裴 星, 韓 勇, 張占華, 李 娜, 施 遙, 張園園, 樊一鋼, 田紅燕
(1西安交通大學醫(yī)學院附屬紅會醫(yī)院內科,陜西西安710054;2西安交通大學醫(yī)學院第一附屬醫(yī)院周圍血管科,陜西西安710061)
芥子酸對高糖誘導下大鼠血管平滑肌細胞增殖和凋亡的影響*
裴 星1, 韓 勇1, 張占華1, 李 娜1, 施 遙1, 張園園1, 樊一鋼1, 田紅燕2△
(1西安交通大學醫(yī)學院附屬紅會醫(yī)院內科,陜西西安710054;2西安交通大學醫(yī)學院第一附屬醫(yī)院周圍血管科,陜西西安710061)
目的:探討芥子酸對高糖誘導下大鼠血管平滑肌細胞(vascular smooth muscle cells,VSMCs)增殖和凋亡的影響及機制。方法:將培養(yǎng)的A7r5細胞隨機分組處理,MTT法檢測細胞活力,BrdU法檢測細胞DNA合成,流式細胞術檢測細胞周期進程和細胞凋亡,ELISA檢測細胞活性氧簇(reactive oxygen species,ROS)生成,Western blot檢測cyclin D1、P21和P27等蛋白的表達,以及蛋白激酶C(PKC)和P38的磷酸化水平。結果:與正常組比較,高糖組細胞活力顯著升高,DNA合成加快,細胞周期加快,P21和P27表達降低,cyclin D1表達增加,ROS水平增加,細胞凋亡率降低,p-PKC和p-P38蛋白水平增加(P<0.05)。而芥子酸(0.1、1和10 μmol/L)處理引起細胞增殖活性降低,DNA合成減弱,細胞周期受阻,P21和P27表達增加,cyclin D1表達降低,ROS水平降低,細胞凋亡率升高,p-PKC和p-P38蛋白水平降低,且呈一定濃度依賴性(P<0.05)。P38抑制劑SB203580和PKC抑制劑chelerythrine均顯著抑制高糖誘導的PKC/P38活化和細胞活力(P<0.05)。結論:芥子酸可通過抑制PKC/P38激活降低高糖誘導的VSMCs增殖,并促進細胞凋亡。
芥子酸;高糖;血管平滑肌細胞;細胞增殖;細胞凋亡
長期髙血糖癥是誘發(fā)糖尿病心血管并發(fā)癥的重要因素。高糖(high glucose,HG)可引起代謝障礙,促進血管平滑肌細胞(vascular smooth muscle cells,VSMCs)增殖,在動脈粥樣硬化發(fā)生發(fā)展中發(fā)揮重要作用。白芥子,又叫白罌粟,為十字花科蕓薹屬植物白芥Sinapis alba L.的干燥成熟種子,主含白芥子苷、芥子堿、多糖、黃酮、芥子酸(sinapic acid,SA)和黏液質等。其中,芥子酸具有較強的抗氧化活性[1],能顯著改善糖尿病大鼠高血糖癥狀[2],并且其衍生物可明顯抑制血管內皮細胞活性氧簇(reactive oxygen species,ROS)生成[3]。因而,本研究擬探討芥子酸對高糖誘導下VSMCs增殖和凋亡的影響,并初步探究其可能的分子機制。
1 材料與試劑
大鼠VSMCs株A7r5購自ATCC;高糖DMEM培養(yǎng)基、低糖DMEM培養(yǎng)基和胎牛血清購自Gibco;胰蛋白酶購自Invitrogen;芥子酸、MTT、甘露醇、二甲基亞砜、碘化丙啶、P38抑制劑SB203580和蛋白激酶C (protein kinase C,PKC)抑制劑chelerythrine購自Sigma;兔抗p-PKC和兔抗p-P38抗體購自Cell Signaling Technology;兔抗cyclin D1、兔抗P21、兔抗P27和兔抗β-actin抗體購自Santa Cruz;HRP-標記的山羊抗兔II抗和ECL發(fā)光試劑盒購自碧云天生物技術研究所;BCA蛋白定量分析試劑盒購自Thermo;BrdU增殖檢測試劑盒購自Millipore;ROS檢測試劑盒購自南京建成生物工程研究所;Annexin V-FITC凋亡試劑盒購自BD Biosciences;細胞培養(yǎng)6孔板購自Corning。
2 方法
2.1 細胞培養(yǎng)和分組 A7r5細胞用含10%胎牛血清、1×105IU/L青霉素和100 mg/L鏈霉素的DMEM培養(yǎng)基,在37℃、5%CO2條件下進行培養(yǎng)。用25 mmol/L高濃度葡萄糖模擬糖尿病患者的體內高糖環(huán)境,設立等滲透壓的甘露醇組作為對照。取對數(shù)生長期細胞用于實驗,分為正常組(5.5 mmol/L葡萄糖)、甘露醇組(5.5 mmol/L葡萄糖+19.5 mmol/L甘露醇)、高糖組(25 mmol/L葡萄糖)、高糖+芥子酸(0.1、1和10 μmol/L)組、高糖+chelerythrine(10 μmol/L)組和高糖+SB203580(10 μmol/L)組。
2.2 MTT法檢測細胞活力 將A7r5細胞按每孔1×104個的密度接種96孔板,每孔200 μL培養(yǎng)基。24 h后,細胞換液,無血清培養(yǎng)。24 h后,按預先的實驗設計分組并更換不同處理的培養(yǎng)基,每組設6個復孔。分別在24 h、48 h和72 h時點,向待測孔加入20 μL MTT溶液(5 g/L),37℃孵育4 h后棄上清,每孔加入150 μL二甲基亞砜,溶解后,用酶標儀檢測490 nm處各孔的吸光度(absorbance,A)值。
2.3 BrdU法檢測細胞DNA合成 將A7r5細胞按每孔2×105個的密度接種6孔板,按之前步驟處理細胞。48 h后,使用BrdU增殖檢測試劑盒,嚴格按說明書步驟,酶標儀檢測450 nm處各孔的吸光度值,間接反映細胞BrdU的摻入率。
2.4 流式細胞儀檢測細胞周期進程 按之前步驟處理細胞48 h后,收集細胞,經70%乙醇4℃固定過夜和0.5 mL碘化丙啶(propidium iodide,PI;50 mg/L)避光孵育30 min,上流式細胞儀檢測,使用MODFIT軟件分析細胞在靜止期/DNA合成前期(G0/G1)、DNA合成期(S)和DNA合成后期/分裂期(G2/M)的分布百分比。
2.5 ELISA檢測ROS水平 按之前步驟處理細胞48 h后,使用ROS檢測試劑盒(DCFH-DA探針法),嚴格按說明書步驟,酶標儀測定細胞內ROS水平。
2.6 流式細胞術檢測細胞凋亡 按之前步驟處理細胞48 h后,收集細胞,預冷PBS洗滌,加入預冷75%乙醇,4℃固定4 h以上。離心棄上清,PBS洗滌2次后,加入500 μL binding buffer懸浮細胞。加入5 μL Annexin V-FITC混勻后,加入5 μL PI,混勻。室溫下避光反應5~15 min。1 h內進行流式細胞術檢測。
2.7 Western blot檢測 按之前步驟處理細胞48 h后,收集細胞,加入RAPI細胞裂解液,提取全蛋白。經BCA定量后、上樣、SDS-PAGE分離、轉膜、封閉、I抗、II抗孵育和ECL顯影等步驟,檢測各組細胞中P21、P27、cyclin D1、p-PKC、p-P38和β-actin等的蛋白水平。
3 統(tǒng)計學處理
實驗數(shù)據(jù)采用SPSS 17.0統(tǒng)計軟件進行統(tǒng)計學分析,計量資料以均數(shù)±標準差(mean±SD)表示,多組間比較采用單因素方差分析,各組均數(shù)間兩兩比較采用SNK-q檢驗。以P<0.05為差異有統(tǒng)計學意義。
1 芥子酸抑制高糖誘導的A7r5細胞增殖活性
MTT結果顯示,48 h后,與正常組比較,高糖組細胞活力顯著升高(P<0.05);而芥子酸(0.1、1和10 μmol/L)抑制細胞活力,且呈一定的濃度依賴性(P<0.05)。流式細胞術結果顯示,48 h后,與正常組比較,高糖組細胞G0/G1期比例明顯減少,S期細胞比例明顯增多;而芥子酸抑制細胞G0/G1期向S期轉換,且呈一定的濃度依賴性。Western blot結果顯示,48 h后,與正常組比較,高糖組細胞P21和P27表達明顯降低,cyclin D1表達明顯增加(P<0.05);而芥子酸逆轉上述蛋白表達,且呈一定的濃度依賴性(P<0.05)。BrdU結果顯示,48 h后,與正常組比較,高糖組細胞DNA合成加快(P<0.05);而芥子酸抑制細胞DNA合成,且呈一定的濃度依賴性(P<0.05)。因此,芥子酸可抑制高糖誘導的A7r5細胞增殖,見圖1。
Figure 1.The effects of SA on HG-induced the proliferation of the A7r5 cells.A:the cell viability was determined by MTT assay;B: the cell cycle progression was determined by flow cytometry;C:the expression of cyclin D1,P21 and P27 was detected by Western blot;D:the DNA synthesis was measured by BrdU assay.Mean±SD.n=4.*P<0.05 vs control group;#P<0.05 vs HG group.圖1 芥子酸對高糖引起的A7r5細胞增殖的影響
2 芥子酸抑制高糖誘導的A7r5細胞ROS生成
48 h后,與正常組比較,高糖組細胞ROS水平明顯增加(P<0.05);而芥子酸(0.1、1和10 μmol/L)降低細胞ROS水平,且呈一定的濃度依賴性(P<0.05),見圖2。
3 芥子酸上調高糖抑制的A7r5細胞凋亡
流式細胞術結果顯示,48 h后,與正常組比較,高糖組細胞凋亡率水平明顯降低(P<0.05);而芥子酸(0.1、1和10 μmol/L)上調細胞凋亡率,且呈一定的濃度依賴性(P<0.05),見圖3。
Figure 2.The effects of SA on HG-induced ROS levels in the A7r5 cells.The ROS levels were detected by ELISA assay.Mean±SD.n=4.*P<0.05 vs control group;#P<0.05 vs HG group.圖2 芥子酸對高糖引起的A7r5細胞ROS水平的影響
Figure 3.The effects of SA on HG-induced cell apoptosis inhibition in A7r5 cells.The cell apoptosis rate was determined by flow cytometry.Mean±SD.n=4.*P<0.05 vs control group;#P<0.05 vs HG group.圖3 芥子酸對高糖引起的A7r5細胞凋亡抑制的影響
4 芥子酸抑制高糖誘導的A7r5細胞PKC/P38活化
Western blot結果顯示,48 h后,與正常組比較,高糖組細胞的p-PKC和p-P38蛋白水平明顯增加;而芥子酸(0.1、1和10 μmol/L)降低細胞p-PKC和p-P38的蛋白水平,且呈一定的濃度依賴性(P<0.05),見圖4。
Figure 4.The effects of SA on HG-induced p-PKC and p-P38 protein expression in the A7r5 cells.The protein levels of p-PKC and p-P38 were evaluated by Western blot.Mean±SD.n=4.*P<0.05 vs control group;#P<0.05 vs HG group.圖4 芥子酸對高糖引起的A7r5細胞p-PKC和p-P38表達的影響
5 芥子酸通過抑制PKC/P38活化降低高糖誘導的A7r5細胞生長
Western blot結果顯示,10 μmol/L SB203580和10 μmol/L chelerythrine均能顯著抑制高糖誘導的PKC/P38的磷酸化水平,并下調高糖誘導的細胞生長(P<0.05),見圖5。
糖尿病患者發(fā)生心血管疾病的風險比正常人顯著增加,高糖環(huán)境下,VSMCs增殖加快,并遷移至動脈內膜,引起動脈壁增厚和管腔狹窄,VSMCs功能亦發(fā)生異常,是導致糖尿病大血管并發(fā)癥的重要機制。長期高血糖誘導ROS生成和氧化應激,ROS生成亦促進VSMCs增殖[4],均與心血管疾病發(fā)生密切相關[5]。芥子酸,又稱白芥酸或3,5-二甲氧基-4-羥基肉桂酸,屬于天然酚酸類化合物,存在于紫山藥、菜籽、柑橘類、醬油中以及炒白芥子、川穹、白芥子等多種藥用植物中。研究表明,芥子酸可抑制結腸癌大鼠模型氧化應激反應[6],通過降低氧化應激治療高血壓性心臟?。?]和保護心臟缺血性損傷[8],并可通過自由基清除活性減輕小鼠腦神經元損傷[9]。本實驗發(fā)現(xiàn),芥子酸高糖可明顯抑制高糖誘導的VSMCs ROS生成,提示芥子酸有可能用于糖尿病并發(fā)癥的預防和治療。
Figure 5.The effects of PKC/P38 inhibition and SA on HG-induced p-PKC and p-P38 proteins,and the changes of the viability in the A7r5 cells.A:the protein levels of p-PKC and p-P38 were evaluated by Western blot;B:the cell viability was determined by MTT assay.Mean±SD.n=4.*P<0.05 vs control group;#P<0.05 vs HG group.圖5 PKC/P38抑制和芥子酸對高糖引起的A7r5細胞p-PKC、p-P38蛋白水平和細胞活力的影響
文獻顯示,高血糖促進VSMCs增殖,并抑制細胞凋亡[10],是糖尿病致動脈粥樣硬化形成過程中的重要環(huán)節(jié),該過程受到P21、P27和cyclin D1等因素調控[11-12]。體外研究表明,包含芥子酸的酚酸混合物能抑制乳腺癌細胞生長[13],芥子酸能抑制結腸癌細胞和Hela細胞生長[14]。本研究通過MTT法和BrdU法發(fā)現(xiàn)芥子酸抑制高糖誘導的VSMCs細胞增殖活性。細胞周期分析發(fā)現(xiàn),在高糖環(huán)境下,VSMCs在細胞周期S期的分布比例明顯增加,但芥子酸可顯著逆轉高糖對細胞周期的調控作用。進一步探討芥子酸在高糖條件下抑制VSMCs增殖的分子機制表明,高糖對P21、P27和cyclin D1蛋白表達的調控作用可被芥子酸所逆轉。因此,芥子酸可通過調控P21、P27和cyclin D1蛋白的表達抑制高糖誘導的促增殖效應。進一步研究表明,芥子酸可通過誘導細胞凋亡抑制HeLa細胞生長[14]。本實驗流式細胞儀檢測亦發(fā)現(xiàn),芥子酸可明顯促進高糖抑制的細胞凋亡。
PKC作為絲氨酸/蘇氨酸激酶家族的成員,參與氧化應激和動脈粥樣硬化等病理過程,并可作為預防和治療糖尿病血管并發(fā)癥的潛在靶點[15]。P38通路激活促進VSMCs增殖遷移,與糖尿病動脈粥樣硬化病理過程密切相關[16-17]。高糖促進VSMCs P38激活[11]和主動脈平滑肌細胞PKC激活,并且PKC抑制顯著降低大鼠主動脈平滑肌細胞增殖活性[18-19]。而且PLC/PKC抑制能逆轉血管緊張素II誘導的小鼠胚胎干細胞增殖和cyclin D1表達增加,以及P21 和P27水平降低[20];P38抑制劑SB203580可阻斷人皮膚鱗狀細胞癌A431細胞cyclin D1表達增加,以及P21和P27表達降低[21]。另有文獻表明,芥子酸可通過抑制PLC-PKC激活提高葡萄糖利用率[2],通過負調節(jié)P38激活抑制炎癥反應[22]。本實驗發(fā)現(xiàn),P38抑制劑SB203580和PKC抑制劑chelerythrine均能顯著抑制高糖誘導的PKC/P38活化和細胞增殖活性,芥子酸亦顯著抑制高糖誘導的VSMCs PKC/P38磷酸化和細胞增殖??梢?,芥子酸可通過抑制PKC/ P38激活降低高糖誘導的VSMCs增殖。
綜上所述,本研究發(fā)現(xiàn)芥子酸可通過抑制PKC/ P38激活降低高糖誘導的VSMCs過度增殖,并促進細胞凋亡,為芥子酸應用于糖尿病血管并發(fā)癥治療提供了一定的理論基礎,但其作用機制和應用前景仍需進一步研究。
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(責任編輯:林白霜,羅 森)
Effects of sinapic acid on proliferation and apoptosis of rat vascular smooth muscle cells induced by high glucose
PEI Xing1,HAN Yong1,ZHANG Zhan-hua1,LI Na1,SHI Yao1,ZHANG Yuan-yuan1,F(xiàn)AN Yi-gang1,TIAN Hong-yan2
(1Department of Internal Medicine,Hong-Hui Hospital,Xi’an Jiaotong University College of Medicine,Xi’an 710054,China;2Department of Peripheral Vascular Medicine,The First Affiliated Hospital,Xi’an Jiaotong University College of Medicine,Xi’an 710061,China.E-mail:tianhongyan21@sina.com)
AIM:To investigate the effects of sinapic acid(SA)on the proliferation and apoptosis of rat vascular smooth muscle cells(VSMCs)induced by high glucose(HG).METHODS:Cultured A7r5 cells were randomly divided and treated as indicated.The cell viability was determined by MTT assay.DNA synthesis was measured by BrdU assay.Cell cycle progression and cell apoptotic rate were determined by flow cytometry analysis.The levels of reactive oxygen species(ROS)were detected by ELISA.The protein levels of cyclin D1,P21,P27,phosphorylated protein kinase C(p-PKC),p-P38 and β-actin were evaluated by Western blot.RESULTS:Compared with control group,the viability of A7r5 cells was significantly enhanced,the DNA synthesis was increased,the cell cycle progression was promoted,the levels of ROS were elevated,the cell apoptotic rate was reduced,the protein expression of P21 and P27 was decreased,and the protein levels of cyclin D1,p-PKC and p-P38 were increased in HG group(all P<0.05).These effects were reversed by SA (0.1,1 and 10 μmol/L)treatment in a dose-dependent manner(all P<0.05).Both P38 inhibitor SB203580 and PKC inhibitor chelerythrine significantly inhibit HG-induced PKC/P38 activation and cell viability(P<0.05).CONCLUSION:SA inhibits HG-induced VSMCs proliferation and promotes cell apoptosis via reducing PKC/P38 activation.
Sinapic acid;High glucose;Vascular smooth muscle cells;Cell proliferation;Apoptosis
R587.1;R363.2
A
10.3969/j.issn.1000-4718.2016.07.004
1000-4718(2016)07-1174-06
2016-02-22
2016-04-05
西安市紅會醫(yī)院2016年院級科研基金資助項目(No.YJ2016003)
△Tel:029-85324128;E-mail:tianhongyan21@sina.com