早期百草枯中毒大鼠的急性肝损伤研究

Early acute liver injury in paraquat poisoning rats

目的 探讨百草枯中毒大鼠肝细胞凋亡、炎症因子表达情况及其发生机制.方法 按随机数字表法将40只Wistar大鼠分为对照组（n=8）与模型组（n=32）.模型组腹腔注射20％百草枯浓缩液30 mg/kg;对照组则给予等量生理盐水.制模后0.5、1、3、7d分别处死8只大鼠,收集下腔静脉血及肝组织,采用酶联免疫吸附试验（ELISA）检测血清白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）水平;采用逆转录-聚合酶链反应（RT-PCR）检测IL-1β、TNF-α、诱导型一氧化氮合酶（iNOS）和p53的mRNA表达;采用底物显色法检测3d时肝组织天冬氨酸特异性半胱氨酸蛋白酶（caspase-3、-8、-9、-12）的活性;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 模型组肝组织出现碎片状坏死、毛细血管扩张、炎性细胞广泛浸润等现象,且随时间延长明显加重.模型组0.5 d时血清IL-1β、TNF-α水平即明显高于对照组[IL-1β（ng/L）：220.13±69.74比0.14±0.03,TNF-α（ng/L）：102.66±26.43比0.16±0.02,P＜0.01和P＜0.05],分别于3d、1d时达高峰[IL-1β：（423.72±153.11） ng/L,TNF-α：（690.35±229.64） ng/L],随后均逐渐降低,7d时仍明显高于对照组[IL-1β：（357.47±87.28） ng/L,TNF-α：（12.39±5.06） ng/L,均P＜ 0.05].模型组肝组织IL-1β、TNF-α和iNOS的mRNA表达均较对照组明显升高,其峰值分别出现在1d、1d、3d时[IL-1β mRNA（灰度值）：1.569±0.057比0.123±0.016,TNF-α mRNA（灰度值）：0.683±0.077比0.261±0.025,iNOS mRNA（灰度值）：3.259±0.135比0.002±0.001,P＜0.05或P＜0.01].模型组早期p53 mRNA表达与对照组无差异,均为低表达,染毒7d时p53 mRNA表达显著增高（灰度值：2.959±0.086比0.263±0.032,P＜0.01）.模型组3d时肝组织caspase活性（pmol/mg）显著高于对照组（caspase-3：857.25±309.26比169.73±48.21,caspase-8：199.18±61.41比32.26±11.09,caspase-9：321.62±80.73比90.38±29.76,caspase-12：413.13±89.77比26.73±9.86,均P＜0.01）.结论 百草枯中毒可导致大鼠发生急性肝损伤,caspase-3、-8、-9、-12活性显著增强;肝损伤的发生与TNF-α、iNOS和p53基因早期高表达有密切关系.

Objective To observe hepatocellular apoptosis and inflammatory cytokines expression and their mechanisms after paraquat poisoning in rat.Methods Forty Wistar rats were divided into control group （n =8） and model group （n =32） by random number table.Rats in model group were intraperitoneally injected with 30 mg/kg 20％ paraquat concentrate,while those in control group were injected with normal saline.0.5,1,3,7 days after reproduction of the model,8 rats were sacrificed,and blood was collected from inferior vena cava and hepatic tissue was harvested.The serum levels of interleukin-1β （IL-1 β） and tumor necrosis factor-α （TNF-α） were determined by enzyme-linked immunosorbent assay （ELISA）.The mRNA expressions of IL-1β,TNF-α,inducible nitric oxide synthase （iNOS） and p53 were determined by reverse transcription-polymerase chain reaction （RT-PCR）.Cysteine-containing aspartate-specific proteases （caspase-3,-8,-9,-12） activity in hepatic tissue was determined on the 3rd day with chromogenic substrate method.The liver histopathological changes were observed after hematoxylin-eosin （HE） staining.Results In model group,hepatic tissue showed extensive necrosis with inflammatory cell infiltration in time dependant manner.Serum IL-1β and TNF-α levels were significantly higher in model group half a day after reproduction than those in control group [IL-1β （ng/L）：220.13 ± 69.74 vs.0.14 ± 0.03,TNF-α （ng/L）：102.66 ± 26.43 vs.0.16 ± 0.02,P＜ 0.01 and P＜0.05],and peaked on the 3rd day and 1st day [IL-1β：（423.72 ± 153.11） ng/L,TNF-α：（690.35 ± 229.64） ng/L].They then decreased gradually,but were still significantly higher than those in control group on the 7th day [IL-1 β：（357.47 ± 87.28） ng/L,TNF-α：（12.39 ± 5.06） ng/L,both P＜0.05].The contents of IL-1β,TNF-α and iNOS mRNA expressions in hepatic tissue were significantly higher than those in control group,and the highest values were seen on the 1st day,the 1st day,and the 3rd day [IL-1β mRNA （gray value）：1.569 ± 0.057 vs.0.123 ± 0.016,TNF-α mRNA （gray value）：0.683 ± 0.077 vs.0.261 ± 0.025,iNOS mRNA （gray value）：3.259 ± 0.135 vs.0.002 ±0.001,P＜0.05 or P＜0.01].There was no difference in p53 mRNA expression between model group and control group at early stage,and both of them showed low expression,and p53 mRNA expression was significantly higher in model group on the 7th day （gray value：2.959 ± 0.086 vs.0.263 ± 0.032,P＜0.01）.In model group,caspase activity （pmol/mg） in liver tissue were significantly higher on the 3rd day than those in control group （caspase-3：857.25 ± 309.26 vs.169.73 ± 48.21,caspase-8：199.18 ± 61.41 vs.32.26 ± 11.09,caspase-9：321.62 ± 80.73 vs.90.38 ± 29.76,caspase-12：413.13 ± 89.77 vs.26.73 ± 9.86,all P＜0.01）.Conclusion Paraquat can cause acute liver injury in rats,with caspase-3,-8,-9,-12 activities markedly enhanced,and liver injury may be associated with an early high expression of TNF-α,iNOS and p53 gene.