胸腺肽β4对H_2O_2诱导的兔角膜基质细胞氧化应激损伤及继发细胞凋亡的抑制作用
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摘要
目的探讨胸腺肽β4(Tβ4)对H_2O_2诱导的兔角膜基质细胞氧化应激损伤及继发的细胞凋亡的抑制作用。方法采用组织块培养法获得原代兔角膜基质细胞,选取传代的兔角膜基质细胞,根据实验目的将细胞分为3组:正常组、H_2O_2组以及治疗组。H_2O_2组细胞用200μmol·L~(-1)的H_2O_2处理4 h,随后更换为不含FBS的DMEM/F-12培养液继续培养细胞48 h。治疗组细胞则是在H_2O_2处理后换用含终浓度为1μg·mL~(-1)Tβ4的无FBS的DMEM/F-12培养液继续培养48 h。同时把按常规方法培养的兔角膜基质细胞设立为正常组。采用CCK-8法检测各组细胞活性;应用2’,7’-二氯荧光黄双乙酸盐探针检测细胞活性氧水平;实时荧光定量PCR检测细胞中过氧化氢酶和锰超氧化物歧化酶mRNA的相对表达量;采用TUNEL法检测细胞凋亡率。ELISA法检测细胞中Caspase-3蛋白的表达。结果组织块培养法培养的兔角膜基质细胞呈多角形,规则排列呈束状。正常组细胞活性为(100.00±0.00)%、H_2O_2组(66.41±9.28)%、治疗组(82.54±7.72)%;H_2O_2组细胞活性明显低于正常组,差异有统计学意义(P<0.01);治疗组细胞活性明显高于H_2O_2组,差异有统计学意义(P<0.01)。正常组细胞活性氧水平为(4.12±0.93)%、H_2O_2组(77.84±6.98)%、治疗组(59.48±8.92)%;与正常组相比,H_2O_2组细胞呈现明显的强阳性着染;H_2O_2组细胞的活性氧水平明显高于正常组,差异有统计学意义(P<0.01);治疗组细胞的活性氧水平明显低于H_2O_2组,差异有统计学意义(P<0.01)。H_2O_2组细胞过氧化氢酶及锰超氧化物歧化酶mRNA的相对表达量较正常组均明显下降,差异均有统计学意义(均为P<0.05);治疗组细胞过氧化氢酶及锰超氧化物歧化酶mRNA的相对表达量较H_2O_2组均显著升高,差异均有统计学意义(均为P<0.05)。正常组细胞凋亡率为(6.81±1.48)%、H_2O_2组(76.14±6.12)%、治疗组(39.04±7.47)%;H_2O_2组细胞凋亡率明显高于正常组,差异有统计学意义(P<0.01);治疗组细胞凋亡率明显低于H_2O_2组,差异有统计学意义(P<0.01)。正常组细胞内Caspase-3蛋白的质量浓度为(0.46±0.09)ng·mL~(-1)、H_2O_2组(0.95±0.08)ng·mL~(-1)、治疗组(0.56±0.17)ng·mL~(-1);H_2O_2组细胞内Caspase-3蛋白的质量浓度较正常组明显升高,差异有统计学意义(P<0.01);治疗组细胞内Caspase-3蛋白的质量浓度明显低于H_2O_2组,差异有统计学意义(P<0.01)。结论 H_2O_2可诱导兔角膜基质细胞发生氧化应激损伤并继发细胞凋亡,而Tβ4对细胞的氧化应激损伤及细胞凋亡有抑制作用,其作用机制可能与Tβ4对细胞内Caspase-3、过氧化氢酶和锰超氧化物歧化酶表达的调节有关。
Objective To investigate the inhibitory effects of thymosin β4(Tβ4) on hydrogen peroxide(H_2O_2)-induced oxidative damage and subsequent cell apoptosis of rabbit corneal keratocytes.Methods Primary rabbit corneal keratocytes were cultured by explant culture technique.After passage,according to the aims of the present study,the rabbit corneal keratocytes were divided into 3 groups:control group,H_2O_2 group and treatment group.The cells of the H_2O_2 group were treated with 200 μmol·L~(-1) H_2O_2 for 4 h,then the cells were cultured with FBS-free DMEM/F-12 medium for 48 h.The cells of the treatment group were cultured with FBS-free DMEM/F-12 medium containing 1 μg·mL~(-1) Tβ4 for 48 h after treatment with H_2O_2.Beyond that,control group were designed in which the cells were cultured with routine methods.The cell activity was detected by CCK-8.The 2',7'-dichlorodihydrofluorescein diacetate was applied to detect the cellular reactive oxygen species(ROS).Detection of the relative level of catalase mRNA and MnSOD mRNA was performed by real-time fluorescent quantitative PCR.The cell apoptosis rate was detected by TUNEL methods.The content of Caspase-3 protein of cells was examined by ELISA assay.Results The primary rabbit corneal keratocytes cultured by explant culture technique were dendritic,and arranged regularly in bundles.The cell activity of control group,H_2O_2 group,and treatment group were(100.00±0.00)%,(66.41±9.28)%,and(82.54±7.72)%,respectively.The cell activity of the H_2O_2 group was markedly reduced in comparison with the control group,and the difference was statistically significant(P<0.01).The cell activity of the treatment group was apparently increased compared to the H_2O_2 group,and the difference was statistically significant(P<0.01).The levels of ROS of control group,H_2O_2 group,and treatment group were(4.12±0.93)%,(77.84±6.98)%,and(59.48±8.92)%,respectively.The cells of the H_2O_2 group were significantly positive staining compared to the control group.The level of ROS of the H_2O_2 group showed an marked increase in comparison with the control group,and the difference was statistically significant(P<0.01).The level of ROS of the treatment group was declined compared with the H_2O_2 group,and the difference was statistically significant(P<0.01).The relative levels of catalase mRNA and MnSOD mRNA expression of the H_2O_2 group were significantly lower than those of the control group,and the differences were statistically significant(both P<0.05),and the relative levels of catalase mRNA and MnSOD mRNA expression in the treatment group were markedly higher than those of the H_2O_2 group,and the differences were also statistically significant(both P<0.05).The cell apoptosis rate of control group,H_2O_2 group,and treatment group were(6.81±1.48)%,(76.14±6.12)%,and(39.04±7.47)%,respectively.Compared with the control group,the cell apoptosis rate of the H_2O_2 group was significantly increased,and the difference was statistically significant(P<0.01).The cell apoptosis rate of the treatment group was significantly decreased in comparison with the H_2O_2 group,and the difference was also statistically significant(P<0.01).The content of Caspase-3 protein of the control group,H_2O_2 group and treatment group were(0.46±0.09)ng·mL~(-1),(0.95±0.08)ng·mL~(-1) and(0.56±0.17)ng·mL~(-1).The content of Caspase-3 protein of the H_2O_2 group was significantly higher than that of the control group,and the difference was statistically significant(P<0.01),and the content of Caspase-3 protein of the treatment group was significantly lower than that of the H_2O_2 group,and the difference was also statistically significant(P<0.01).Conclusion H_2O_2 can induce oxidative stress damage and subsequent cell apoptosis on rabbit corneal keratocytes,and Tβ4 can inhibit the oxidative stress damage and cells apoptosis,of which mechanism might be related to the regulation of the expression of Caspase-3,catalase and MnSOD.
Objective To investigate the inhibitory effects of thymosin β4(Tβ4) on hydrogen peroxide(H_2O_2)-induced oxidative damage and subsequent cell apoptosis of rabbit corneal keratocytes.Methods Primary rabbit corneal keratocytes were cultured by explant culture technique.After passage,according to the aims of the present study,the rabbit corneal keratocytes were divided into 3 groups:control group,H_2O_2 group and treatment group.The cells of the H_2O_2 group were treated with 200 μmol·L~(-1) H_2O_2 for 4 h,then the cells were cultured with FBS-free DMEM/F-12 medium for 48 h.The cells of the treatment group were cultured with FBS-free DMEM/F-12 medium containing 1 μg·mL~(-1) Tβ4 for 48 h after treatment with H_2O_2.Beyond that,control group were designed in which the cells were cultured with routine methods.The cell activity was detected by CCK-8.The 2',7'-dichlorodihydrofluorescein diacetate was applied to detect the cellular reactive oxygen species(ROS).Detection of the relative level of catalase mRNA and MnSOD mRNA was performed by real-time fluorescent quantitative PCR.The cell apoptosis rate was detected by TUNEL methods.The content of Caspase-3 protein of cells was examined by ELISA assay.Results The primary rabbit corneal keratocytes cultured by explant culture technique were dendritic,and arranged regularly in bundles.The cell activity of control group,H_2O_2 group,and treatment group were(100.00±0.00)%,(66.41±9.28)%,and(82.54±7.72)%,respectively.The cell activity of the H_2O_2 group was markedly reduced in comparison with the control group,and the difference was statistically significant(P<0.01).The cell activity of the treatment group was apparently increased compared to the H_2O_2 group,and the difference was statistically significant(P<0.01).The levels of ROS of control group,H_2O_2 group,and treatment group were(4.12±0.93)%,(77.84±6.98)%,and(59.48±8.92)%,respectively.The cells of the H_2O_2 group were significantly positive staining compared to the control group.The level of ROS of the H_2O_2 group showed an marked increase in comparison with the control group,and the difference was statistically significant(P<0.01).The level of ROS of the treatment group was declined compared with the H_2O_2 group,and the difference was statistically significant(P<0.01).The relative levels of catalase mRNA and MnSOD mRNA expression of the H_2O_2 group were significantly lower than those of the control group,and the differences were statistically significant(both P<0.05),and the relative levels of catalase mRNA and MnSOD mRNA expression in the treatment group were markedly higher than those of the H_2O_2 group,and the differences were also statistically significant(both P<0.05).The cell apoptosis rate of control group,H_2O_2 group,and treatment group were(6.81±1.48)%,(76.14±6.12)%,and(39.04±7.47)%,respectively.Compared with the control group,the cell apoptosis rate of the H_2O_2 group was significantly increased,and the difference was statistically significant(P<0.01).The cell apoptosis rate of the treatment group was significantly decreased in comparison with the H_2O_2 group,and the difference was also statistically significant(P<0.01).The content of Caspase-3 protein of the control group,H_2O_2 group and treatment group were(0.46±0.09)ng·mL~(-1),(0.95±0.08)ng·mL~(-1) and(0.56±0.17)ng·mL~(-1).The content of Caspase-3 protein of the H_2O_2 group was significantly higher than that of the control group,and the difference was statistically significant(P<0.01),and the content of Caspase-3 protein of the treatment group was significantly lower than that of the H_2O_2 group,and the difference was also statistically significant(P<0.01).Conclusion H_2O_2 can induce oxidative stress damage and subsequent cell apoptosis on rabbit corneal keratocytes,and Tβ4 can inhibit the oxidative stress damage and cells apoptosis,of which mechanism might be related to the regulation of the expression of Caspase-3,catalase and MnSOD.
引文
[1] FINKEL T.Oxidant signals and oxidative stress[J].Curr Opin Cell Biol,2003,15(2):247-254.
[2] BEHNDIG A,KARLSSON K,JOHANSSO B O,BRANNSTROM T,MARKLUND S L.Superoxide dismutase isoenzymes in the normal and diseased human cornea[J].Invest Ophthalmol Vis Sci,2001,42(10):2293-2296.
[3] BUDDI R,LIN B,ATILANO S R,ZORAPAPEL N C,KENNEY M C,BROWN D J.Evidence of oxidative stress in human corneal diseases[J].J Histochem Cytochem,2002,50(3):341-351.
[4] TOPRAK I,KUCUKATAY V,YILDIRIM C,KILIC-TOPRAK E,KILIC-ERKEK O.Increased systemic oxidative stress in patients with keratoconus[J].Eye (Lond),2014,28(3):285-289.
[5] FRIDOVICH I.The biology of oxygen radicals[J].Science,1978,201(4359):875-880.
[6] LOW T L,GOLDSTEIN A L.Chemical characterization of thymosin beta 4[J].J Biol Chem,1982,257(2):1000-1006.
[7] CHEN C,LI M,YANG H,CHAI H,FISHER W,YAO Q.Roles of thymosins in cancers and other organ systems[J].World J Surg,2005,29(3):264-270.
[8] SOSNE G,SZLITER E A,BARRETT R,KERNACKI K A,KLEINMAN H,HAZLETT L D.Thymosin beta 4 promotes corneal wound healing and decreases inflammation in vivo following alkali injury[J].Exp Eye Res,2002,74(2):293-299.
[9] CAVASIN D M A.Therapeutic potential of thymosin-β4 and its derivative n -acetyl-seryl-aspartyl-lysyl-proline (AC-SDKP) in cardiac healing after infarction[J].Am J Cardiovasc Drug,2006,6(5):305-311.
[10] LI X,JIANG Z X,HAO P,TANG X.Protective effect of thy-mosinβ4 against oxidative damage in cultured rabbit corneal epithelial cells[J].Chin J Ophthalmol,2013,49(8):716-722. 李轩,姜志昕,郝朋,汤欣.胸腺素β4对体外培养的兔角膜上皮细胞氧化损伤的保护作用[J].中华眼科杂志,2013,49(8):716-722.
[11] XUAN M,WANG S,LIU X,HE Y,LI Y,ZHANG Y.Proteins of the corneal stroma:importance in visual function[J].Cell Tissue Res,2016,364(1):9-16.
[12] HO J H,TSENG K C,MA W H,CHEN K H,LEE O K,SU Y.Thymosin beta-4 upregulates anti-oxidative enzymes and protects human cornea epithelial cells against oxidative damage[J].Br J Ophthalmol,2008,92(7):992-997.
[13] PORTER A G,JANICKE R U.Emerging roles of Caspase-3 in apoptosis[J].Cell Death Differ,1999,6(2):99-104.
[14] SHALINI S,DORSTYN L,DAWAR S,KUMAR S.Old,new and emerging functions of caspases[J].Cell Death Differ,2015,22(4):526-539.
[2] BEHNDIG A,KARLSSON K,JOHANSSO B O,BRANNSTROM T,MARKLUND S L.Superoxide dismutase isoenzymes in the normal and diseased human cornea[J].Invest Ophthalmol Vis Sci,2001,42(10):2293-2296.
[3] BUDDI R,LIN B,ATILANO S R,ZORAPAPEL N C,KENNEY M C,BROWN D J.Evidence of oxidative stress in human corneal diseases[J].J Histochem Cytochem,2002,50(3):341-351.
[4] TOPRAK I,KUCUKATAY V,YILDIRIM C,KILIC-TOPRAK E,KILIC-ERKEK O.Increased systemic oxidative stress in patients with keratoconus[J].Eye (Lond),2014,28(3):285-289.
[5] FRIDOVICH I.The biology of oxygen radicals[J].Science,1978,201(4359):875-880.
[6] LOW T L,GOLDSTEIN A L.Chemical characterization of thymosin beta 4[J].J Biol Chem,1982,257(2):1000-1006.
[7] CHEN C,LI M,YANG H,CHAI H,FISHER W,YAO Q.Roles of thymosins in cancers and other organ systems[J].World J Surg,2005,29(3):264-270.
[8] SOSNE G,SZLITER E A,BARRETT R,KERNACKI K A,KLEINMAN H,HAZLETT L D.Thymosin beta 4 promotes corneal wound healing and decreases inflammation in vivo following alkali injury[J].Exp Eye Res,2002,74(2):293-299.
[9] CAVASIN D M A.Therapeutic potential of thymosin-β4 and its derivative n -acetyl-seryl-aspartyl-lysyl-proline (AC-SDKP) in cardiac healing after infarction[J].Am J Cardiovasc Drug,2006,6(5):305-311.
[10] LI X,JIANG Z X,HAO P,TANG X.Protective effect of thy-mosinβ4 against oxidative damage in cultured rabbit corneal epithelial cells[J].Chin J Ophthalmol,2013,49(8):716-722. 李轩,姜志昕,郝朋,汤欣.胸腺素β4对体外培养的兔角膜上皮细胞氧化损伤的保护作用[J].中华眼科杂志,2013,49(8):716-722.
[11] XUAN M,WANG S,LIU X,HE Y,LI Y,ZHANG Y.Proteins of the corneal stroma:importance in visual function[J].Cell Tissue Res,2016,364(1):9-16.
[12] HO J H,TSENG K C,MA W H,CHEN K H,LEE O K,SU Y.Thymosin beta-4 upregulates anti-oxidative enzymes and protects human cornea epithelial cells against oxidative damage[J].Br J Ophthalmol,2008,92(7):992-997.
[13] PORTER A G,JANICKE R U.Emerging roles of Caspase-3 in apoptosis[J].Cell Death Differ,1999,6(2):99-104.
[14] SHALINI S,DORSTYN L,DAWAR S,KUMAR S.Old,new and emerging functions of caspases[J].Cell Death Differ,2015,22(4):526-539.