索艷，谷優(yōu)優(yōu)，王肅

(天津市第五中心醫(yī)院，天津300451)

α-平滑肌肌動(dòng)蛋白對(duì)高糖誘導(dǎo)的腎小管上皮細(xì)胞增殖、凋亡的影響

索艷，谷優(yōu)優(yōu)，王肅

(天津市第五中心醫(yī)院，天津300451)

目的 探討α-平滑肌肌動(dòng)蛋白(α-SMA)對(duì)高糖誘導(dǎo)的腎小管上皮細(xì)胞增殖、凋亡的影響及機(jī)制。方法 培養(yǎng)人腎小管上皮細(xì)胞，分為低糖組、高糖組和干擾組。干擾組將α-SMA siRNA轉(zhuǎn)染至腎小管上皮細(xì)胞中，高糖組、低糖組細(xì)胞均不進(jìn)行α-SMA轉(zhuǎn)染。轉(zhuǎn)染完成后，高糖組、干擾組細(xì)胞用高糖DMEM細(xì)胞培養(yǎng)液培養(yǎng)，低糖組細(xì)胞用低糖DMEM細(xì)胞培養(yǎng)液培養(yǎng)，均培養(yǎng)48 h。采用MTT法檢測(cè)細(xì)胞增殖情況。采用流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡情況，計(jì)算細(xì)胞凋亡率。采用Western blotting法檢測(cè)細(xì)胞含半胱氨酸的天冬氨酸蛋白水解酶3(Caspase-3)、Bcl-2相關(guān)X蛋白(Bax)、Toll樣受體4(TLR4)、核因子-κB(NF-κB) 蛋白表達(dá)，采用二氯二氫熒光素-乙酰乙酸酯(DCFH-DA)法檢測(cè)細(xì)胞中ROS水平，采用ELISA法檢測(cè)上清液TNF-α、IL-8水平。結(jié)果 高糖組、干擾組細(xì)胞增殖能力均低于低糖組(P均<0.01)，干擾組細(xì)胞增殖能力高于高糖組(P<0.01)。高糖組、干擾組細(xì)胞凋亡率均高于低糖組(P均<0.01)，干擾組細(xì)胞凋亡率低于高糖組(P<0.01)。高糖組、干擾組細(xì)胞Caspase-3、Bax、TLR4、NF-κB蛋白表達(dá)均高于低糖組(P均<0.01)，干擾組細(xì)胞Caspase-3、Bax、TLR4、NF-κB蛋白表達(dá)均低于高糖組(P均<0.01)。高糖組、干擾組細(xì)胞中ROS水平及上清液TNF-α、IL-8水平均高于低糖組(P均<0.01)，干擾組細(xì)胞中ROS水平及上清液TNF-α、IL-8水平均低于高糖組(P均<0.01)。結(jié)論 干擾α-SMA后能夠逆轉(zhuǎn)高糖對(duì)腎小管上皮細(xì)胞的增殖抑制和凋亡促進(jìn)作用，其機(jī)制可能與降低細(xì)胞內(nèi)ROS水平及抑制TLR4/NF-κB信號(hào)通路有關(guān)。

糖尿病腎病；α-平滑肌肌動(dòng)蛋白；細(xì)胞增殖；細(xì)胞凋亡；炎癥因子

糖尿病腎病是由糖尿病引發(fā)的慢性疾病，是終末期腎病的主要原因之一。腎小管上皮萎縮及腎組織纖維化是糖尿病腎病的主要病理變化，而腎小管上皮細(xì)胞的增殖、凋亡是導(dǎo)致腎小管上皮萎縮的重要原因[1]。α-平滑肌肌動(dòng)蛋白(α-SMA)是腎組織纖維化的重要標(biāo)志蛋白[2]。研究表明，糖尿病腎病患者腎組織中α-SMA表達(dá)增多，腎小管上皮細(xì)胞凋亡增多[3]。腎組織炎癥也是糖尿病損害腎組織的重要原因。研究發(fā)現(xiàn)，腎組織中炎癥反應(yīng)與Toll樣受體4(TLR4)有關(guān)，TLR4激活后可導(dǎo)致核因子-κB(NF-κB)活化，誘導(dǎo)炎癥反應(yīng)的發(fā)生，進(jìn)而影響細(xì)胞的生長(zhǎng)及凋亡[4]。2015年12月～2016年11月，我們采用小干擾RNA(siRNA)技術(shù)抑制人腎小管上皮細(xì)胞α-SMA蛋白表達(dá)，觀察對(duì)高糖環(huán)境下腎小管上皮細(xì)胞增殖、凋亡的影響并探討其機(jī)制，為明確α-SMA對(duì)腎小管上皮細(xì)胞增殖、凋亡影響的機(jī)制提供實(shí)驗(yàn)依據(jù)。

1 材料與方法

1.1 細(xì)胞及試劑 人腎小管上皮細(xì)胞HK-2購(gòu)自上海中喬新舟生物科技有限公司。α-SMA多克隆抗體、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Caspase-3)單克隆抗體、Bcl-2相關(guān)X蛋白(Bax)單抗、TLR4多抗、NF-κB多抗、甘油醛-3-磷酸脫氫酶(GAPDH)單抗均購(gòu)自美國(guó)Abcam公司；低糖DMEM培養(yǎng)基(含5.6 mmol/L的D-葡萄糖)、高糖DMEM培養(yǎng)基(含25 mmol/L的D-葡萄糖)均購(gòu)自美國(guó)Gibco公司；活性氧(ROS)檢測(cè)試劑盒、二喹啉甲酸(BCA)蛋白濃度檢測(cè)試劑盒購(gòu)自碧云天生物技術(shù)研究所；TNF-α、IL-8 ELISA檢測(cè)試劑盒均購(gòu)自上海心語(yǔ)生物科技有限公司。

1.2 細(xì)胞培養(yǎng) 液氮中保存的腎小管上皮細(xì)胞在37 ℃融化，用含有10%胎牛血清的低糖DMEM培養(yǎng)液懸浮細(xì)胞，接種到細(xì)胞瓶中，37 ℃、飽和濕度、5%CO2培養(yǎng)箱培養(yǎng)。待細(xì)胞融合度達(dá)到90%時(shí)，用0.25%的胰蛋白酶消化，按照1∶3比例接種至細(xì)胞瓶中繼續(xù)培養(yǎng)。

1.3 細(xì)胞分組、轉(zhuǎn)染及干預(yù)方法 取培養(yǎng)至對(duì)數(shù)生長(zhǎng)期的人腎小管上皮細(xì)胞，胰蛋白酶消化后，以2×105個(gè)/孔細(xì)胞接種至6孔細(xì)胞培養(yǎng)板中，用不含胎牛血清的低糖培養(yǎng)液同步化24 h后，分為低糖組、高糖組和干擾組。干擾組按照Lipofectamine 2000轉(zhuǎn)染試劑說明書將α-SMA siRNA轉(zhuǎn)染至腎小管上皮細(xì)胞中，α-SMA siRNA 正義鏈為5′-GAGGAUGAACUGUUUCAAGTT-3′，反義鏈為5′-CUUGAAACAGUUCAUCCUCTT-3′。并設(shè)立對(duì)照細(xì)胞。Western blotting法檢測(cè)結(jié)果示干擾組細(xì)胞α-SMA蛋白表達(dá)明顯低于對(duì)照細(xì)胞，表明α-SMA siRNA轉(zhuǎn)染成功。高糖組、低糖組細(xì)胞均不進(jìn)行α-SMA轉(zhuǎn)染。待轉(zhuǎn)染完成后，高糖組、干擾組細(xì)胞用D-葡萄糖濃度為25 mmol/L的高糖DMEM細(xì)胞培養(yǎng)液培養(yǎng)，低糖組細(xì)胞用D-葡萄糖濃度為5.6 mmol/L的低糖DMEM細(xì)胞培養(yǎng)液培養(yǎng)，均培養(yǎng)48 h。

1.4 細(xì)胞增殖情況觀察 采用MTT法。取各組細(xì)胞，每組設(shè)置6個(gè)復(fù)孔，在每孔中加入5 mg/mL的MTT溶液20 μL，放在37 ℃孵育4 h后，棄上清液，加入二甲基亞砜150 μL，搖床震蕩反應(yīng)10 min。酶標(biāo)儀檢測(cè)490 nm處的光密度(OD)值，分析細(xì)胞增殖情況。

1.5 細(xì)胞凋亡情況觀察 采用流式細(xì)胞術(shù)。取各組細(xì)胞，加入胰蛋白酶消化細(xì)胞，棄上清液，用PBS懸浮細(xì)胞，調(diào)整細(xì)胞密度為1×106/mL。吸取1 mL細(xì)胞懸浮液，離心，加入500 μL的結(jié)合緩沖液混合后，加入碘化丙啶(PI)和膜聯(lián)蛋白-V各5 μL，放置于室溫條件下反應(yīng)10 min，流式細(xì)胞儀檢測(cè)細(xì)胞凋亡情況，計(jì)算細(xì)胞凋亡率。

1.6 細(xì)胞 Caspase-3、Bax、TLR4、NF-κB 蛋白表達(dá)檢測(cè) 采用Western blotting法。取各組細(xì)胞，提取細(xì)胞總蛋白，Western blotting法檢測(cè)細(xì)胞中 Caspase-3、Bax、TLR4、NF-κB蛋白表達(dá)。

1.7 細(xì)胞中ROS水平檢測(cè) 采用二氯二氫熒光素-乙酰乙酸酯(DCFH-DA)熒光探針法。取各組細(xì)胞，胰蛋白酶消化，收集1×106個(gè)細(xì)胞，用PBS反復(fù)洗滌后，加入2′7′-DCFH-DA，放在37 ℃孵育30 min。PBS洗滌3次，熒光酶標(biāo)儀檢測(cè)熒光強(qiáng)度，檢測(cè)的發(fā)射波長(zhǎng)為530 nm，激發(fā)波長(zhǎng)為485 nm，熒光強(qiáng)度越強(qiáng)代表ROS水平越高。

1.8 細(xì)胞上清液TNF-α、IL-8水平檢測(cè) 采用ELISA法。取各組細(xì)胞，吸取培養(yǎng)液上清，按照ELISA檢測(cè)試劑盒說明書檢測(cè)培養(yǎng)液上清中TNF-α、IL-8水平。

2 結(jié)果

2.1 各組細(xì)胞增殖情況比較 低糖組、高糖組、干擾組OD值分別為0.63±0.06、0.34±0.04、0.49±0.04。高糖組、干擾組細(xì)胞增殖能力均低于低糖組(P均<0.01)，干擾組細(xì)胞增殖能力高于高糖組(P<0.01)。

2.2 各組細(xì)胞凋亡率比較 低糖組、高糖組、干擾組細(xì)胞凋亡率分別為11.32%±1.02%、39.25%±2.12%、19.36%±1.85%。高糖組、干擾組細(xì)胞凋亡率均高于低糖組(P均<0.01),干擾組細(xì)胞凋亡率低于高糖組(P<0.01)。

2.3 各組細(xì)胞 Caspase-3、Bax、TLR4、NF-κB 蛋白表達(dá)比較 高糖組、干擾組細(xì)胞中 Caspase-3、Bax、TLR4、NF-κB蛋白表達(dá)均高于低糖組(P均<0.01),干擾組細(xì)胞中 Caspase-3、Bax、TLR4、NF-κB蛋白表達(dá)均低于高糖組(P均<0.01)。見表1。

表1 各組細(xì)胞 Caspase-3、Bax、TLR4、NF-κB蛋白表達(dá)比較±s)

注：與低糖組比較，*P<0.01；與高糖組比較，#P<0.01。

2.4 各組細(xì)胞中ROS及上清液TNF-α、IL-8水平比較 高糖組、干擾組細(xì)胞中ROS水平及上清中TNF-α、IL-8水平均高于低糖組(P均<0.01)，干擾組細(xì)胞中ROS水平及上清中TNF-α、IL-8水平均低于高糖組(P均<0.01)。見表2。

表2 各組細(xì)胞中ROS及上清液TNF-α、IL-8水平比較

注：與低糖組比較，*P<0.01；與高糖組比較，#P<0.01。

3 討論

糖尿病腎病是一種常見的發(fā)生于腎組織的慢性疾病。腎小管上皮細(xì)胞凋亡及腎組織中的炎癥是導(dǎo)致腎組織損傷的重要途徑[5]。α-SMA是肌動(dòng)蛋白家族的成員之一，在糖尿病腎病腎組織損傷中發(fā)揮重要作用[6]。研究表明，在糖尿病腎病患者腎組織中α-SMA表達(dá)增多，與腎組織的損傷程度有關(guān)[7]。目前對(duì)于α-SMA在糖尿病發(fā)病中的作用機(jī)制尚不明確，對(duì)其在高糖環(huán)境下對(duì)腎小管上皮細(xì)胞增殖、凋亡的影響及機(jī)制尚不明確。

腎小管上皮細(xì)胞的增殖、凋亡與糖尿病腎病腎組織損傷密切相關(guān)，而細(xì)胞凋亡受到一系列基因的嚴(yán)格調(diào)控[8]。α-SMA能夠影響成纖維細(xì)胞、腎小管上皮細(xì)胞等細(xì)胞凋亡過程[9]。本研究發(fā)現(xiàn)，高糖組、干擾組細(xì)胞增殖能力均低于低糖組，干擾組細(xì)胞增殖能力高于高糖組，提示高糖對(duì)腎小管上皮細(xì)胞的增殖具有抑制作用，抑制α-SMA表達(dá)后，則可逆轉(zhuǎn)高糖對(duì)腎小管上皮細(xì)胞的增殖抑制作用；高糖組細(xì)胞凋亡率均高于低糖組，干擾組細(xì)胞凋亡率低于高糖組，提示高糖可明顯促進(jìn)腎小管上皮細(xì)胞的凋亡，抑制α-SMA表達(dá)后，則可逆轉(zhuǎn)高糖對(duì)腎小管上皮細(xì)胞的促凋亡作用。以上結(jié)果表明，高糖能夠誘導(dǎo)腎小管上皮細(xì)胞凋亡、抑制腎小管上皮細(xì)胞增殖，干擾α-SMA表達(dá)后可使高糖環(huán)境下的腎小管上皮細(xì)胞增殖能力降低、凋亡增加。

Caspase-3和Bcl-2在細(xì)胞凋亡過程中發(fā)揮促進(jìn)作用[10]。Bax是Bcl-2蛋白家族的成員之一，其表達(dá)升高后能夠促進(jìn)細(xì)胞凋亡的發(fā)生，是一種促凋亡蛋白。細(xì)胞內(nèi)的ROS水平與細(xì)胞凋亡也有密切關(guān)系[11]。研究[12,13]表明，在糖尿病腎病腎組織中腎小管上皮細(xì)胞凋亡增多，ROS水平增高，促凋亡蛋白Bax表達(dá)增多，Caspase-3活化水平升高。α-SMA一方面能夠提高細(xì)胞中ROS水平，使細(xì)胞內(nèi)的脂質(zhì)發(fā)生過氧化，造成細(xì)胞膜通透性改變，影響細(xì)胞內(nèi)外平衡；另一方面可以促進(jìn)細(xì)胞中凋亡相關(guān)蛋白的表達(dá)，激活細(xì)胞中Caspase級(jí)聯(lián)反應(yīng)，使Caspase活化成為 Caspase-3，而 Caspase-3是細(xì)胞凋亡進(jìn)入不可逆階段的標(biāo)志，從而發(fā)揮促進(jìn)細(xì)胞凋亡的作用；同時(shí)還能夠促進(jìn)Bax的表達(dá)，進(jìn)一步促進(jìn)細(xì)胞凋亡[14]。本研究發(fā)現(xiàn)，高糖組細(xì)胞中 Caspase-3、Bax蛋白及ROS表達(dá)水平均高于低糖組，提示高糖能夠通過影響細(xì)胞中凋亡相關(guān)蛋白及ROS水平促進(jìn)腎小管上皮細(xì)胞凋亡；干擾組細(xì)胞中 Caspase-3、Bax蛋白及ROS表達(dá)水平均低于高糖組，提示抑制α-SMA表達(dá)后，能夠使高糖誘導(dǎo)的腎小管上皮細(xì)胞中 Caspase-3、Bax蛋白表達(dá)降低，減輕氧化應(yīng)激反應(yīng)，可能是其拮抗高糖對(duì)腎小管上皮細(xì)胞的增殖抑制作用、促凋亡的機(jī)制之一。

糖尿病腎病本質(zhì)上也是腎組織中的炎癥反應(yīng)。研究發(fā)現(xiàn)，糖尿病腎病患者的腎組織中TNF-α、IL-8表達(dá)上升[15,16]。本研究發(fā)現(xiàn)，高糖組細(xì)胞上清液TNF-α、IL-8水平均高于低糖組，干擾組細(xì)胞上清液TNF-α、IL-8水平均低于高糖組，提示高糖能夠誘導(dǎo)腎小管上皮細(xì)胞分泌TNF-α、IL-8導(dǎo)致炎癥反應(yīng)，而抑制α-SMA表達(dá)后能夠拮抗高糖誘導(dǎo)的腎小管上皮細(xì)胞分泌TNF-α、IL-8。

Toll樣受體是一種跨膜蛋白，能夠?qū)⒓?xì)胞外的抗原信息傳遞至細(xì)胞內(nèi)，在免疫反應(yīng)中發(fā)揮重要作用[17,18]。在腎組織中，Toll除了參與免疫反應(yīng)外，還能夠?qū)⑿盘?hào)傳遞給NF-κB，而NF-κB在細(xì)胞內(nèi)多種轉(zhuǎn)錄過程中都發(fā)揮關(guān)鍵作用，進(jìn)而影響細(xì)胞的生長(zhǎng)[19]。TLR4/NF-κB信號(hào)轉(zhuǎn)導(dǎo)通路在糖尿病腎病中發(fā)揮重要作用[20]。研究表明，在糖尿病腎病大鼠腎組織中發(fā)現(xiàn)TLR4和NF-κB的異常升高，高糖環(huán)境能夠誘導(dǎo)大鼠腎小管上皮細(xì)胞TLR4和NF-κB表達(dá)升高[21,22]。本研究發(fā)現(xiàn)，高糖組細(xì)胞中TLR4、NF-κB蛋白表達(dá)均高于低糖組，干擾組細(xì)胞中TLR4、NF-κB蛋白表達(dá)均低于高糖組，這提示高糖能夠誘導(dǎo)腎小管上皮細(xì)胞中TLR4和NF-κB升高，而抑制α-SMA表達(dá)后，腎小管上皮細(xì)胞中TLR4和NF-κB表達(dá)降低，表明抑制α-SMA表達(dá)能夠拮抗高糖誘導(dǎo)的腎小管上皮細(xì)胞過表達(dá)TLR4和NF-κB。

綜上所述，高糖可抑制腎小管上皮細(xì)胞增殖，誘導(dǎo)腎小管上皮細(xì)胞凋亡；抑制α-SMA表達(dá)能夠拮抗高糖對(duì)腎小管上皮細(xì)胞增殖的抑制作用及促凋亡作用，其機(jī)制可能與抑制TLR4/NF-κB信號(hào)通路及降低細(xì)胞內(nèi)ROS水平有關(guān)。

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Effects of α -SMA on proliferation and apoptosis of renal tubular epithelial cells induced by high glucose

SUOYan,GUYouyou,WAMGSu

(TianjinFifthCentralHospital,Tianjin300451,China)

Objective To investigate the effects and mechanism of α-smooth muscle actin (α-SMA) on the proliferation and apoptosis of renal tubular epithelial cells induced by high glucose. Methods We cultured the human renal tubular epithelial cells and divided them into the low glucose group, high glucose group, and interference group. In the interference group, we transfected the α-SMA siRNA into the renal tubular epithelial cells. After transfection, the high glucose group and the interference group were cultured in the high glucose DMEM cell culture medium with D-glucose concentration of 25 mmol/L, the low sugar group was cultured in the low glucose DMEM cell culture medium with D-glucose concentration of 5.6 mmol/L, and they were all cultured for 48 h. MTT assay was used to detect cell proliferation. The apoptosis was detected by flow cytometry and apoptosis rate was calculated. Western blotting was used to detected the Caspase-3, Bcl-2-related X protein (Bax) and Toll-like receptor 4 (TLR4) and nuclear factor (NF)-κB protein expression. The DCFH-DA was used to detected ROS level. The levels of TNF-α and IL-8 in the supernatant were detected by ELISA.Results The cell proliferation in the high glucose group and the interference group were lower than that in the low sugar group (P<0.01), the ability of cell proliferation in the interference group was higher than that in the high glucose group (P<0.01). The apoptosis rate in the high glucose group and the interference group was higher than that in the low sugar group (P<0.01), and the rate of apoptosis in the interference group was lower than that in the high glucose group (P<0.01). The expression of Caspase-3, Bax, TLR4, and NF- κB protein in the high glucose group and the interference group was higher than that in the low sugar group (allP<0.01), the expressions of Caspase-3, Bax, TLR4, and NF- κ B protein in the interference group was lower than that in the high glucose group (allP<0.01). The levels of ROS and TNF-α, IL-8 in the supernatant in the high glucose group and the interference group were higher than those in the low sugar group (allP<0.01), the levels of ROS, and TNF-α, IL-8 in the supernatant of the interference group were lower than those in the high glucose group (allP<0.01). Conclusion Interfering with α-SMA can partially reverse the effect of high glucose on the proliferation and apoptosis of renal tubular epithelial cells by decreasing the intracellular ROS level and inhibiting the TLR4/NF- κB signaling pathway.

diabetic nephropathy; α-smooth muscle actin; cell proliferation; cell apoptosis; inflammation

10.3969/j.issn.1002-266X.2017.30.004

R692.6；R587.1

A

1002-266X(2017)30-0014-04

2017-03-10)