[Early acute liver injury in paraquat poisoning rats].
[Early acute liver injury in paraquat poisoning rats].
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DOI：
10.3760/cma.j.issn.2095-4352.2014.06.002
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发表时间：
2014-06
影响因子：
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通讯作者：
Hongxing Guo;Ke Gao;L. Luo;Qingwen Deng;Yanping Zhang;Jie Luo;Lian-Zhong Liu
中科院分区：
文献类型：
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作者：
Hongxing Guo;Ke Gao;L. Luo;Qingwen Deng;Yanping Zhang;Jie Luo;Lian-Zhong Liu
目的观察百草枯中毒大鼠肝细胞凋亡和炎性细胞因子的表达及其机制。方法40只Wistar大鼠按随机数字表法分为对照组（n=8）和模型组（n=32）。模型组大鼠腹腔注射20%百草枯浓缩液30 mg/kg，对照组腹腔注射生理盐水。模型复制后0.5、1、3、7天处死8只大鼠，下腔静脉采血并采集肝组织。采用酶联免疫吸附法（ELISA）测定血清白细胞介素-1β（IL-1β）和肿瘤坏死因子-α（TNF-α）水平。通过逆转录聚合酶链反应（RT-PCR）测定IL-1β、TNF-α、诱导型一氧化氮合酶（iNOS）和p53的mRNA表达。第3天用显色底物法测定肝组织中含有半胱氨酸的天冬氨酸特异性蛋白酶(caspase-3、-8、-9、-12)活性。苏木精-伊红（HE）染色观察肝脏组织病理学变化。结果模型组肝组织出现广泛坏死，并呈时间依赖性炎症细胞浸润。模型组复制后半天血清IL-1β、TNF-α水平显着高于对照组（IL-1β：220.13±69.74 ng/L比0.14±0.03 ng/L，TNF-α：102.66±26.43 ng/L比0.16±0.02 ng/L，P<0.01 P<0.05），并在第3天和第1天达到峰值（IL-1β：423.72±153.11ng/L，TNF-α：690.35±229.64ng/L）。随后逐渐下降，但第7天仍显着高于对照组（IL-1β：357.47±87.28ng/L，TNF-α：12.39±5.06ng/L，均P<0.05）。肝组织中 IL-1β、TNF-α、iNOS mRNA 表达量显着高于对照组，且第 1 天、第 1 天、第 3 天最高值[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 vs. 0.261±0.025，iNOS mRNA（灰度值）：3.259±0.135 vs. 0.002±0.001，P<0.05或P<0.01]。早期模型组与对照组p53 mRNA表达量无差异，均呈低表达，第7天模型组p53 mRNA表达量显着升高（灰度值：2.959±0.086比0.263±0.032，P<0.01）。模型组第3天肝组织中caspase活性显着高于对照组（caspase-3：857.25±309.26 pmol/mg vs. 169.73±48.21 pmol/mg，caspase-8：199.18±61.41 pmol/mg vs. 32.26±11.09 pmol/mg， caspase-9：321.62±80.73 pmol/mg 对比 90.38±29.76 pmol/mg，caspase-12：413.13±89.77 pmol/mg 对比 26.73±9.86 pmol/mg，均 P<0.01)。结论百草枯可引起大鼠急性肝损伤，caspase-3、-8、-9、-12活性明显增强，肝损伤可能与TNF-α、iNOS和p53基因早期高表达有关。
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-1β) 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: 220.13 ± 69.74 ng/L vs. 0.14 ± 0.03 ng/L, TNF-α: 102.66 ± 26.43 ng/L vs. 0.16 ± 0.02 ng/L, 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-1β: 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 in liver tissue were significantly higher on the 3rd day than those in control group (caspase-3: 857.25±309.26 pmol/mg vs. 169.73±48.21 pmol/mg, caspase-8: 199.18±61.41 pmol/mg vs. 32.26±11.09 pmol/mg, caspase-9: 321.62±80.73 pmol/mg vs. 90.38±29.76 pmol/mg, caspase-12: 413.13±89.77 pmol/mg vs. 26.73±9.86 pmol/mg, all P<0.01). CONCLUSIONS 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.