选择性磷酸酶抑制剂Salubrinal对急性百草枯中毒大鼠肺组织细胞自噬和凋亡的影响

The effect of selective phosphatase inhibitors Salubrinal on autophagy and apoptosis in the lung tissue of rats with acute paraquat poisoning

目的 观察选择性磷酸酶抑制剂Salubrinal治疗对急性百草枯（PQ）中毒大鼠肺组织细胞自噬和凋亡的影响,并探讨其治疗机制.方法 将200只Wistar大鼠按计算机生成的随机排列表分为4组,每组50只.一次性灌胃40 mg/kg PQ溶液1 mL制备PQ染毒模型,之后每日1次腹腔注射1 mL生理盐水（NS）;对照组一次性给予1 mL NS灌胃,之后每日2次腹腔注射1 mL NS;Sal 0.5和Sal 1.0组分别于PQ染毒后1、3、5d腹腔注射0.5 mg/kg或1.0 mg/kg Salubrinal 1 mL,每日1次.染毒后7d取肺组织,苏木素-伊红（HE）染色后观察肺组织形态学改变;免疫组化染色观察肺组织自噬相关蛋白LC3-Ⅱ阳性表达;蛋白质免疫印迹试验（Western Blot）检测肺组织LC3-Ⅱ和天冬氨酸特异性半胱氨酸蛋白酶3（caspase-3）的蛋白表达.结果 HE染色结果显示：PQ染毒组肺组织结构部分破坏,肺泡壁塌陷,肺泡腔扩大,局部炎性细胞浸润,肺泡隔显著增厚,局部明显出血;Sal 0.5组和Sal 1.0组肺组织病理改变较PQ染毒组明显减轻.免疫组化染色显示：与对照组比较,PQ染毒组、Sal 0.5组和Sal 1.0组肺组织LC3-Ⅱ阳性表达均明显降低（A值：78.34±10.71、76.52±8.21、77.48±9.11比117.58±15.26,均P＜0.05）;而后3组间两两比较差异无统计学意义（均P＞0.05）.Western Blot结果显示,与对照组比较,PQ染毒组肺组织LC3-Ⅱ、caspase-3蛋白表达均明显升高[LC3-Ⅱ（A值）：0.22±0.05比0.14±0.03,caspase-3（A值）：0.115 ±0.013比0.023±0.006,均P＜0.05].与PQ染毒组比较,Sal 0.5组和Sal 1.0组肺组织LC3-Ⅱ、caspase-3蛋白表达均明显降低[LC3-Ⅱ（A值）：0.19±0.05、0.18±0.04比0.22±0.05,caspase-3（A值）：0.078±0.012、0.076±0.010比0.115±0.013,均P＜0.05].结论 急性PQ中毒大鼠肺组织发生了内质网应激-自噬;Salubrinal可降低急性PQ中毒大鼠肺组织发生细胞自噬,抗细胞凋亡,从而起到治疗作用.

Objective To investigate the effect of selective phosphatase inhibitors Salubrinal on autophagy and apoptosis in the lung tissue of rats with acute paraquat （PQ） poisoning,and to explore its mechanism.Methods 200 Wistar rats were randomly divided into four groups by randomized arrangement table formed by computer,with 50 rats in each group.PQ poisoning model was reproduced by one time gastric lavage with 1 mL of 40 mg/kg PQ solution followed by intraperitoneal injection of 1 mL normal saline （NS） once a day.The rats in control group were lavaged once with 1 mL of NS followed by intraperitoneal injection of 1 mL NS twice a day.The rats in Sal 0.5 and Sal 1.0 groups were intraperitoneal injected with 1 mL Salubrinal 0.5 mg/kg or 1.0 mg/kg on the 1st,3rd,and 5th day after PQ poisoning once a day.The lung tissue was harvested on the 7th day after poisoning,and the changes in histomorphology were observed using hematoxylin and eosin （HE） staining.The positive expression of autophagy-related protein LC3-Ⅱ in lung tissue was observed after immunohistochemistry staining,and LC3-Ⅱ and caspase-3 protein expressions were determined by Western Blot.Results HE staining results showed partial abnormal pulmonary structure in the PQ poisoning group：collapse of pulmonary alveoli,enlargement of the cavity,local infiltration of inflammatory cells,increasing thickness in the alveoli wall and obvious bleeding in the local lung tissue.Compared with the PQ poisoning group,the above changes in Sal 0.5 and Sal 1.0 groups were obviously relieved.It was shown by immunohistochemistry staining that compared with control group,the positive expression of LC3-Ⅱ was obviously decreased in the PQ poisoning group,Sal 0.5,and Sal 1.0 groups （A value：78.34 ± 10.71,76.52 ± 8.21,77.48 ± 9.11 vs.117.58 ± 15.26,all P〈0.05）.There was no significant difference in positive expression of LC3-Ⅱ between each of the later three groups （all P〉0.05）.Western Blot results showed：compared with the control group,the protein expressions of LC3-Ⅱ and caspase-3 were significantly increased in PQ poisoning group [LC3-Ⅱ （A value）：0.22 ±0.05 vs.0.14 ±0.03,caspase-3 （A value）：0.115 ± 0.013 vs.0.023 ± 0.006,both P〈0.05].Compared with PQ poisoning group,the protein expressions of LC3-Ⅱ and caspase-3 were obviously decreased in the Sal 0.5 and Sal 1.0 groups [LC3-Ⅱ （A value）：0.19 ±0.05,0.18 ±0.04 vs.0.22 ±0.05; caspase-3 （A value）：0.078 ±0.012,0.076 ±0.010 vs.0.115 ±0.013,all P〈0.05].Conclusions The endoplasmic reticulum stress-autophagy is activated in the pulmonary cell of acute PQ poisoning rats.Salubrinal can decrease the autophagy and apoptosis in the lung of rats with acute PQ poisoning,which play a role in the treatment.