维拉帕米对糖基化终产物诱导人晶状体上皮细胞凋亡的保护作用

Protection of Verapamil against AGE induced apoptosis of human lens epithelial cell

目的研究维拉帕米对糖基化终产物（AGE）诱导人晶状体上皮细胞（HLEC）凋亡的保护作用。方法实验研究。培养HLEC系SRAOI／04，传至第3代。实验分为4组，A组为对照组LEC细胞；B组为AGE组，LEC细胞用20 μmol／LAGE处理；C组为AGE＋SB202190组，15 μmol／LSB202190预处理2h后的LEC细胞用AGE处理；D组为AGE＋维拉帕米组，50 μmol／L维拉帕米预处理2h后的细胞用AGE处理。应用MTT法检测各组细胞活力，AnnexinV试剂盒处理细胞后用流式细胞仪检测各组细胞的凋亡率，通过Westernblot法检测各组细胞p-p38、Caspase3的表达情况。统计学方法为单因素方差分析，组间的两两比较均采用LSD-t检验。结果作用24h后，HLEC活力AGE组为0．28±0．08，较对照组的0．97±0．05明显下降（LSD-t检验，P=0．008），AGE＋SB202190组（0．79±0．06）及AGE＋维拉帕米组（0．62±0．07）较AGE组（0．28±0．08）明显增加（F=34．52，P=0．001）；细胞凋亡率AGE组为（19．9±1．1）％，较对照组的（2．5±0．6）％明显上升（LSD．t检验，P=0．003），AGE＋SB202190组（4．2±1．2）％及AGE＋维拉帕米组（5．8±1．8）％较AGE组（19．9±1．1）％明显减少（F=371．61，P〈0．01）；LECp-p38、Caspase3蛋白表达量AGE组分别为223．35±20．15及256．77±19．88，较对照组106．44±10．74及100．26±18．65升高，AGE＋SB202190组两种蛋白表达量为139．17±19．10，142．75±23．36，AGE＋维拉帕米组则为154．79±21．87，139．79±25．73，较对照组降低（F=248．01，F=76．68；P〈0．01）。结论维拉帕米可干预p38细胞通路，部分抑制AGE诱导晶状体上皮细胞凋亡作用。

Objective To investigate the protection of Verapamil against advanced glycation end products （AGE） induced human lens epithelial cells （HLEC） apoptosis. Methods Experiment study. SRA01/04 （HLEC line） was cultivated and passaged to the third generation and then divided into four groups. A group was named as control group, and B group was named as AGE group （ LEC was treated by 20 μ mol/L AGE） . C group was AGE＋SB202190 group （ LEC was treated 2 hours by SB2012190 and then treated by 15 p, mol/L AGE）. D group was AGE＋ Verapamil group （LEC was treated 2 hours by 50 μmol/L Verapamil and then treated by AGE）. MTT was used to evaluate the cell viability. Flow cytometry with Annexin V-FITC apoptosis detection was used to assess cell apoptosis.The expression of p-p38 and caspase3 was detected by Western blot between groups. One way Chi-square analysis was used for data analysis. LSD-t test was used as comparison between every two groups. Results After 24 hours, LEC viability （A570） was （0.28±0.08） in B group, which was significantly lower than A group （0.97±0.05） （LSD-t test, P= 0.008）. LEC viability in C and D group was （0.79±0.06） and （0.62±0.07） separately, which can partly higher than it was in B group （F=34.52, P=0.001）. The apoptosis ceils were （19.9±1.1）% in B group, which were significantly higher than they were in A group （2.5±0.6）% （P=0.003）. The apoptosis cells in C and D group were （4.23±1.20） and （5.79±1.75） separately, which were significant lower than they were in B group （F= 371.61, P〈0.01）. In additional, expressions of p-p38, Caspase3 proteins in the cells of group B were （223.35±20.15） and （256.77±19.88） separately, which were higher than it were in A group,which were （106.44±10.74） and （100.26±18.65） separately. However, they were （139.17±19.10） and （142.75±23.36） in group C and （154.79±21.87） and （139.79±25.73） in group D （F=248.01, F=76.68;P〈0.01）, which were lower than they were in A group. Conclusion p38 pathway is involved in the apoptotic procedure of LEC induced by AGE. Verapamil can interdict the p38 signal pathway and protect LEC apoptosis.