硝苯地平诱发儿童肝损伤的分子机制

Molecular mechanism of Nifedipine inducing liver injury in children

目的建立硝苯地平肝损伤的细胞分子模型，研究硝苯地平诱导儿童肝细胞损伤的分子机制。方法采用肝癌细胞系HepG2建立硝苯地平肝细胞损伤细胞模型，确定硝苯地平处理细胞的最佳浓度及时间。应用Western blot及实时定量PCR（RT－PCR）方法检测细胞特异性肝功能蛋白水平[丙氨酸氨基转移酶（ALT）、天冬氨酸转氨酶（AST）、γ－谷氨酰转肽酶（γ－GT）和碱性磷酸酶（ALP）]及其mRNA变化，利用流式细胞学技术及细胞克隆形成实验确定硝苯地平对细胞周期及其生长增殖的影响。结果1.硝苯地平体外致肝细胞损伤的最佳浓度为20 mg/L，最佳时间为21 d。2.Western blot结果：硝苯地平组细胞内ALT水平低于对照组，而硝苯地平处理组培养基内ALT、AST、γ－GT及ALP蛋白水平（3.55±0.05、4.91±0.06、3.51±0.05、3.08±0.08）均高于对照组（0.96±0.02、1.03±0.02、1.00±0.05、0.90±0.13），差异均有统计学意义（t＝－85.695、－117.582、－47.371、－33.260，均P〈0.05）。3.RT－PCR结果：硝苯地平组细胞内ALT、γ－GT及ALP mRNA水平（0.26±0.02、0.05±0.04、0.05±0.02）均低于对照组（1.13±0.21、0.94±0.10、1.03±0.06），差异均有统计学意义（t＝7.233、127.436、25.687，均P〈0.05）；而硝苯地平组培养基内mRNA水平（5.95±0.05、3.13±0.10、3.32±0.08）均高于对照组（1.01±0.08、1.00±0.05、1.00±0.05），差异均有统计学意义（t＝－92.339、－31.250、－43.007，均P〈0.05）。4.细胞流式检测结果：硝苯地平组G0/G1期细胞比例[（84.09±0.43）%]高于对照组[（30.93±0.32）%]，差异有统计学意义（t＝173.084，P＝0.000）。5.硝苯地平组细胞克隆形成率和细胞增殖速率[（38.56±1.51）%、（25.00±1.71）%]均低于对照组[（97.10±1.17） %、（56.37±2.06）%]，差异均有统计学意义（t＝92.088、20.285，均P＝0.000）。结论硝苯地平可能通过调节细胞周期相关蛋白促进肝细胞损伤。

ObjectiveTo establish the Nifedipine-induced liver cell damage model, and to investigate the cellular or molecular mechanism for children′s liver cell damage.MethodsThe HepG2 cells were utilized to establish liver cell damage models.The optimal concentration and the optimal pretreatment time of Nifedipine-induced liver cell injury were confirmed.Western blot and real-time PCR （RT-PCR） were used to check the alteration in proteins and mRNAs level of the liver function-associated classic markers, which contained alkaline phosphatase （ALP）, aspartate amino transferase （AST）, glutamyl transpeptidase （γ-GT） and alanine aminotransferase （ALT）. Additionally, flow cytometry （FCM）, colony formation assay （CFA） and cell wound healing assay （CWHA） were utilized to check the effect of Nifedipine on the cell cycle progression and proliferation of HepG2.Results（1）The optimal concentration of Nifedipine was 20 mg/L and the optimal treatment period was 21 days for liver damage.（2）Western blot： intracellular ALT protein content after Nifedipine group was less than that of control group, while the protein levels of AST, γ-GT and ALP in the culture medium after Nifedipine addition （3.55±0.05, 4.91±0.055, 3.51±0.05, 3.08±0.08） were higher than those of control group（0.96±0.02, 1.03±0.02, 1.00±0.05, 0.90±0.13）, and the differences were all significant （t =-85.695, -117.582, -47.371, -33.260, all P〈0.05）. （3）The findings of RT-PCR showed that the mRNA levels of intracellular ALT, γ-GT and ALP in Nifedipine group （0.26±0.02, 0.05±0.04, 0.05±0.02） were lower than those of control group （1.13±0.21, 0.94±0.10, 1.03±0.06）, and the differences were all significant （t=7.233, 127.436, 25.687, all P〈0.05）. However, the mRNAs levels in purified culture me-dium in Nifedipine group （5.95±0.05, 3.13±0.10, 3.32±0.08） were higher than those in control group （1.01±0.08, 1.00±0.05, 1.00±0.05）, and the differences were all significant （t=-92.339, -31.250, -43.007, all P〈0.05）. （4）The portion of G0/G1 phase in Nifedipine group [（84.09±0.43）%] was more than that of control group[（30.93±0.32）%], which had statistical significance （t=173.084, P=0.000）. （5）In contrast with control group, the colony formation of cells in Nifedipine group declined from （97.10±1.17）% to （38.56±1.51）% （t=92.088, P=0.000） and the migration rate of cells wound healing was （56.37±2.06）%, （25.00±1.71）% separately（ t=20.285, P=0.000）.ConclusionNifedipine may promote children′s liver injury through regulating cell cycle related proteins.