褪黑素对百草枯诱导小鼠急性肝损伤的干预作用及其机制

Effect of melatonin on paraquat-induced acute liver injury in mice and its mechanism

目的探讨褪黑素（MT）对百草枯（PQ）染毒小鼠肝损伤的保护作用及其可能机制。方法将48只雄性BALB／c小鼠按随机数字表法分为正常组（n=6）、MT对照组（n=6）、PQ染毒组（n=18）、MT干预组（n=18，PQ染毒1h＋MT）；腹腔注射给药，MT为15mg／kg，PQ为30mg／kg；后两组再按给药后不同时间点分为12、24、72h亚组，每个亚组6只。干预后不同时间点于小鼠内眦取血并取肝组织，用碘比色法检测血清丙氨酸转氨酶（ALT）和天冬氨酸转氨酶（AST）含量，用双抗夹心酶联免疫吸附试验（ELISA）测定肝组织肿瘤坏死因子-α（TNF-α）、白细胞介素-1B（IL-1β）水平，用蛋白质免疫印迹试验（Western Blot）检测肝细胞核内核转录因子-κB p65（NF-κB p65）的蛋白表达；经苏木素-伊红（HE）染色后光镜下观察肝组织病理学改变。结果与正常组比较，PQ染毒组在染毒后12h血清ALT、AST水平和肝组织TNF-α水平即明显升高，24h达峰值[ALT（U／L）：417．88±76．16比41．76±3．63，AST（U／L）：469．79±69．81比53．19±6．31，TNF—α（pg／mg）：46．39±9．81比13．01±3．19，均P〈0．05]，之后逐渐下降；肝组织IL-1β水平和细胞核内NF-κBp65表达随时间延长持续升高，72h达峰值[IL-1β（pg／mg）：30．74±5．81比3．81±0．71，NF-κB p65蛋白（灰度值）：1．70±0．14比0．85±0．08，均P〈0．05]。MT干预可明显抑制PQ染毒导致的上述指标异常升高，与PQ染毒组比较，MT干预组血清AST和肝组织TNF—α、IL-1β水平于12h起即明显降低[AST（U／L）：269．35±11．34比391．11±8．71，TNF-α（pg／mg）：15．10±5．03比28．77±5．96，IL-1β（pg／mg）：6．23±1．03比10．89±3．02，均P〈0．05]，血清ALT水平和细胞核内NF-κB p65蛋白表达于24h起明显降低[ALT（U／L）：249．38±21．71比417．88±76．16，NF-κB p65蛋白表达（灰度值）：1．13±0．07比1．45±0．09，均P〈0．05]。光镜下观察显示，正常组肝细胞无病理学改变；PQ染毒组肝组织出现炎性细胞广泛浸润，中心静脉、血窦出现扩张、充血，并有肝细胞坏死；MT干预组肝细胞病理学改变较PQ染毒组明显减轻。正常组与MT对照组上述各指标比较均无差异。结论MT可通过抑制NF-κB p65的过度活化和减少TNF—α的释放，减轻PQ中毒引起的肝细胞坏死程度，

Objective To observe the protective effect of melatonin （MT） on paraquat （PQ）-induced acute liver injury in mice and its possible mechanism. Methods A total of 48 male BALB/c mice were randomly divided into normal group （n=6）, MT control group （n = 6）, PQ poisoning group （n = 18）, and MT treatment group （n = 18, 1 hour-PQ poisoning ± MT）. The drugs were intraperitoneally injected, MT 15 mg/kg, PQ 30 mg/kg. The mice in the later two groups were subdivided into 12, 24, 72 hours subgroups according to different time points after administration, with 6 mice in each subgroup. Blood from medial angle of eye and liver tissue were collected at different time points after intervention to determine the serum levels of alanine transaminase （ALT） and aspartate aminotransferase （AST） with iodine colorimetry. The levels of tumor necrosis factor-α （TNF-α ） and interleukin-1β （IL-1β ） in liver tissue were determined with double-antibody sandwich enzyme linked immunosorbent assay （ELISA）. The protein expression of nuclear factor-κB p65 （NF-κB p65） in liver nuclei was determined by Western Blot. The pathological Changes in liver tissue were observed with light microscope using hematoxylin and eosin （HE） staining. Results Compared with the normal group, the levels of serum ALT and AST, and TNF-α in liver tissue were significantly increased from 12 hours after poisoning, and peaked at 24 hours [ALT （U/L）： 417.88±76.16 vs. 41.76±3.63, AST （U/L）： 469.79±69.81 vs. 53.19±6.31, TNF-α （pg/mg）： 46.39±9.81 vs. 13.01 ±3.19, all P 〈 0.05], then they were gradually decreased; the levels of IL-1β in liver tissue and NF-κB p65 in nucleus were continuously rose with time prolongation, peaked at 72 hours [IL-1β （pg/mg）： 30.74±5.81 vs. 3.81±0.71, NF-κB p65 protein （gray value）： 1.70±0.14 vs. 0.85±0.08, both P 〈 0.05]. MT intervention could significantly inhibit the above parameters with abnormal increase induced by PQ poisoning. Compared with the PQ poisoning group, the levels of AST in serum and TNF-α and IL-1β in liver tissue were significantly decreased from 12 hours [AST （U/L）： 269.35± 11.34 vs. 391.11±8.71, TNF-α （pg/mg）： 15.10±5.03 vs. 28.77±5.96, IL-1β （pg/mg）： 6.23± 1.03 vs. 10.89±3.02, all P 〈 0.05], and the levels of serum ALT and NF-κB p65 in nucleus were significantly decreased from 24 hours [ALT （U/L）： 249.38±21.71 vs. 417.88±76.16, NF-κB p65 protein expression （gray value）： 1.13±0.07 vs. 1.45±0.09, both P 〈 0.05]. It was shown by light microscope that no pathological changes in hepatocyte were found in normal group. The pathological changes in hepatocyte were obvious in PQ poisoning group as following： inflammatory cell infiltrates, central venous and blood sinus dilation, hyperemia, and necrosis of liver cells. The pathological changes in hepatocyte in MT treatment group were lessened as compared with those of PQ poisoning group. No significant differences were found in above parameters between normal group and MT control group. Conclusion MT can lessen the necrosis of hepatocyte induced by PQ poisoning via inhibiting the excessive activity of NF-κB p65 and decreasing the TNF-α releasing, and play a role of liver protection.