丹酚酸A(SalA)对H_2O_2诱导的氧化应激损伤人晶状体上皮细胞增殖、凋亡、超微结构及炎症反应的作用
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摘要
目的探讨丹酚酸A(salvianolic acid A,Sal A)对H_2O_2诱导的氧化应激损伤人晶状体上皮细胞(human lens epithelial cells,HLEC)增殖、凋亡、超微结构及炎症反应的作用。方法将HLEC SRA01/04细胞传代培养后,用不同浓度(50μmol·L~(-1)、100μmol·L~(-1)、200μmol·L~(-1)、400μmol·L~(-1)、800μmol·L~(-1))H_2O_2处理培养24 h,通过MTT法检测细胞存活率,筛选H_2O_2最佳作用浓度,作为细胞模型组使用浓度。对照组细胞用正常完全培养液培养。Sal A干预组将细胞先在含50μmol·L~(-1)Sal A培养液中预孵育24 h,后加入最佳作用浓度H_2O_2继续培养24 h。而后采用MTT法检测各组细胞增殖情况,流式细胞术检测细胞凋亡率,电镜观察细胞超微结构,Western blot检测各组细胞炎症相关蛋白IL-1β、IL-18、TNF-α的表达情况。结果MTT法检测结果显示不同浓度H_2O_2(50μmol·L~(-1)、100μmol·L~(-1)、200μmol·L~(-1)、400μmol·L~(-1)、800μmol·L~(-1))处理组细胞存活率分别为61.26%±3.35%、47.81%±2.63%、29.53%±2.54%、21.47%±2.26%、14.97%±1.82%,各浓度处理组细胞存活率和对照组相比,差异均有统计学意义(均为P<0.05)。其中,100μmol·L~(-1)H_2O_2处理组细胞存活率最接近半数致死量,故选择100μmol·L~(-1)H_2O_2作为氧化应激损伤模型浓度用于后续实验。Sal A干预组细胞存活率为82.54%±3.07%,比100μmol·L~(-1)H_2O_2模型组明显升高,差异有统计学意义(P<0.05)。流式细胞术检测结果显示,对照组细胞凋亡率为4.06%±0.41%,H_2O_2模型组细胞凋亡率为20.52%±3.29%,Sal A干预组细胞凋亡率为12.35%±1.48%,H_2O_2模型组和Sal A干预组细胞凋亡率差异有统计学意义(P<0.05)。电镜观察结果显示H_2O_2诱导产生了明显的细胞超微结构损伤,线粒体、内质网数目减少,分泌颗粒减少,胞核体积缩小,可见少数细胞核内异染色质增多甚至聚集成块状。Sal A预处理后,细胞损伤的超微结构出现一定程度改善,如细胞形态较规则,细胞器线粒体、内质网数目增多,空泡数量减少,染色质分布基本均匀。Western blot检测结果显示,H_2O_2引起的氧化损伤会显著提高IL-1β、IL-18、TNF-α表达,Sal A可以在一定程度上降低IL-1β、IL-18、TNF-α的表达。结论Sal A可以有效增加H_2O_2诱导的氧化应激损伤HLEC的增殖,抑制细胞凋亡,减小细胞超微结构损伤性改变,降低IL-1β、IL-18、TNF-α表达。
Objective To explore the protective effects of salvianolic acid A( Sal A)on the proliferation,a poptosis,ultrastructural damage and inflammatory reaction of human lens epithelial cells SRA01/04( HLEC SRA01/04) induced by H_2O_2. Methods After subculture of HLEC SRA01/04,we cultured cells with different concentrations( 50μmol·L~(-1), 100 μmol·L~(-1), 200 μmol·L~(-1), 400 μmol·L~(-1), 800 μmol·L~(-1)) of H_2O_2 for 24 h and detected cell survival rate by MTT in order to screen of the optimal concentration of H_2O_2 as the cell model group. For Sal A intervention group,c ells were pre-incubated in medium containing 50 μmol·L~(-1) Sal A for 24 h,and then cultured in this medium after the addition of the optimal concentration of H_2O_2 for another 24 h. And then,MTT was used to detect cell proliferation,flow cytometry instrument was used for cell apoptosis rate,electron microscopic was used toobserve cell ultrastructure and western blot was used to detect the expression of inflammatory factor IL-1β, IL-18,TNF-α of all groups. Results The results of MTT method showed that cell survival rates of each experimental groups treated by different concentrations of H_2O_2( 50 μmol·L~(-1),100 μmol·L~(-1),200 μmol·L~(-1),400 μmol·L~(-1),800 μmol·L~(-1)) were 61. 26% ±3. 35%, 47. 81% ± 2. 63%, 29. 53% ± 2. 54%, 21. 47% ± 2. 26% and 14. 97% ± 1. 82%,r espectively.The survival rate of each experimental group was statistically significant compared with the control group( P < 0. 05). Among them,the cell survival rate( 47. 81% ± 2. 63%) induced by 100 μmol·L~(-1) H_2O_2 was closest to the lethal dose,which was selected as the oxidative damage model. The results of cell apoptosis by flow cell technology show ed that the cell apoptosis rate of control group,H_2O_2 model group and Sal A intervention group was 4. 06% ± 0. 41%, 20. 52% ± 3. 29%, 12. 35% ± 1. 48%,and the difference was statistically significant between the latter two groups( P <0. 05). Observation of cell ultrastructure by electron microscopy show ed that obvious cell ultrastructure damages induced by H_2O_2,such as the decreased number of mitochondria and endoplasmic reticulum,the decreased secretion of granule,the reduced cell nucleus volume,and the increased heterochromatin in the few nuclei or even clumped. After the pretreatment of Sal A,the cells showed improvement in ultrastructural damage at a certain degree,and the cell morphology was more regular,the number of mitochondria and endoplasmic reticulum increased,the number of vacuoles decreased and the chromatin distribution was basically even. Detection of inflammatory factors including IL-1β, IL-18 and TNF-α by Western blot indicated that oxidative damage caused by H_2O_2 could significantly upregulate the expression of IL-1β, IL-18 and TNF-α,and their expression levels were reduced to a certain extent through Sal A intervention. Conclusion Sal A can effectively protect cell proliferation inhibition,c ell apoptosis injury,c ell ultrastructural damage and decrease enhancement of expression of IL-1β, IL-18 and TNF-α of human lens epithelial cells SRA01/04 induced by H_2O_2.
Objective To explore the protective effects of salvianolic acid A( Sal A)on the proliferation,a poptosis,ultrastructural damage and inflammatory reaction of human lens epithelial cells SRA01/04( HLEC SRA01/04) induced by H_2O_2. Methods After subculture of HLEC SRA01/04,we cultured cells with different concentrations( 50μmol·L~(-1), 100 μmol·L~(-1), 200 μmol·L~(-1), 400 μmol·L~(-1), 800 μmol·L~(-1)) of H_2O_2 for 24 h and detected cell survival rate by MTT in order to screen of the optimal concentration of H_2O_2 as the cell model group. For Sal A intervention group,c ells were pre-incubated in medium containing 50 μmol·L~(-1) Sal A for 24 h,and then cultured in this medium after the addition of the optimal concentration of H_2O_2 for another 24 h. And then,MTT was used to detect cell proliferation,flow cytometry instrument was used for cell apoptosis rate,electron microscopic was used toobserve cell ultrastructure and western blot was used to detect the expression of inflammatory factor IL-1β, IL-18,TNF-α of all groups. Results The results of MTT method showed that cell survival rates of each experimental groups treated by different concentrations of H_2O_2( 50 μmol·L~(-1),100 μmol·L~(-1),200 μmol·L~(-1),400 μmol·L~(-1),800 μmol·L~(-1)) were 61. 26% ±3. 35%, 47. 81% ± 2. 63%, 29. 53% ± 2. 54%, 21. 47% ± 2. 26% and 14. 97% ± 1. 82%,r espectively.The survival rate of each experimental group was statistically significant compared with the control group( P < 0. 05). Among them,the cell survival rate( 47. 81% ± 2. 63%) induced by 100 μmol·L~(-1) H_2O_2 was closest to the lethal dose,which was selected as the oxidative damage model. The results of cell apoptosis by flow cell technology show ed that the cell apoptosis rate of control group,H_2O_2 model group and Sal A intervention group was 4. 06% ± 0. 41%, 20. 52% ± 3. 29%, 12. 35% ± 1. 48%,and the difference was statistically significant between the latter two groups( P <0. 05). Observation of cell ultrastructure by electron microscopy show ed that obvious cell ultrastructure damages induced by H_2O_2,such as the decreased number of mitochondria and endoplasmic reticulum,the decreased secretion of granule,the reduced cell nucleus volume,and the increased heterochromatin in the few nuclei or even clumped. After the pretreatment of Sal A,the cells showed improvement in ultrastructural damage at a certain degree,and the cell morphology was more regular,the number of mitochondria and endoplasmic reticulum increased,the number of vacuoles decreased and the chromatin distribution was basically even. Detection of inflammatory factors including IL-1β, IL-18 and TNF-α by Western blot indicated that oxidative damage caused by H_2O_2 could significantly upregulate the expression of IL-1β, IL-18 and TNF-α,and their expression levels were reduced to a certain extent through Sal A intervention. Conclusion Sal A can effectively protect cell proliferation inhibition,c ell apoptosis injury,c ell ultrastructural damage and decrease enhancement of expression of IL-1β, IL-18 and TNF-α of human lens epithelial cells SRA01/04 induced by H_2O_2.
引文
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[9]DING C,ZHAO Y,SHI X,ZHANG N,ZU G,LI Z,et al.New insights into salvianolic acid A action:Regulation of the TXNIP/NLRP3 and TXNIP/ChREBP pathw ays ameliorates HFDinduced NAFLD in rats[J].Sci Rep,2016,6:28734.
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[12]LI G,SONG H,CHEN L,YANG W,NAN K,LU P.TUG1 promotes lens epithelial cell apoptosis by regulating miR-421/caspase-3 axis in age-related cataract[J].Exp Cell Res,2017,356(1):20-27.
[13]LIAN-NIANG L,RUI T,WEI-MING C.Salvianolic acid a,a new depside from roots of salvia miltiorrhiza[J].Planta Med,1984,50(3):227-228.
[14]TANG Y,HE L Q,ZHU W,ZHANG Y.The regulating role of salvianolic acid a on bmp-7/smads/tgf-β1 signaling pathw ay in chronic renal failure rats[J].Chin J Integ Trad West Nephrol,2017,18(1):9-13.唐英,何立群,朱祎,张翼.丹酚酸A对慢性肾衰竭大鼠BMP-7/Smads/TGF-β_1信号通路的调控作用[J].中国中西医结合肾病杂志,2017,18(1):9-13.
[15]PEI R,SI T,LU Y,ZHOU J X,J IANG L.Salvianolic acid A,a novel PI3K/Akt inhibitor,induces cell apoptosis and suppresses tumor grow th in acute myeloid leukemi[J].Leuk Lymphoma,2017,22:1-9.
[16]MA Q X,CHEN X Z,RONG Y L,HUANG J J,ZHU K Y,CHEN C,et al.Effect of salvianolic acid A on retinopathy and its mechanism in ZDF rats[J].Chin J Compar Med,2017,27(10):40-46.马全鑫,陈小真,戎亦骊,黄俊杰,朱科燕,陈诚,等.丹酚酸A改善ZDF大鼠视网膜病变及其作用机制[J].中国比较医学杂志,2017,27(10):40-46.
[17]MAO K,SHU W,QIU Q,GU Q,WU X.Salvianolic acid A protects retinal pigment epithelium from OX-LDL-induced inflammation in an age-related macular degeneration model[J].Discov Med,2017,23(125):129-147.
[18]ZHANG H,LIU Y Y,J IANG Q,LI K R,ZHAO Y X,CAO C,et al.Salvianolic acid A protects RPE cells against oxidative stress through activation of Nrf2/HO-1 signaling[J].Free Radic Biol Med,2014,69(4):219-228.
[2]VANNER E A,STEWART M W.Meta-analysis comparing sameday versus delayed vitrectomy clinical outcomes for intravitreal retained lens fragments after age-related cataract surgery[J].Clin Ophthalmol,2014,8:2261-2276.
[3]ZHANG S Y.Epidemiological investigation of cataract in China[J].Chin J Ophthalmol,1999,35(5):336-340.张士元.我国白内障的流行病学调查资料分析[J].中华眼科杂志,1999,35(5):336-340.
[4]OLOKOBA L,ISLAM T,NAHAR N,MAHMOUD A,OLOKOBA A.A 3-year review of the outcome of pars plana vitrectomy for dropped lens fragments after cataract surgery in a tertiary eye hospital in Dhaka[J].Ethiop J Health Sci,2017,27(4):427-432.
[5]ZHANG J,YAN H,LOU M F.Does oxidative stress play any role in diabetic cataract formation?Re-evaluation using a thioltransferase gene knockout mouse model[J].Exp Eye Res,2017,161:36-42.
[6]WU C,LIU Z,MA L,PEI C,QIN L,GAO N,et al.MiRNAs regulate oxidative stress related genes via binding to the 3’UTR and TATA-box regions:a new hypothesis for cataract pathogenesis[J].BMC Ophthalmol,2017,17(1):142.
[7]KRUK J,KUBASIK-KLADNA K,ABOUL-ENEIN H Y.The role oxidative stress in the pathogenesis of eye diseases:current status and a dual role of physical activity[J].Mini Rev Med Chem,2015,16(3):241-257.
[8]ZHANG J Y,SUN G B,LUO Y,WANG M,WANG W,DU Y Y,et al.Salvianolic acid a protects H9c2 cells from arsenic trioxideinduced injury via inhibition of the MAPK signaling pathw ay[J].Cell Physiol Biochem,2017,41(5):1957-1969.
[9]DING C,ZHAO Y,SHI X,ZHANG N,ZU G,LI Z,et al.New insights into salvianolic acid A action:Regulation of the TXNIP/NLRP3 and TXNIP/ChREBP pathw ays ameliorates HFDinduced NAFLD in rats[J].Sci Rep,2016,6:28734.
[10]EHMANN D S,HO A C.Cataract surgery and age-related macular degeneration[J].Curr Opin Ophthalmol,2017,28(1):58-62.
[11]OU Y,LIAO G Y,WU W T.Potential use of hirudin in diabetic cataract:a study of galactose mediated human lens epithelial cells injury[J].Chem Biol Interact,2008,173(2):141-147.
[12]LI G,SONG H,CHEN L,YANG W,NAN K,LU P.TUG1 promotes lens epithelial cell apoptosis by regulating miR-421/caspase-3 axis in age-related cataract[J].Exp Cell Res,2017,356(1):20-27.
[13]LIAN-NIANG L,RUI T,WEI-MING C.Salvianolic acid a,a new depside from roots of salvia miltiorrhiza[J].Planta Med,1984,50(3):227-228.
[14]TANG Y,HE L Q,ZHU W,ZHANG Y.The regulating role of salvianolic acid a on bmp-7/smads/tgf-β1 signaling pathw ay in chronic renal failure rats[J].Chin J Integ Trad West Nephrol,2017,18(1):9-13.唐英,何立群,朱祎,张翼.丹酚酸A对慢性肾衰竭大鼠BMP-7/Smads/TGF-β_1信号通路的调控作用[J].中国中西医结合肾病杂志,2017,18(1):9-13.
[15]PEI R,SI T,LU Y,ZHOU J X,J IANG L.Salvianolic acid A,a novel PI3K/Akt inhibitor,induces cell apoptosis and suppresses tumor grow th in acute myeloid leukemi[J].Leuk Lymphoma,2017,22:1-9.
[16]MA Q X,CHEN X Z,RONG Y L,HUANG J J,ZHU K Y,CHEN C,et al.Effect of salvianolic acid A on retinopathy and its mechanism in ZDF rats[J].Chin J Compar Med,2017,27(10):40-46.马全鑫,陈小真,戎亦骊,黄俊杰,朱科燕,陈诚,等.丹酚酸A改善ZDF大鼠视网膜病变及其作用机制[J].中国比较医学杂志,2017,27(10):40-46.
[17]MAO K,SHU W,QIU Q,GU Q,WU X.Salvianolic acid A protects retinal pigment epithelium from OX-LDL-induced inflammation in an age-related macular degeneration model[J].Discov Med,2017,23(125):129-147.
[18]ZHANG H,LIU Y Y,J IANG Q,LI K R,ZHAO Y X,CAO C,et al.Salvianolic acid A protects RPE cells against oxidative stress through activation of Nrf2/HO-1 signaling[J].Free Radic Biol Med,2014,69(4):219-228.