Src-家族酪氨酸激酶特异性抑制剂对氧化损伤下晶状体内Ca~（2+）的影响
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摘要
研究背景
我们的前期研究证实,晶状体脱离缺氧的生理状态,在体外培养条件下出现从赤道部向中央部的混浊,在这种皮质性白内障模型中Src-家族酪氨酸激酶(SFK)被异常激活,而用特异性SFK抑制剂PP1可以有效阻止晶状体的混浊,保持晶状体透明。此结果提示SFK异常激活在皮质性白内障形成过程中起关键作用。SFK可在不同细胞被高浓度H2O2激活,参与氧化应激引起的一系列级联反应。Ca2+稳态对维持晶状体透明至关重要,皮质性白内障的晶状体内钙离子显著增加,有关氧化损伤白内障模型的研究表明钙离子激活了钙蛋白酶继而引起晶状体蛋白降解导致晶状体的混浊。钙离子作为细胞内的第二信使参与了许多信号转导途径的激活,钙离子激活的转导通路与氧化应激激活SFK引起的一系列信号转导通路之间存在一定交联。实验性皮质性白内障是否通过SFK的激活,导致晶状体内钙离子浓度的增加,从而参与白内障的形成是本研究关注的问题。
目的
本研究观察Src-家族酪氨酸激酶抑制剂PP1对氧化损伤下晶状体内钙离子浓度的影响,探讨PP1阻止晶状体混浊的机理。
方法
用钙离子荧光探针Fluo-3/AM负载人晶状体细胞HLE-B3,用激光扫描共聚焦显微镜观察在不同浓度H2O2刺激后细胞内钙离子的荧光强度变化;进一步用0.1nmol/L PP1、0.3u/L过氧化氢酶和DMSO分别预处理细胞,观察在常规钙、低钙和高钙离子培养基的培养条件下,H2O2刺激后细胞内钙离子荧光强度的变化。将胚胎10天(E10)的鸡全晶状体分离后培养在含10%FBS的M199培养基中,连续培养10天并以此为对照组;在培养液中分别加入0.1nmol/LPP1、0.2u/L过氧化氢酶和50μmol/LVitamin C作为实验组。每天换液,观察、拍照记录并测量晶状体的混浊面积,计算晶状体混浊面积的百分比;检测体外培养第1、5、10天晶状体培养液中的钙离子浓度;用免疫印迹法检测体外培养10天晶状体囊膜p-Src表达水平的变化。
结果
不同浓度H2O2刺激后细胞内游离钙离子浓度升高,有剂量依赖效应。用0.1 mM H2O2刺激细胞,在常规钙培养基中,PP1组和过氧化氢酶组细胞内钙离子荧光强度增长幅度与DMSO组比分别降低28.5%±4.2%,33.8%±3.7% (t=4.281, 5.740; P<0.01);在低钙培养基中,三组细胞内钙离子荧光强度增强均不明显;在高钙培养基中,PP1组、catalase组的荧光强度增加幅度较DMSO组分别降低13.5%±1.8%和21.3%±2.4% (t=2.901, 3.219; P<0.01)。常规和高钙培养基中,PP1组和过氧化氢酶组细胞内钙离子荧光强度增长幅度均无明显差异(t=0.803, 1.47; P>0.05)。体外培养10天后,对照组晶状体出现了明显的皮质部的混浊,混浊面积百分比在第10天为52%,维生素C、过氧化氢酶和PP1处理组分别为10%、19%和13%,四组间有统计学差异(P<0.01)。晶状体在体外培养10天,培养液中钙离子浓度均增加,维生素C组、过氧化氢酶组和PP1组的钙离子浓度与对照组比明显降低(P<0.01),PP1组更明显,晶状体囊膜的p-Src的表达维生素C、过氧化氢酶和PP1处理组也比对照组明显降低(P<0.01),维生素C和PP1处理组抑制p-Src的作用较过氧化氢酶强(P<0.01)。
结论
晶状体上皮细胞在H2O2刺激下,出现钙离子从细胞外向胞内的流动增加,使细胞内游离钙离子浓度升高,Src-家族酪氨酸激酶特异性抑制剂PP1能有效预防钙离子的内流,提示PP1通过抑制氧化应激引起的钙内流增加,阻断了依赖钙离子激活的信号转导途径,阻止皮质性白内障发生。维生素C、过氧化氢酶与PP1一样能抑制实验性皮质性白内障的发生,还可以抑制晶状体中钙离子弄的升高,可能是通过抑制p-Src的活性保持晶状体上皮层的完整性来实现的。
Background
Our previous study proved that cortical cataract formed in chick embryo lens mechanically removed from the eye, the opacities began in the equatorial region and developed gradually toward the inner part of the lens. The cortical cataract formed in this model was involed with the activation of SFK, then the inhibition of SFK in the cultured lens would block the formation of cataract.These results suggest that the activation of SFK play a vital role in the cataract formation. The activation of SFK by high H202 concentration has been suggested in different cell types, and was required for a series of signal transduction mechanisms, especially those stimulated by oxidant stress. Ca2+ homeostasis is highly significant in terms of lens transparency, and Ca2+ increseases sharply in cortial cataract. The studies on H202-induced cataract demonstrated that Ca2+-activated protease calpain induced opacification. Ca2+ have fumdamental importance as a second messenger in cell signalling mechanisms, the signalling path moduated by Ca2+ must be interacted with that initiated from H202-induced-SFK-activation.So we focus on whether the formation of cortical cataract is involed with the increasement of Ca2+ in lens that caused by SFK activation.
Aim
To investigate the effects of specific inhibitor of Src-family tyrosin kinase (SFK) on calcium increasement induced by hydrogen peroxide in human lens.
Methods
The isolated LECs were loaded with Fluo-3/AM and then treated with H2O2 at various concentrations. The alteration of intracellular calcium was observed by confocal laser scanning microscope.To investigate the effects of PP1 on intracellular calcium, we employed three sets of cells uploaded with Fluo-3/AM and pretreated with 0.1nmol/L PP1, 0.3u/L catalase, and DMSO. These three different sets of cells were then treated with 0.1mM H2O2 and cultured in medium DMEM with normal, low, or high concentration of calcium, respectively. The intracellular free calcium was determined by confocal laser scanning microscope.The embryonic lenses were cultured in medium 199,containing 10% fetal bovine serum with the condition of vitaminC,catalase and PP1,the control was added the vehicle DMSO.Culture media containing inhibitor or control media were renewed every day throughout the 10-day culture period.Lens were observed in a dissectiong microscope and images captured daily.The total area of each lens and the area of the lenses that were opaque were also determined;The calcium concentration in culture medium of lens respectively cultured for 1,5,10 days was detected by colotimetric method.After cultued for 10 days, the expression of p-src in lens capsule was determined by Western blot.
Results
The fluorescent intensity of calcium in LECs increased with the concentration of H2O2 . In culture with normal DMEM, after treatment with 0.1mM H2O2, the increased amplitudes of fluorescent intensity in PP1 pretreated cells and catalase pretreated cells, were reduced 28.5%±4.2% and 33.8%±3.7% compared with DMSO-pretreated cells (P<0.01). While in the cells cultured with DMEM with low calcium, there were no obvious changes of fluorescent intensity of calcium in all three sets of cells after treatment with H2O2. In contrast with them, in the cells cultured in medium with high calcium, the fluorescent intensity increased dramatically induced by H2O2 in all three sets of cells, but the increased amplitudes in PP1 and catalase pretreated cells decreased 13.5%±1.8% and 21.3%±2.4% compared with DMSO-pretreated cells P<0.01), respectively. There were no significant differences between PP1 and catalase pretreated cells at reduced amplitudes when cultured in normal and high calcium medium (P>0.05).; Almost all lens in the control group developed cortical opacities covering approximately 52% of the lens area by day 10,while that of vitamin C,catalase and PP1-treated groups was 10%,19%,13%(P<0.01). After cultued for 10 days in vitro,the calcium concentration in culture mdium of four groups all raised, vitamin C,catalase and PP1-treated group was remarkably decreased(P<0.01),and the PP1- treated group had a most remarkable decreasing effect.The expressin of p-src in lens capsule was significantly low after treatment with vitamin C,catalase and PP1( P<0.01), and the effect of vitamin C,PP1 was superior to catalase( P<0.01).
Conclusions
The specific inhibitor of SFK, PP1 can block the calcium influx induced by oxidative stress, PP1 may prevent formation of cortical cataract by inhibiting Ca-dependent signaling pathways involved in cataractgenise.
我们的前期研究证实,晶状体脱离缺氧的生理状态,在体外培养条件下出现从赤道部向中央部的混浊,在这种皮质性白内障模型中Src-家族酪氨酸激酶(SFK)被异常激活,而用特异性SFK抑制剂PP1可以有效阻止晶状体的混浊,保持晶状体透明。此结果提示SFK异常激活在皮质性白内障形成过程中起关键作用。SFK可在不同细胞被高浓度H2O2激活,参与氧化应激引起的一系列级联反应。Ca2+稳态对维持晶状体透明至关重要,皮质性白内障的晶状体内钙离子显著增加,有关氧化损伤白内障模型的研究表明钙离子激活了钙蛋白酶继而引起晶状体蛋白降解导致晶状体的混浊。钙离子作为细胞内的第二信使参与了许多信号转导途径的激活,钙离子激活的转导通路与氧化应激激活SFK引起的一系列信号转导通路之间存在一定交联。实验性皮质性白内障是否通过SFK的激活,导致晶状体内钙离子浓度的增加,从而参与白内障的形成是本研究关注的问题。
目的
本研究观察Src-家族酪氨酸激酶抑制剂PP1对氧化损伤下晶状体内钙离子浓度的影响,探讨PP1阻止晶状体混浊的机理。
方法
用钙离子荧光探针Fluo-3/AM负载人晶状体细胞HLE-B3,用激光扫描共聚焦显微镜观察在不同浓度H2O2刺激后细胞内钙离子的荧光强度变化;进一步用0.1nmol/L PP1、0.3u/L过氧化氢酶和DMSO分别预处理细胞,观察在常规钙、低钙和高钙离子培养基的培养条件下,H2O2刺激后细胞内钙离子荧光强度的变化。将胚胎10天(E10)的鸡全晶状体分离后培养在含10%FBS的M199培养基中,连续培养10天并以此为对照组;在培养液中分别加入0.1nmol/LPP1、0.2u/L过氧化氢酶和50μmol/LVitamin C作为实验组。每天换液,观察、拍照记录并测量晶状体的混浊面积,计算晶状体混浊面积的百分比;检测体外培养第1、5、10天晶状体培养液中的钙离子浓度;用免疫印迹法检测体外培养10天晶状体囊膜p-Src表达水平的变化。
结果
不同浓度H2O2刺激后细胞内游离钙离子浓度升高,有剂量依赖效应。用0.1 mM H2O2刺激细胞,在常规钙培养基中,PP1组和过氧化氢酶组细胞内钙离子荧光强度增长幅度与DMSO组比分别降低28.5%±4.2%,33.8%±3.7% (t=4.281, 5.740; P<0.01);在低钙培养基中,三组细胞内钙离子荧光强度增强均不明显;在高钙培养基中,PP1组、catalase组的荧光强度增加幅度较DMSO组分别降低13.5%±1.8%和21.3%±2.4% (t=2.901, 3.219; P<0.01)。常规和高钙培养基中,PP1组和过氧化氢酶组细胞内钙离子荧光强度增长幅度均无明显差异(t=0.803, 1.47; P>0.05)。体外培养10天后,对照组晶状体出现了明显的皮质部的混浊,混浊面积百分比在第10天为52%,维生素C、过氧化氢酶和PP1处理组分别为10%、19%和13%,四组间有统计学差异(P<0.01)。晶状体在体外培养10天,培养液中钙离子浓度均增加,维生素C组、过氧化氢酶组和PP1组的钙离子浓度与对照组比明显降低(P<0.01),PP1组更明显,晶状体囊膜的p-Src的表达维生素C、过氧化氢酶和PP1处理组也比对照组明显降低(P<0.01),维生素C和PP1处理组抑制p-Src的作用较过氧化氢酶强(P<0.01)。
结论
晶状体上皮细胞在H2O2刺激下,出现钙离子从细胞外向胞内的流动增加,使细胞内游离钙离子浓度升高,Src-家族酪氨酸激酶特异性抑制剂PP1能有效预防钙离子的内流,提示PP1通过抑制氧化应激引起的钙内流增加,阻断了依赖钙离子激活的信号转导途径,阻止皮质性白内障发生。维生素C、过氧化氢酶与PP1一样能抑制实验性皮质性白内障的发生,还可以抑制晶状体中钙离子弄的升高,可能是通过抑制p-Src的活性保持晶状体上皮层的完整性来实现的。
Background
Our previous study proved that cortical cataract formed in chick embryo lens mechanically removed from the eye, the opacities began in the equatorial region and developed gradually toward the inner part of the lens. The cortical cataract formed in this model was involed with the activation of SFK, then the inhibition of SFK in the cultured lens would block the formation of cataract.These results suggest that the activation of SFK play a vital role in the cataract formation. The activation of SFK by high H202 concentration has been suggested in different cell types, and was required for a series of signal transduction mechanisms, especially those stimulated by oxidant stress. Ca2+ homeostasis is highly significant in terms of lens transparency, and Ca2+ increseases sharply in cortial cataract. The studies on H202-induced cataract demonstrated that Ca2+-activated protease calpain induced opacification. Ca2+ have fumdamental importance as a second messenger in cell signalling mechanisms, the signalling path moduated by Ca2+ must be interacted with that initiated from H202-induced-SFK-activation.So we focus on whether the formation of cortical cataract is involed with the increasement of Ca2+ in lens that caused by SFK activation.
Aim
To investigate the effects of specific inhibitor of Src-family tyrosin kinase (SFK) on calcium increasement induced by hydrogen peroxide in human lens.
Methods
The isolated LECs were loaded with Fluo-3/AM and then treated with H2O2 at various concentrations. The alteration of intracellular calcium was observed by confocal laser scanning microscope.To investigate the effects of PP1 on intracellular calcium, we employed three sets of cells uploaded with Fluo-3/AM and pretreated with 0.1nmol/L PP1, 0.3u/L catalase, and DMSO. These three different sets of cells were then treated with 0.1mM H2O2 and cultured in medium DMEM with normal, low, or high concentration of calcium, respectively. The intracellular free calcium was determined by confocal laser scanning microscope.The embryonic lenses were cultured in medium 199,containing 10% fetal bovine serum with the condition of vitaminC,catalase and PP1,the control was added the vehicle DMSO.Culture media containing inhibitor or control media were renewed every day throughout the 10-day culture period.Lens were observed in a dissectiong microscope and images captured daily.The total area of each lens and the area of the lenses that were opaque were also determined;The calcium concentration in culture medium of lens respectively cultured for 1,5,10 days was detected by colotimetric method.After cultued for 10 days, the expression of p-src in lens capsule was determined by Western blot.
Results
The fluorescent intensity of calcium in LECs increased with the concentration of H2O2 . In culture with normal DMEM, after treatment with 0.1mM H2O2, the increased amplitudes of fluorescent intensity in PP1 pretreated cells and catalase pretreated cells, were reduced 28.5%±4.2% and 33.8%±3.7% compared with DMSO-pretreated cells (P<0.01). While in the cells cultured with DMEM with low calcium, there were no obvious changes of fluorescent intensity of calcium in all three sets of cells after treatment with H2O2. In contrast with them, in the cells cultured in medium with high calcium, the fluorescent intensity increased dramatically induced by H2O2 in all three sets of cells, but the increased amplitudes in PP1 and catalase pretreated cells decreased 13.5%±1.8% and 21.3%±2.4% compared with DMSO-pretreated cells P<0.01), respectively. There were no significant differences between PP1 and catalase pretreated cells at reduced amplitudes when cultured in normal and high calcium medium (P>0.05).; Almost all lens in the control group developed cortical opacities covering approximately 52% of the lens area by day 10,while that of vitamin C,catalase and PP1-treated groups was 10%,19%,13%(P<0.01). After cultued for 10 days in vitro,the calcium concentration in culture mdium of four groups all raised, vitamin C,catalase and PP1-treated group was remarkably decreased(P<0.01),and the PP1- treated group had a most remarkable decreasing effect.The expressin of p-src in lens capsule was significantly low after treatment with vitamin C,catalase and PP1( P<0.01), and the effect of vitamin C,PP1 was superior to catalase( P<0.01).
Conclusions
The specific inhibitor of SFK, PP1 can block the calcium influx induced by oxidative stress, PP1 may prevent formation of cortical cataract by inhibiting Ca-dependent signaling pathways involved in cataractgenise.
引文
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[49]. Churchill GC, Louis CF.Roles of Ca2+,isositol trisphosphate and cyclic ADP -ribose in mediating intercellular Ca2+ signaling in sheep lens cells.Cell Sci, 1998; 111:1217-1225
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[53]. Jacob TJC.A direct measurement of intracellular with free calcium within the lens. Exp Eye Res,1983;36:451-453
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[61]. Hu RG, Suzukiker H, Webb KF, et al.Molecular and functional mapping of regional defferences in P2Y receptor expression in the rat lens. Exp Eye Res, 2008;10:108-116
[62]. Ireland ME.Activated epidermal growth factor receptors in the adult human lens. Exp Eye Res,2005;80:443-445
[63]. Collison DJ, Coleman RA, James RS, et al.Characterisation of muscarinic receptors in human lens cells by pharmalogic and molecular techniques. Invest Ophthalmol Vis Sci,2000;41:2633-2641
[64]. Duncan G, Webb SF, Dawson AP, et al.Calcium mobilisation modulates growth of lens cells.Cell Calcium,1996;19:83-89
[65]. Collison DJ, Wang L, Wormstone IM, et al.Spatial characteristics of receptor induced calcium signaling in human lens capsular bags. Invest Ophthalmol Vis Sci,2004;45:200-205
[66]. Rosado JA, Redondo PC, Salido GM. Hydrogen Peroxide Generation Induces pp60-src Activation in Human Platelets. JBC,2004,10:1665-1675
[67]. Taylor A, Nowell T. Oxidative stress and antioxidant function in relation to risk for cataract.Adv N Pharmacol,1997,38:515-536
[68]. Bogeski I, Bozem M, Sternfeld L, et al Inhibition of protein tyrosine phosphatase 1B by reactive oxyen species leads to maintenance of Ca2+ influx following store depletion in HEK 293 cells . Cell Calcium,2006,40(1):1-10
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[70]. Jacques PF.The potential preventive effects of vitamins for cataract and age-related macular degeneration.Vita Nutr Res,1999;69:198-205
[71]. Varma SD.Scientific basis for medical therapy of cataracts by antioxidants. Am J C lin Nutri,1991;53:335-345
[72]. Fu S, Ming L, Edward DK, et al.Vitamin C and vitamina E restore the resist ance of GSH-depleted lens cells to H2O2.Free Radic. Biol.Med ,2000; 28:1456 -1462
[73]. Zhang JT, Xing D, Gao XJ.Low-power laser irradiation activates Src tyrosine kinase through reactive oxygen species-mediated signaling pathway.Cell Physiol,2008;217(2):518-528
[74]. Chylack LT, Brown NP, Bron A,et al.The roche european american cataract:a randomized clinical trial to investigate the efficacy of an oral antioxidant micronutrient mixture to slow progression of age-related cataract.Ophthalmic Epidemiol,2002(1):49-80