H2O2对人晶状体上皮细胞AQP1和AQP5表达影响及其机制

EFFECTS OFH_(2)O_(2) ON EXPRESSION OF AQP1 AND AQP5 IN HUMAN LENS EPITHELIAL CELLS AND THE UNDERLYING MECHANISMS

目的探究氧化应激对晶状体上皮细胞(HLECs)水通道蛋白(AQPs)表达的影响及其机制。方法取健康人群和白内障病人的晶状体前囊膜,应用实时荧光定量PCR(RT-PCR)和免疫组织化学染色方法检测两组AQP1和AQP5表达与分布情况。应用RT-PCR方法检测不同浓度的H_(2)O_(2)作用HLECs不同时间后AQP1和AQP5 mRNA表达情况;用400μmol/L的H_(2)O_(2)刺激HLECs不同时间,Westernblot方法检测AQP1和AQP5蛋白表达,RT-PCR和Westernblot分别检测丝裂酶原激活的蛋白激酶(MAPK)抑制剂联合H_(2)O_(2)(400μmol/L)共同作用于HLECs后AQP1和AQP5的mRNA与蛋白的表达情况。结果白内障组晶状体前囊膜中AQP1和AQP5的mRNA表达与分布明显低于健康组(t=10.71、5.46,P<0.05)。H_(2)O_(2)刺激HLECs引起的AQP1和AQP5 mRNA表达下调具有浓度和时间依赖性(F_(浓度)=80.38、436.20,F_(时间)=46.95、175.00,F_(交互)=7.99、17.52,P<0.01)。其中以400μmol/L的H_(2)O_(2)作用最为明显,分别在作用4、24h时AQP1和AQP5的mRNA与蛋白表达降至最低水平(t=3.32~5.31,P<0.05)。p38和细胞外信号调节激酶(ERK)抑制剂可以抑制H_(2)O_(2)诱导的AQP1的mRNA和蛋白表达下调(t=4.37~7.28,P<0.05),JNK、p38和ERK的抑制剂对AQP5的表达量无明显作用(P>0.05)。结论氧化应激使HLECsAQP1和AQP5表达下降;在氧化应激条件下,MAPK(p38和ERK)途径参与AQP1表达调控,但MAPK途径不参与AQP5的表达调控。

Objective To explore the effects of oxidative stress on the expression of aquaporins(AQPs)in human lens epithelial cells(HLECs)and the underlying mechanisms.Methods The anterior lens capsules from healthy people and patients with cataract were collected.RT-PCR and immunohistochemistry were used to determine the expression and distribution of AQP1 and AQP5 in both groups.After stimulating HLECs by different concentrations of H_(2)O_(2)for different lengths of time,the mRNA expression of AQP1 and AQP5 was measured by RT-PCR.With H_(2)O_(2)at 400μmol/L stimulating HLECs for different lengths of time,the protein expression of AQP1 and AQP5 was measured by Western blot.After using mitogen-activated protein kinase(MAPK)inhibitors and H_(2)O_(2)(400μmol/L)combined to stimulate HLECs,the mRNA and protein expression of AQP1 and AQP5 was determined by RT-PCR and Western blot,respectively.Results The cataract group showed significantly lower mRNA expression and distribution of AQP1 and AQP5 in the anterior lens capsule compared with the healthy group(t=10.71,5.46;P<0.05).H_(2)O_(2)down-regulated the mRNA expression of AQP1 and AQP5 in a concentration-and time-dependent manner(_(Fconcentration)=80.38,436.20;F_(time)=46.95,175.00;F_(interaction)=7.99,17.52;P<0.01)H_(2)O_(2)showed the greatest effects at 400μmol/L,with the lowest mRNA and protein expression of AQP1 and AQP5 at 4 h and 24 h,respectively(t=3.32-5.31,P<0.05).Inhibitors of p38 and extracellular signal-regulated kinase(ERK)could significantly inhibit H_(2)O_(2)-induced down-regulation of AQP1 mRNA and protein(t=4.37-7.28,P<0.05).Inhibitors of c-Jun N-terminal kinase,p38,and ERK had no significant effects on the expression of AQP5(P>0.05).Conclusion Oxidative stress reduces the expression of AQP1 and AQP5 in HLECs.Under oxidative stress,the MAPK pathways(p38 and ERK)are involved in the regulation of AQP1 expression,but not in the regulation of AQP5 expression.