p38在体外培养的鸡胚胎晶状体半乳糖性白内障中的作用
摘要
研究背景
白内障是多因素引起的晶状体混浊,是第一位致盲眼病。丝裂原激活蛋白激酶(MAPK)信号通路参与多种应激反应, p38是其家族成员之一,它主要参与高渗透压、高糖、炎症等引起的细胞应激反应。凋亡是各种非先天性白内障共同的细胞基础。虽然有研究显示凋亡存在于半乳糖性白内障的晶状体上皮细胞中,半乳糖激活p38,尚没有研究证实半乳糖诱导的p38激活与凋亡直接相关。
目的
本研究将利用体外培养的胚胎鸡晶状体培养系统,建立半乳糖性白内障模型,探讨p38激活与晶状体上皮细胞凋亡的关系,及其在白内障形成中的作用。
方法
用30mmol/L半乳糖在体外培养的鸡胚胎晶状体上诱导半乳糖性白内障模型(对照组),在培养的10天里,通过每日观察、照相、计算晶状体的混浊面积,并用Western Blot检测晶状体中磷酸化p38的表达;用p38特异性抑制剂SB203580抑制p38活性,观察其对晶状体混浊程度的影响;通过对晶状体细胞膜(WGA)及细胞核(DAPI)染色,观察晶状体纤维结构和分布变化;利用TUNEL凋亡细胞染色法观察白内障发生中晶状体切片上的凋亡细胞的数量变化,并检测晶状体上皮细胞内caspase-3活性变化。
结果
30 mmol/L半乳糖能诱导体外培养的鸡胚胎晶状体产生周边部皮质性混浊,随着半乳糖暴露时间的延长,晶状体的混浊面积增加,p38抑制剂SB203580能有效减轻半乳糖性晶状体的混浊。半乳糖性白内障晶状体切片显示赤道部晶状体纤维结构紊乱、晶状体纤维细胞分化异常,而p38抑制剂能够减轻这些组织学改变。TUNEL原位凋亡细胞检测显示,在半乳糖处理的d2、d5和d10天,晶状体凋亡细胞比例明显增加,分别为1.7%、4.5%和12%,而SB203580明显阻止晶状体细胞的凋亡,在相应时间点凋亡细胞比例降至0.7%、1.5%和1.4%。与此结果对应,caspase-3活性在半乳糖性白内障发生中明显升高,在培养的第d2,d5和d10天,分别是胚胎10天正常晶状体(E10)的8.5,19.5和41.9倍,而抑制P38的活性后caspase-3活性明显减低,为E10的3,7.8和26.9倍。
结论
抑制p38的激活能减轻半乳糖性白内障的发生。p38诱导caspase-3激活的细胞凋亡途径参与半乳糖性白内障的形成。
Background
Cataract characterized by opacification of lens is caused by multi-factors. MAPK participate in many signaling pathways of stress. P38, a member of MAPK, mediates in the signaling pathways in various cells induced by osmotic stress, high glucose, and inflammation and so on. Apoptosis of lens epithelial cell is considered a common cellular basis for non-congenital cataract development in humans and animals. Although it has shown that activation of p38 and apoptosis of lens epithelial cells appeared in galactose cataracts, there is no direct evidence to show that P38 involves in signaling pathway of apoptosis in galactose cataract.
Aim
The goal of this study is to determine the signaling pathway of apoptosis mediated by p38 in formation of galactose cataract, developed by chicken embryonic lenses culture system in vitro.
Methods
The cataract model was developed by 30mmol/L galactose in cultured chicken embryonic lenses. To examine the role of P38 in formation of cataract, lenses were grown for ten days with or without inhibitor of p38, SB203580. Lens was observed and photographed daily, and the degree of opacification was quantified by using imagine software. Phospho-p38 in the lenses treated with or without p38 inhibitor was determined by western blot assay. Histomorphological changes of the cultured lenses were observed after immunostaining with WGA for cellular membrane and DAPI for cellular nucleus in the frozen sections. Apoptosis in the sections was examined by TUNEL assay and the activity of caspase-3 in lens epithelium of cultured lens was measured by CaspACETM Assay System.
Results
p38 activation was found in cultured lens treated with 30mmol/L galactose during the 10-day-culture period, and inhibited by SB203580. The cultured lens developed opacification at the periphery cortex, the degree of opacification increased with time during ten-day-culture period. SB203580-treated lenses developed similar cortical opacity, but the degree of opacification was attenuated. By immunostaining with WGA and DAPI, we found the lens opacities appeared to result from gross abnormalities in the shape and organization of cells in the equatorial and cortical fiber zones. Culturing the lenses in the presence of SB203580 prevented these lens cell aberrations as well as the development of lens opacity. In the section of galactose cataract lenses, the percentage of apoptotic cells was 1.7%, 4.5% and 12% at cultured day 2, 5 and 10, but in SB203580-treated lenses it was decreased dramatically to 0.7%, 1.5% and 1.4% respectively. Further more, the activity of caspase-3 in galactose cataract lenses increased to 8.5,19.5 and 41.9 folds to E10 lens at culture day 2, 5 and 10, while in SB203580-treated lenses, caspase-3 activity was inhibited to 3,7.8 and 26.9 folds to E10 lens, respectively. It suggested that activation of p38 participated in the signaling pathway of apoptosis in lens epithelial cells.
Conclusion
The activation of p38 may involve in formation of galactose cataract by mediating the signaling pathway of apoptosis in lens epithelial cell. Inhibition of p38 activity can inhibit the activation of the caspase-3, prevent the lens epithelial cells from apoptosis and attenuate formation of cataract.
白内障是多因素引起的晶状体混浊,是第一位致盲眼病。丝裂原激活蛋白激酶(MAPK)信号通路参与多种应激反应, p38是其家族成员之一,它主要参与高渗透压、高糖、炎症等引起的细胞应激反应。凋亡是各种非先天性白内障共同的细胞基础。虽然有研究显示凋亡存在于半乳糖性白内障的晶状体上皮细胞中,半乳糖激活p38,尚没有研究证实半乳糖诱导的p38激活与凋亡直接相关。
目的
本研究将利用体外培养的胚胎鸡晶状体培养系统,建立半乳糖性白内障模型,探讨p38激活与晶状体上皮细胞凋亡的关系,及其在白内障形成中的作用。
方法
用30mmol/L半乳糖在体外培养的鸡胚胎晶状体上诱导半乳糖性白内障模型(对照组),在培养的10天里,通过每日观察、照相、计算晶状体的混浊面积,并用Western Blot检测晶状体中磷酸化p38的表达;用p38特异性抑制剂SB203580抑制p38活性,观察其对晶状体混浊程度的影响;通过对晶状体细胞膜(WGA)及细胞核(DAPI)染色,观察晶状体纤维结构和分布变化;利用TUNEL凋亡细胞染色法观察白内障发生中晶状体切片上的凋亡细胞的数量变化,并检测晶状体上皮细胞内caspase-3活性变化。
结果
30 mmol/L半乳糖能诱导体外培养的鸡胚胎晶状体产生周边部皮质性混浊,随着半乳糖暴露时间的延长,晶状体的混浊面积增加,p38抑制剂SB203580能有效减轻半乳糖性晶状体的混浊。半乳糖性白内障晶状体切片显示赤道部晶状体纤维结构紊乱、晶状体纤维细胞分化异常,而p38抑制剂能够减轻这些组织学改变。TUNEL原位凋亡细胞检测显示,在半乳糖处理的d2、d5和d10天,晶状体凋亡细胞比例明显增加,分别为1.7%、4.5%和12%,而SB203580明显阻止晶状体细胞的凋亡,在相应时间点凋亡细胞比例降至0.7%、1.5%和1.4%。与此结果对应,caspase-3活性在半乳糖性白内障发生中明显升高,在培养的第d2,d5和d10天,分别是胚胎10天正常晶状体(E10)的8.5,19.5和41.9倍,而抑制P38的活性后caspase-3活性明显减低,为E10的3,7.8和26.9倍。
结论
抑制p38的激活能减轻半乳糖性白内障的发生。p38诱导caspase-3激活的细胞凋亡途径参与半乳糖性白内障的形成。
Background
Cataract characterized by opacification of lens is caused by multi-factors. MAPK participate in many signaling pathways of stress. P38, a member of MAPK, mediates in the signaling pathways in various cells induced by osmotic stress, high glucose, and inflammation and so on. Apoptosis of lens epithelial cell is considered a common cellular basis for non-congenital cataract development in humans and animals. Although it has shown that activation of p38 and apoptosis of lens epithelial cells appeared in galactose cataracts, there is no direct evidence to show that P38 involves in signaling pathway of apoptosis in galactose cataract.
Aim
The goal of this study is to determine the signaling pathway of apoptosis mediated by p38 in formation of galactose cataract, developed by chicken embryonic lenses culture system in vitro.
Methods
The cataract model was developed by 30mmol/L galactose in cultured chicken embryonic lenses. To examine the role of P38 in formation of cataract, lenses were grown for ten days with or without inhibitor of p38, SB203580. Lens was observed and photographed daily, and the degree of opacification was quantified by using imagine software. Phospho-p38 in the lenses treated with or without p38 inhibitor was determined by western blot assay. Histomorphological changes of the cultured lenses were observed after immunostaining with WGA for cellular membrane and DAPI for cellular nucleus in the frozen sections. Apoptosis in the sections was examined by TUNEL assay and the activity of caspase-3 in lens epithelium of cultured lens was measured by CaspACETM Assay System.
Results
p38 activation was found in cultured lens treated with 30mmol/L galactose during the 10-day-culture period, and inhibited by SB203580. The cultured lens developed opacification at the periphery cortex, the degree of opacification increased with time during ten-day-culture period. SB203580-treated lenses developed similar cortical opacity, but the degree of opacification was attenuated. By immunostaining with WGA and DAPI, we found the lens opacities appeared to result from gross abnormalities in the shape and organization of cells in the equatorial and cortical fiber zones. Culturing the lenses in the presence of SB203580 prevented these lens cell aberrations as well as the development of lens opacity. In the section of galactose cataract lenses, the percentage of apoptotic cells was 1.7%, 4.5% and 12% at cultured day 2, 5 and 10, but in SB203580-treated lenses it was decreased dramatically to 0.7%, 1.5% and 1.4% respectively. Further more, the activity of caspase-3 in galactose cataract lenses increased to 8.5,19.5 and 41.9 folds to E10 lens at culture day 2, 5 and 10, while in SB203580-treated lenses, caspase-3 activity was inhibited to 3,7.8 and 26.9 folds to E10 lens, respectively. It suggested that activation of p38 participated in the signaling pathway of apoptosis in lens epithelial cells.
Conclusion
The activation of p38 may involve in formation of galactose cataract by mediating the signaling pathway of apoptosis in lens epithelial cell. Inhibition of p38 activity can inhibit the activation of the caspase-3, prevent the lens epithelial cells from apoptosis and attenuate formation of cataract.
引文
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2. Lou MF, X.G., Cui XL. Further studies on the dynamic changes of glutathione and protein-thiol mixed disulfides in H2O2 induced cataract in rat lenses: distributions and effect of aging. Curr Eye Res. 1995;14: 951–958.
3. Hightower KR, M.P. The relationship between osmotic stress and calcium elevation: in vitro and in vivo rat lens models. Exp Eye Res. 1998; 66: 775–781.
4. Marcantonio JM, D.G., Rink H. Calcium-induced opacification and loss of protein in the organ-cultured bovine lens. Exp Eye Res. 1986; 42: 617–630.
5. Oriowo OM, C.A., Chou BR. Action spectrum and recovery for in vitro UV-induced cataract using whole lenses. Invest Ophthalmol Vis Sci. 2001; 42: 2596–2602.
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