西红花酸对百草枯中毒大鼠的肝保护效应

Liver protection of crocetin against paraquat poisoning in rats

目的：探讨西红花酸对百草枯（PQ）中毒大鼠急性肝损伤的保护机制。方法按随机数字表法将54只雄性Wistar大鼠分为对照组、染毒组和治疗组，每组再分为染毒后0.5、2、6d亚组（n＝6）。采用腹腔注射20%PQ20mg/kg制备PQ中毒急性肝衰竭模型；对照组注射等量生理盐水。治疗组0.5d后腹腔注射西红花酸50mg/kg，每日1次，直至处死动物；另两组注射等量生理盐水。各组制模后于相应时间点处死大鼠，收集下腔静脉血和肝组织。苏木素-伊红（HE）染色后光镜下观察6d肝组织病理学改变；采用酶联免疫吸附试验（ELISA）检测血清肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）水平；采用反转录-聚合酶链反应（RT-PCR）检测诱导型一氧化氮合酶（iNOS）、核转录因子-κB（NF-κB）的mRNA表达；采用底物显色法检测6d肝组织凋亡相关因子天冬氨酸特异性半胱氨酸蛋白酶（caspase-8、-9、-12）活性。结果染毒组肝组织炎性细胞广泛浸润，弥漫性碎片状坏死，肝细胞再生不明显，且随时间延长而加重；治疗组肝小叶结构尚存，可见点状坏死、少量充血和炎性细胞浸润。染毒组和治疗组0.5、2、6d血清IL-6、TNF-α水平和肝组织iNOS、NF-κB的mRNA表达，以及6dcaspase-8、-9、-12活性均较对照组显著升高；而治疗组各指标则较染毒组显著降低〔IL-6（ng/L）：0.5d为188.37±64.21比376.61±82.42，2d为287.18±58.69比432.77±96.28，6d为234.24±10.17比375.41±37.59；TNF-α（ng/L）：0.5d为472.36±76.43比688.33±102.19，2d为189.32±87.54比296.21±89.77，6d为99.28±16.13比168.41±66.78；iNOSmRNA（灰度值）：0.5d为2.998±0.801比3.453±0.026，2d为3.126±0.306比5.259±0.153，6d为0.841±0.135比1.225±0.057；NF-κBmRNA（灰度值）：0.5d为1.569±0.818比2.361±0.063，2d为2.345±0.489比4.668±0.368，6d为2.348±0.316比3.972±0.449；caspase-8（pmol/mg）：6d 为126.77±9.97比199.18±66.48；caspase-9（pmol/mg）：6d 为213.12±69.06比321.62±89.39；caspase-12（pmol/mg）：6d为183.46±70.52比219.68±53.93，均P＜0.05〕。结论西红花酸对PQ中毒大鼠具有肝保护效应，其作用可能是通过降低血中炎性因子水平，抑制肝组织caspase-8、-9、-12活性及iNOS、NF-κB基因表达来实现的。

Objective To study the liver protection of crocetin against paraquat （PQ） poisoning induced acute liver injury in rats. Methods Fifty-four male Wistar rats were randomly divided into control group, exposure group and treatment group, and the rats in each group were subdivided into the 0.5th, 2nd, and 6th day after exposure subgroups （n = 6）. The model of acute liver failure induced by PQ poisoning was reproduced by intraperitoneal injection of 20 mg/kg of 20% PQ, and the rats in control group was injected with the same amount of normal saline. The rats in treatment group were given with intraperitoneal injection of 50 mg/kg crocetin after 0.5 day, once a day until they were sacrificed; the other two groups were injected with the same amount of normal saline. The rats in all groups were sacrificed at the corresponding time points, and blood was collected from inferior vena cava and hepatic tissue was harvested. Hematoxylin and eosin （HE） staining was used to observe the pathological changes in liver tissue on the 6th day under light microscope. Enzyme linked immunosorbent assay （ELISA） was used to detect the serum tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） levels. Reverse transcription-polymerase chain reaction （RT-PCR） was used to detect the mRNA expressions of inducible nitric oxide synthase （iNOS） and nuclear factor-κB （NF-κB）. The activities of apoptosis related factors, including caspase-8, -9, -12, in hepatic tissue were determined on the 6th day with chromogenic substrate method. Results In the liver tissue of exposed group, extensive infiltration of the inflammatory cells and the diffuse fragments necrosis were visible, and the regeneration of the liver cells was not obvious, and severity of the injury in a time dependent way. In the treatment group, the structure of hepatic artery was visible, and the infiltration of necrosis, congestion and inflammatory cells were not obvious. On the 0.5th, 2nd, and 6th day, serum levels of IL-6 and TNF-α, the mRNA expressions of iNOS and NF-κB in liver tissue, and the caspase-8, -9, -12 activities on the 6th day in the exposure group and treatment group were significantly higher than those in the control group. And the parameters in treatment group were significantly lower than those of the exposure group [IL-6 （ng/L）： 188.37±64.21 vs. 376.61±82.42 on the 0.5th day, 287.18±58.69 vs. 432.77±96.28 on the 2nd day, 234.24±10.17 vs. 375.41±37.59 on the 6th day; TNF-α （ng/L）： 472.36±76.43 vs. 688.33±102.19 on the 0.5th day, 189.32±87.54 vs. 296.21±89.77 on the 2nd day, 99.28±16.13 vs. 168.41±66.78 on the 6th day; iNOS mRNA （gray value）： 2.998±0.801 vs. 3.453±0.026 on the 0.5th day, 3.126±0.306 vs. 5.259±0.153 on the 2nd day, 0.841±0.135 vs. 1.225±0.057 on the 6th day; NF-κB mRNA （gray value）： 1.569±0.818 vs. 2.361±0.063 on the 0.5th day, 2.345±0.489 vs. 4.668±0.368 on the 2nd day, 2.348±0.316 vs. 3.972±0.449 on the 6th day; caspase-8 （pmol/mg）： 126.77±9.97 vs. 199.18±66.48 on the 6th day; caspase-9 （pmol/mg）： 213.12±69.06 vs. 321.62±89.39 on the 6th day; caspase-12 （pmol/mg）： 183.46±70.52 vs. 219.68±53.93 on the 6th day, all P 〈 0.05]. Conclusion Crocetin has protective effect on liver in rats with PQ poisoning, which role is related with reducing the blood level of inflammatory factors, inhibiting the hepatic caspase-8, -9, -12 activities and gene expressions of iNOS and NF-κB.