雷帕霉素调控Nrf2通路抑制百草枯中毒致肺纤维化作用

Rapamycin Inhibits the Paraquat Poisoning Induced-pulmonary Fibrosis by Regulating Nrf2 Signaling Pathway

目的评估雷帕霉素(RAPA)对大鼠百草枯(PQ)中毒导致肺纤维化的治疗作用,并初步探讨其作用机制。方法 6～8周龄SD大鼠随机分为对照组、染毒(PQ)组、染毒+Nrf2抑制剂(鸭胆子苦醇)组、染毒+RAPA、染毒+Nrf2抑制剂+RAPA组,每组10只。分别在7 d,28 d处死大鼠。HE染色及Masson’s染色法观察肺组织病理结构变化,用酶联免疫吸附测定(ELISA)试剂盒检测肺组织中的羟脯氨酸(HYP)含量。RT-PCR和Western blot检测Nrf2的表达,同时检测抗氧化物SOD、CAT和GSH。结果 (1)肺组织病理染色结果和HYP结果显示,Nrf2抑制剂(鸭胆子苦醇)治疗后明显加重肺纤维化程度,给与RAPA治疗能缓解肺纤维化,同时给与鸭胆子苦醇后肺纤维化程度重于单独的RAPA治疗;(2) RT-PCR和western blot结果均显示Nrf2在百草枯中毒后7 d,28 d均明显升高。而给与Nrf2抑制剂鸦胆子苦醇治疗后Nrf2表达明显减弱,雷帕霉素治疗明显增加Nrf2的表达,同时联合使用后Nrf2表达低于雷帕霉素单独治疗;(3)抗氧化物SOD、CAT和GSH在染毒组表达下降,Nrf2抑制剂组明显下降,给予雷帕霉素明显促进SOD、CAT和GSH表达。结论雷帕霉素可能通过调节Nrf2通路的表达减轻百草枯中毒引起的肺纤维化。

Objective To evaluate the therapeutic effect and possible mechanism of rapamycin (RAPA) in pulmonary fibrosis induced by paraquat (PQ) intoxication in rats. Methods SD rats aged 6-8 weeks were randomly divided into a control group,a Paraquat exposure group,a Paraquat +Nrf2 inhibitor(gallinol) group, a Paraquat + RAPA, a Paraquat +Nrf2 inhibitor + RAPA group,and 10 rast in each group. Rats were killed on 7th and 28th day. HE staining and Masson's staining was used to observe the pathological changes of lung tissues,and the content of hydroxyproline (HYP) in lung tissues was measured. Expression of Nrf2 was detected by RT-PCR and Western blot,while antioxidant SOD,CAT and GSH were detected. Results(1)Lung histopathological staining results and HYP results showed that Nrf2 inhibitor (gallbladder bitter alcohol) significantly increased the degree of pulmonary fibrosis after treatment,and RAPA treatment could alleviated pulmonary fibrosis, while the degree of pulmonary fibrosis after gallbladder bitter alcohol treatment was more than that of RAPA alone.(2)RT-PCR and western blot results showed that Nrf2 was significantly increased on 7th and 28th day after paraquat poisoning. However,the expression of Nrf2 was significantly decreased after treatment with the Nrf2 inhibitor bittersweet,and the expression of Nrf2 was significantly increased after treatment with rapamycin. Meanwhile,the expression of Nrf2 was lower than that of rapamycin alone after combined treatment. 3. The expressions of antioxidant SOD,CAT and GSH were decreased in the toxic group, and significantly decreased in the Nrf2 inhibitor group,and the expression of SOD,CAT and GSH were significantly promoted by the administration of rapamycin. Conclusion Rapamycin could alleviate the paraquat poisoning induced-pulmonary fibrosis by regulating the expression of Nrf2 pathway.