游 莉 姜 輝 朱敏敏

(復(fù)旦大學(xué)附屬腫瘤醫(yī)院麻醉科 上海 200032)

異丙酚抑制高糖誘導(dǎo)的臍靜脈內(nèi)皮細(xì)胞黏附分子表達(dá)

游 莉▲姜 輝▲朱敏敏△

(復(fù)旦大學(xué)附屬腫瘤醫(yī)院麻醉科 上海 200032)

目的 研究異丙酚抑制高糖誘導(dǎo)的臍靜脈內(nèi)皮細(xì)胞黏附分子的表達(dá)并探討其可能的機(jī)制。方法 采用Histopaque-1077溶液提取人外周血單核細(xì)胞。采用一氧化氮(NO)試劑盒檢測臍靜脈內(nèi)皮細(xì)胞NO生成。采用Western blot檢測內(nèi)皮細(xì)胞黏附分子、內(nèi)皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)(總蛋白,單體及雙體)、eNOS磷酸化水平及caveolin-1表達(dá)。結(jié)果 高糖上調(diào)血管內(nèi)皮細(xì)胞黏附分子1(vascular cell adhesion molecule,VCAM-1)的表達(dá),促進(jìn)單核細(xì)胞-內(nèi)皮黏附,并減少NO生成。異丙酚改善高糖環(huán)境下NO生成,并抑制VCAM-1表達(dá)及單核細(xì)胞-內(nèi)皮黏附。異丙酚的作用可被eNOS抑制劑L-NAME所拮抗。異丙酚能上調(diào)高糖環(huán)境下eNOS-Ser1177磷酸化水平及雙體/單體比值,下調(diào)高糖環(huán)境下eNOS-Thr495磷酸化水平及caveolin-1表達(dá)。結(jié)論 異丙酚通過調(diào)節(jié)高糖環(huán)境下eNOS的磷酸化水平、單體/雙體比值及caveolin-1表達(dá),改善內(nèi)皮細(xì)胞NO生成,進(jìn)而抑制內(nèi)皮細(xì)胞黏附分子的表達(dá)及單核細(xì)胞-內(nèi)皮細(xì)胞的黏附。

丙泊酚; 高糖; 臍靜脈; 內(nèi)皮細(xì)胞; 黏附分子

圍術(shù)期常見高血糖,主要由圍術(shù)期的生理性應(yīng)激反應(yīng)及糖輸注過多引起[1-2]。高糖可上調(diào)血管內(nèi)皮細(xì)胞黏附分子(vascular cell adhesion molecule,VCAM)的表達(dá)[3-5],如VCAM-1。黏附分子表達(dá)增加誘導(dǎo)單核細(xì)胞-內(nèi)皮黏附[3],從而導(dǎo)致內(nèi)皮損傷。對(duì)于圍術(shù)期高血糖患者,抑制VCAM表達(dá)可改善內(nèi)皮損傷,從而改善高血糖患者預(yù)后。

一氧化氮(NO)是調(diào)節(jié)內(nèi)皮功能重要信號(hào)分子,在維持血管內(nèi)皮功能中發(fā)揮關(guān)鍵作用。高糖環(huán)境下內(nèi)皮細(xì)胞生成NO的功能受損。研究表明NO生成減少可導(dǎo)致VCAM大量表達(dá)并促進(jìn)單核細(xì)胞-內(nèi)皮黏附,誘導(dǎo)內(nèi)皮損傷[6-7],而改善NO生成可使VCAM的表達(dá)減少[8]。異丙酚能改善缺氧再灌注誘導(dǎo)的VCAM表達(dá)[9],但其是否能改善高糖誘導(dǎo)的VCAM表達(dá)尚不明確。本文旨在研究異丙酚是否能改善高糖誘導(dǎo)的VCAM表達(dá),并探討其可能的調(diào)節(jié)機(jī)制(圖1)。

圖1 科學(xué)假說示意圖:異丙酚調(diào)節(jié)高糖誘導(dǎo)的VCAM表達(dá)

材料和分組 將臍靜脈內(nèi)皮細(xì)胞分為5組:第1組(對(duì)照組)用5mmol/L葡萄糖培養(yǎng)細(xì)胞4 h;第2組(高糖組)用30 mmol/L葡萄糖培養(yǎng)細(xì)胞4 h;第3組(丙泊酚+高糖組)用5μmol/L丙泊酚預(yù)處理細(xì)胞30 min,再用30 mmol/L葡萄糖培養(yǎng)細(xì)胞4 h;第4組[丙泊酚+內(nèi)皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)抑制劑+高糖組]用5μmol/L丙泊酚、eNOS抑制劑L-NAME 100μmol/L預(yù)處理細(xì)胞30 min,再用30 mmol/L葡萄糖培養(yǎng)細(xì)胞4 h;第5組(eNOS抑制劑對(duì)照組)用L-NAME 100μmol/L預(yù)處理細(xì)胞30 min,再用5 mmol/L葡萄糖培養(yǎng)細(xì)胞4 h。

單核細(xì)胞-內(nèi)皮細(xì)胞黏附實(shí)驗(yàn) 用Histopaque-1077 (美國Sigma公司)試劑分離單核細(xì)胞。將5 mL含有單核細(xì)胞的肝素化血液轉(zhuǎn)移到含有Histopaque-1077 (5 mL)的離心管中,室溫下400×g離心30 min,吸出單核細(xì)胞并用PBS清洗,離心后用DEMN培養(yǎng)基重懸單核細(xì)胞。將單核細(xì)胞懸液加入各處理組含內(nèi)皮細(xì)胞的培養(yǎng)皿中,37 ℃孵育30 min。細(xì)胞用PBS清洗3遍,去除未與內(nèi)皮細(xì)胞發(fā)生黏附的單核細(xì)胞,顯微鏡下觀察并計(jì)數(shù)。

NO生成檢測 采用NO試劑盒(南京建成生物工程研究所)檢測臍靜脈內(nèi)皮細(xì)胞中NO的生成。

Western blot檢測 提取細(xì)胞蛋白,等量蛋白經(jīng)6% SDS-PAGE膠分離,轉(zhuǎn)移到PVDF膜上。室溫下用含5%脫脂奶粉的TBST溶液封閉1 h,加入相應(yīng)的一抗,4 ℃孵育過夜。一抗分別為VCAM-1、p-eNOS-Ser1177、p-eNOS-Thr495、eNOS、caveolin-1和β-actin。次日用TBST溶液于室溫下洗膜3次,每次10 min,加入相應(yīng)的二抗室溫孵育1h,再用TBST洗膜3次,每次10 min,最后曝光顯影。二抗分別為鼠二抗和兔二抗。各組對(duì)應(yīng)蛋白條帶的密度使用scan-gel-it軟件進(jìn)行分析,β-actin作為內(nèi)參。對(duì)照組中目的蛋白與β-actin蛋白條帶密度的比值設(shè)定為1。

結(jié) 果

高糖促進(jìn)單核細(xì)胞-內(nèi)皮細(xì)胞的黏附(P<0.000 1),其作用可被異丙酚所抑制,并與異丙酚濃度存在劑量依賴關(guān)系(圖2A)。高糖可誘導(dǎo)內(nèi)皮細(xì)胞表面VCAM-1表達(dá)(P =0.000 2),其作用可被異丙酚預(yù)處理所抑制,并同異丙酚預(yù)處理的濃度存在劑量依賴關(guān)系(圖2B、2C)。5 μmol/L異丙酚預(yù)處理可抑制高糖誘導(dǎo)單核細(xì)胞-內(nèi)皮細(xì)胞的黏附(P<0.000 1)及VCAM-1表達(dá)(P=0.001 3)。

(1)5mmol/L glucose;(2)30 mmol/L glucose.

圖2 異丙酚抑制高糖誘導(dǎo)的單核細(xì)胞-內(nèi)皮黏附及VCAM-1表達(dá)

Fig 2 Propofol inhibited high glucose induced monocyte endothelial adhesion and VCAM-1 expression

與對(duì)照相比,高糖抑制內(nèi)皮細(xì)胞NO生成(P<0.001)。與高糖處理組相比,異丙酚預(yù)處理可以改善NO生成,并與異丙酚的預(yù)處理濃度存在劑量依賴關(guān)系(圖3)。5μmol/L異丙酚預(yù)處理可以改善高糖誘導(dǎo)的NO生成減少(P<0.000 1)。

(1)5 mmol/L glucose;(2)30 mmol/L glucose.

圖3 異丙酚改善高糖環(huán)境下臍靜脈內(nèi)皮細(xì)胞NO生成

Fig 3 Propofol improved NO production in umbilical vein endothelial cell with high glucose

相比于5 mmol/L葡萄糖,30 mmol/L高糖可誘導(dǎo)內(nèi)皮細(xì)胞表面VCAM-1的表達(dá)。異丙酚預(yù)處理可抑制高糖環(huán)境下VCAM-1表達(dá),其作用可被eNOS抑制劑L-NAME所拮抗(P=0.002 9,圖4A、4B)。

相比于5mmol/L葡萄糖,30 mmol/L高糖抑制p-eNOS-Ser1177磷酸化水平(P<0.000 1)、上調(diào)p-eNOS-Thr495磷酸化水平(P=0.0005)及eNOS表達(dá)(P=0.000 4,圖5A、5B)、下調(diào)eNOS雙體/單體(P<0.000 1,圖5A、5C)、上調(diào)caveolin-1表達(dá)(P<0.000 1,圖5A、5D)。5μmol/L異丙酚預(yù)處理可以上調(diào)高糖環(huán)境下p-eNOS-Ser1177磷酸化水平(P=0.001 4)、下調(diào)p-eNOS-Thr495磷酸化水平(P=0.001 3,圖5A、5B)、上調(diào)eNOS雙體/單體(P=0.000 4,圖5A、5C)、下調(diào)caveolin-1表達(dá)(P=0.000 3,圖5A、5D)、對(duì)eNOS總體表達(dá)無影響(P=0.480 8,圖5A、5B)。

討 論

以上研究結(jié)果表明30 mmol/L高糖處理上調(diào)臍靜脈內(nèi)皮細(xì)胞黏附分子表達(dá),增加單核細(xì)胞-內(nèi)皮細(xì)胞的黏附。5μmol/L異丙酚的預(yù)處理通過改善高糖環(huán)境下內(nèi)皮細(xì)胞NO生成,抑制高糖誘導(dǎo)的內(nèi)皮黏附分子表達(dá)及單核細(xì)胞-內(nèi)皮細(xì)胞的黏附。高糖環(huán)境下,5μmol/L異丙酚通過調(diào)節(jié)內(nèi)皮細(xì)胞eNOS的磷酸化水平、單體/雙體比值及抑制caveolin-1表達(dá)來改善NO生成。

以上實(shí)驗(yàn)結(jié)果支持了高糖上調(diào)內(nèi)皮黏附分子表達(dá),從而促進(jìn)單核細(xì)胞與內(nèi)皮細(xì)胞的黏附,誘導(dǎo)內(nèi)皮損傷[3-5];內(nèi)皮細(xì)胞NO生成減少可誘導(dǎo)黏附分子的表達(dá)[6-7];改善NO生成可以抑制黏附分子的表達(dá),且可被eNOS抑制劑L-NAME所阻斷[8];高糖通過抑制內(nèi)皮細(xì)胞NO生成,上調(diào)黏附分子的表達(dá)并促進(jìn)單核細(xì)胞-內(nèi)皮細(xì)胞的黏附,誘導(dǎo)內(nèi)皮損傷。

(1)5 mmol/L glucose;(2)30 mmol/L glucose.

圖 4 eNOS抑制劑L-NAME拮抗異丙酚的作用

Fig 4 Effect of propofol converted by the eNOS inhibitor of L-NAME

(1)5 mmol/L glucose;(2)30 mmol/L glucose.

圖5 異丙酚對(duì)高糖環(huán)境下eNOS磷酸化水平、雙體/單體比值、總表達(dá)及caveolin-1表達(dá)的影響

Fig 5 Effects of propofol on eNOS phpsphorylation level,morphom/disome ratio,expressions of total eNOS and caveolin-1

eNOS是內(nèi)皮細(xì)胞生成NO的主要來源。eNOS的功能主要由eNOS的表達(dá)[10]、磷酸化水平[11]、單體/雙體比值[12]及caveolin-1[13]調(diào)節(jié)。本研究發(fā)現(xiàn)高糖雖然上調(diào)eNOS的表達(dá),但其可以抑制eNOS正性調(diào)節(jié)位點(diǎn)Ser1177磷酸化水平,上調(diào)抑制性磷酸化位點(diǎn)Thr495磷酸化水平,增加eNOS單體/雙體的比值,并上調(diào)caveolin-1的表達(dá)。eNOS主要以雙體形式存在,生成NO而發(fā)揮正常的生理功能;但在高糖環(huán)境下eNOS發(fā)生脫偶聯(lián),此時(shí)eNOS不再生產(chǎn)NO,而生成超氧陰離子[14-15]。以上實(shí)驗(yàn)研究一方面表明高糖可影響eNOS的磷酸化水平而減少內(nèi)皮細(xì)胞NO的生成;另一方面,在內(nèi)皮細(xì)胞中大量存在eNOS大量富集的caveolae結(jié)構(gòu),調(diào)控并保持適當(dāng)?shù)腘O水平,在I型糖尿病中caveolin-1表達(dá)上調(diào)減少NO生成[13],caveolin-1是eNOS上游的負(fù)調(diào)控因子,與eNOS形成復(fù)合物,此時(shí)eNOS失去了催化NO產(chǎn)生活性的能力。

異丙酚是臨床上常用的靜脈全麻藥,除鎮(zhèn)靜、催眠外,還具有抗氧化、抗凋亡、抗炎等作用。本研究發(fā)現(xiàn)異丙酚具有改善高糖環(huán)境下內(nèi)皮細(xì)胞生成NO的作用,而該作用主要通過改善eNOS的功能來實(shí)現(xiàn)。首先,5μmol/L異丙酚能改善高糖環(huán)境下eNOS的磷酸化水平及單體/雙體比值。一方面支持高糖激活內(nèi)皮細(xì)胞PKC-βⅡ,上調(diào)抑制性磷酸化位點(diǎn)eNOS-Thr495的磷酸化水平[16];另一方面支持高糖可以激活PP2A,下調(diào)正性磷酸化位點(diǎn)eNOS-Ser1177的磷酸化水平[17];同時(shí)支持異丙酚可以抑制PKC-βⅡ及PP2A的活性[18],從而改善高糖環(huán)境下eNOS的磷酸化水平。高糖環(huán)境下生成的過氧亞硝基陰離子可誘導(dǎo)eNOS發(fā)生脫偶聯(lián),異丙酚是強(qiáng)大的過氧亞硝基陰離子清除劑[19],可改善高糖誘導(dǎo)的eNOS脫偶聯(lián)。其次,5μmol/L異丙酚能抑制高糖環(huán)境下caveolin-1的表達(dá),在平滑肌細(xì)胞中異丙酚對(duì)eNOS/caveolin-1信號(hào)通路有調(diào)節(jié)作用,能促進(jìn)NO生成[20]。本研究發(fā)現(xiàn),5μmol/L異丙酚在臍靜脈內(nèi)皮細(xì)胞中對(duì)eNOS/caveolin-1信號(hào)通路也有調(diào)節(jié)作用,并能促進(jìn)NO生成。因此,5μmol/L異丙酚通過調(diào)節(jié)高糖環(huán)境下eNOS的磷酸化水平、單體/雙體比值及caveolin-1表達(dá),改善內(nèi)皮細(xì)胞NO生成,進(jìn)而抑制VCAM-1的表達(dá)及單核細(xì)胞-內(nèi)皮細(xì)胞的黏附。雖然eNOS是內(nèi)皮細(xì)胞生成NO的重要來源之一,但是既往研究表明異丙酚不影響高糖環(huán)境下eNOS的表達(dá)及偶聯(lián)狀態(tài)[21]。

本研究的不足之處在于,在人臍靜脈內(nèi)皮細(xì)胞中進(jìn)行的體外實(shí)驗(yàn)不同于體內(nèi)實(shí)驗(yàn),尤其是藥物有效性和毒性,且未觀察丙泊酚和高糖對(duì)單核細(xì)胞的影響。因此,需要進(jìn)一步確定丙泊酚抑制高糖誘導(dǎo)的單核-內(nèi)皮細(xì)胞黏附作用可否通過單核細(xì)胞起作用。

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Propofol inhibits high glucose induced expression of endothelial adhesion molecule in umbilical vein endothelial cells

YOU Li▲, JIANG Hui▲, ZHU Min-min△

(DepartmentofAnesthesiology,ShanghaiCancerCenter,FudanUniversity,Shanghai200032,China)

Objective To study high glucose induced expressiont of endothelial adhesion molecule inhibited by propofol in umbilical vein endothelial cells,and to investigate its mechanism. Methods Human peripheral mononuclear cells were prepared with Histopaque-1077 solution.Nitric oxide (NO) production was measured with an assay kit.Vascular cell adhesion molecule 1 (VCAM-1) expression,endothelial nitric oxide synthase (eNOS) total protein,dimer and monomer expression,eNOS phosphorylation and caveolin-1 were measured by Western blot. Results High glucose induced VCAM-1 expression,increased mononuclear-endothelial adhesion and reduced NO production.Propofol improved NO level,and inhibited VCAM-1 expression and mononuclear-endothelial adhesion.The protective effect of propofol would be blocked by an eNOS inhibitor of L-NAME.Propofol increased high glucose-mediated eNOS-Ser1177phosphrylation and dimmer/monomer ratio,and attenuated high glucose-induced eNOS-Thr495phosphrylation and caveolin-1 expression. Conclusions Propofol improved high glucose mediated eNOS phosphrylation,dimer/monomer ratio and caveolin-1 expression,so that NO production was improved and VCAM-1 expression and mononuclear-endothelial interaction were inhibited.

propofol; high glucose; umbilical vein; endithelial cell; adhesion molecule

R34,R614.1

A

10.3969/j.issn.1672-8467.2017.01.013

2016-01-18;編輯:段佳)

▲YOU Li and JIANG Hui contributed equally to this article

△Corresponding author E-mail:zhu_mm@126.com