硫辛酸通过Nrf2-ARE信号通路对百草枯中毒大鼠急性肺损伤保护作用的研究

Lipoic acid protects the lungs of rats against acute injury induced by paraquat poisoning through Nrf2-ARE signaling pathway

目的研究硫辛酸（LA）对百草枯中毒大鼠急性肺损伤抗氧化相关转录因子核因子相关因子2（nuclear factor—erythroid 2-related factor，Nrf-2）和亚铁血红素氧化酶1（heine oxygenase1，HO-1）表达的影响，探讨硫辛酸保护百草枯肺损伤的作用机制。方法SPF级SD雄性大鼠随机（随机数字法）分为正常组（n=12）、百草枯中毒组（PQ组，n=30）和百草枯中毒＋硫辛酸干预组（LA组，n=30），正常组分为6h和72h亚组，PQ组和LA组大鼠分为6h、12h、24h、48h、72h五个亚组（每个亚组各6只）。PQ组以腹腔注射百草枯25mg／kg，LA组在给予百草枯25mg／kg染毒0．5h后尾静脉给予LA100mg／kg。正常组给予等量生理盐水腹腔注射。测定体质量及肺系数，观察各组肺部组织病理变化（HE染色）及Nrf-2，HO-1的表达（免疫组化法）；同时检测酶学指标谷胱甘肽过氧化物酶（GSH—Px）、超氧化物歧化酶（SOD）活力变化。采用组间单因素方差分析，组间两两比较采用SNK-q检验。结果LA组体质量在48h和72h时点[（191．02±0．82）g和（183．37±7．74）g]降低明显少于PQ组同时点[（183．85±2．07）g和（173．13±4．34）g]（P〈0．01或P〈0．05）；LA组肺系数在24h，48h，72h时点[（6．83±0．48）mg／g、（7．61±0．28）mg／g和（8．29±0．36）mg／g]明显低于PQ组同时点[（7．39±0．53）mg／g、（8．48±0．23）mg／g和（9．06±0．10）mg／g]（P〈0．01或P〈0．05）；Nrf-2蛋白水平的表达LA组在48h，72h时点[（3．99±0．50）和（3．51±0．12）]高于PQ组[（1．33±0．22）和（1．62±0．41）]（均P〈0．01）；LA组HO-1蛋白水平的表达在72h时点（1．76±0．17）高于PQ组（1．31±0．15）（p〈0．01）；LA组GSH—Px活力在24h，48h，72h时点明显高于PQ组同时点（P〈0．01或P〈0．05）；SOD活力在5个时点均明显高于pQ组同时点（均P〈0．01）。结论Nff2-ARE通路参与百草枯中毒致急性肺损伤病理生理过程，硫辛酸可能通过Nrt2-ARE通路保护百草枯中毒大鼠急性肺损伤。

Objective To investigate the nuclear factor-erythroid 2-related factor （ Nrf-2）, and heine oxidase 1 （HO-1） expression in acute lung injury induced by paraquat poisoning in rats and explore the mechanism of lipoic acid acting on protection of lung from paraquat poisoning. Methods Seventy-two adult healthy male Sprague Dawley （SD） rats were randomly divided into three groups with different treatments designated as： control group （control group, n = 12）, paraquat group （PQ group, n =30） and paraquat ± lipoic acid group （LA group, n = 30）. PQ group and LA group were randomly divided into five subgroups （n =6 in each） according to 6 h, 12 h, 24 h,48 h and 72 h after modeling and treatment. The rats in PQ group and PQ ± LA group were treated with intra-peritoneal injection （ip） of PQ （25 mg/kg）, while the rats in control group were treated with the equal volume of saline instead. Half an hours after intra-peritoneal injection of PQ, lipoic acid （ 100 mg/kg） was injected into caudal vein of rats once a day until they were sacrificed. The body weight was measured everyday. The rats of each group were sacrificed at the given intervals, and lung tissues were harvested to measure lung coefficient of rats. The same part of left lung of rats in each group was taken for HE staining and immunohistocbemistry in order to detect the expressions of Nrf2 and HO-1. The right lung of rats in each group was taken for the detection of GSH-Px and SOD activity. All data were analyzed by using the One-way analysis of variance （ANOVA） and SNK-q test. Results The body weight reduction in LA group （191.02 ±0.82） g, （183.37 ± 7.74） g was significantly less than that in PQ group （ 183.85 ±2. 07） g, （ 173.13 ±4. 34） g at 48 h and 72 h after PQ poisoning, respectively （P〈0.01, P〈0.05） .The lung coefficient in LA group 6.83 ±0.48） rag/g, （7. 61 ± 0. 28） mg/g, （ 8.29 ± 0. 36） mg/g was less compared with PQ group 7. 39 ± 0. 53 ） mg/g, （8.48±0.23） mg/g, （9.06±0.10） mg/g at 24 h, 48 h, and72 h, respectively （P〈0.01, P〈 0. 05）. The immunohistochemical expressions of Nrf-2 in LA group （3.99 ±0. 50）, （3.51 ±0. 12） were higher than those in PQ group （ 1.33 ±0. 22）, （ 1.62 ±0. 41 ） at 48 h and 72 h. The immunohistochemieal expression of HO-1 in LA group （ 1.76 ±0. 17） was higher than that in PQ group （ 1.31 ±0. 15） at 72 h. The levels of GSH-Px activity in LA group were significantly higher in comparison with PQ group at 24h, 48h, and 72h （P 〈 0. 01, P 〈 0. 05 ）. The levels of SOD activity in the LA group were significantly higher in comparison with PQ group at 6 h, 12 h, 24 h, 48 h, and 72 h after PQ administration （ P 〈 0.01 ）. Conclusions Nrf2-ARE （antioxidant response element） signaling pathway is involved in the pathogenesis of acute lung injury induced by paraquat poisoning, and lipoic acid may protect acute lung injury in rats induced by paraquat poisoning through Nrf2-ARE signaling pathway.