(dá)而減輕心肌細(xì)胞的凋亡。
目前關(guān)于SGLT2i在心力衰竭中的益處已被證實(shí)[20-22],而SGLT2i在MI中的作用仍不明確。本研究在動(dòng)物實(shí)驗(yàn)中初步說(shuō)明了CANA可以通過(guò)抑制MI后心臟炎癥反應(yīng)及心肌細(xì)胞凋亡,從而改善心臟功能改善預(yù)后,為MI治療過(guò)程中使用SGLT2i提供了一定支持,有潛力成為MI治療過(guò)程中的新型藥物,不足的是CANA發(fā)揮作用的具體分子機(jī)制需要進(jìn)一步研究。
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收稿日期:2023-11-29