血必净干预下百草枯中毒大鼠肺组织线粒体融合蛋白2及超微结构变化的研究

The interventional effect of Xuebijing injection on expression of mitochondrial fusion protein 2 and the ultrastructure changes in lung tissues in rats with paraquat poisoning

目的 探讨百草枯（PQ）中毒致肺纤维化的发生机制以及血必净在PQ中毒时的治疗作用.方法 72只雄性Wistar大鼠按随机数字表法分为对照组、PQ中毒组和血必净干预组3组,每组24只.一次性灌胃50 mg/kg PQ染毒致肺纤维化,对照组灌胃1 mL蒸馏水;血必净组在染毒后30 min腹腔注射血必净注射液4 mL/kg,12h1次;PQ组和对照组则腹腔注射等量生理盐水;给药周期均为14 d.各组于染毒后1、3、7、14d末次干预后30 min各处死6只大鼠,取肺组织,通过苏木素-伊红（HE）染色、马松（Masson）染色,光镜下观察肺组织病理学及肺纤维化改变;透射电镜下观察肺组织超微结构变化;采用碱水解法测定羟脯氨酸（HYP）含量;采用蛋白质免疫印迹试验（Western Blot）检测线粒体融合蛋白2（Mfn2）的表达.结果 ①HE染色：PQ组3d时炎症反应最重;7d时肺泡腔内渗出液逐渐机化,由肥大的成纤维细胞分泌纤细的胶原纤维,肺泡腔内可见到纤维化;14d时成纤维细胞大量增生,肺泡结构破坏并萎陷,胶原沉积,肺纤维化初步形成.血必净组各时间点肺组织病理变化较PQ组轻.②Masson染色：14d时血必净组肺泡炎、肺纤维化程度较PQ组减轻.③电镜下观察：PQ组14d时肺组织线粒体相对少,多数发生变性、肿胀和破坏;可见基膜卷折,肺泡塌陷,间质内胶原纤维增多,纤维化明显,且均较血必净组严重.④HYP含量（μg/g）：PQ组、血必净组3d时肺组织HYP含量即明显高于对照组（743.3±50.2、718.1±34.0比665.8±6.6,均P＜0.05）,随后逐渐升高;但血必净组7d、14d时HYP含量明显低于PQ组（790.5±23.8比876.7±42.0、812.9±72.3比931.3±33.0,均P＜0.05）.⑤Mfn2表达：对照组Mfn2表达量相对较低;PQ组Mfn2表达量随时间延长呈应激性逐渐升高,但升高幅度较小;血必净组1d时Mfn2表达（A值）即明显高于PQ组（0.731±0.035比0.618±0.029,P＜0.05）,并持续高表达,7d时达峰值（0.732±0.037比0.669±0.034,P＜0.05）,而14 d时明显低于PQ组（0.708±0.034比0.765±0.041,P＜0.05）.结论 血必净可减轻PQ中毒引起的肺部炎症反应及肺纤维化程度,其作用机制可能为血必净调节并增加了肺组织中Mfn2的表达.

Objective To investigate the mechanism of pulmonary fibrosis induced by paraquat （PQ）,and the effect of Xuebijing injection in treatment of PQ poisoning.Methods Seventy-two male Wistar rats were randomly divided into control group,PQ poisoning group,and Xuebijing intervention group,with 24 rats in each group.Pulmonary fibrosis was induced by single garage at the dosage of 50 mg/kg of PQ,while 1 mL of distilled water was given by gavage in control group.Xuebijing injection at the dosage of 4 mL/kg were given intraperitoneally at 30 minutes after exposure to PQ in Xuebijing group,and it was repeated every 12 hours; same amount of physiological saline was given intraperitoneally in PQ group and control group.The experiment lasted for 14 days.Six rats in each group were sacrificed on 1,3,7,14 days,respectively,after insult,and 30 minutes after the last intervention.The lung tissues were harvested,the changes in pathology in lung tissue and the degree of pulmonary fibrosis were observed with optical microscope with hematoxylin-eosin （HE） staining and Masson stain.The ultrastructure changes in lung tissues were observed with transmission electron microscopic,and the content of hydroxyproline （HYP） in the lung tissue was determined by alkaline hydrolysis.The expression of mitochondrial fusion protein 2 （Mfn2） was determined by Western Blot.Results ① HE staining：in PQ group,inflammation was most marked on the 3rd day.On the 7th day,exudates in the alveoli started to be organized,and hypertrophic fibroblasts were seen to secrete slim collagen fibers,and fibrosis could be seen in alveoli.On the 14th day,intensive hyperplasia of fibroblasts could be observed,and the alveolar structure was destroyed and collapsed,with deposition of collagen deposited with formation of pulmonary fibrosis.At the same time,pathologic changes were milder in Xuebijing group than those in PQ group.② Masson staining：the degree of inflammation in alveoli and pulmonary fibrosis were less marked in Xuebijing group than those of PQ group on the 14th day.③ Under the transmission electron microscopy,it was found that the mitochondria of lung tissue cells was relatively less in number on the 14th day in PQ group,and the majority of them underwent degeneration,swelling and damage.Basement membrane became folded,alvcoli were collapsed,and fibrosis was obvious.These changes were less serious in Xuebijing group.④ Content of HYP （μg/g）：contents of HYP in lung tissues on the 3rd day in PQ group and Xuebijing group were significantly higher than those in control group （743.3 ± 50.2,718.1 ± 34.0 vs.665.8± 6.6,both P＜0.05）,it then increased gradually,but the contents of HYP in Xuebijing group were significantly lower on the 7th day and 14th day than those in PQ group （790.5 ± 23.8 vs.876.7 ± 42.0,812.9 ± 72.3 vs.931.3 ± 33.0,both P＜0.05）.⑤ Expression of Mfn2：the expression of Mfn2 in control group was relatively lower.The expression of Mfn2 in PQ group was increased gradually under stress,but its rate was low.The expression of Mfn2 （A value） in Xuebijing group was significantly higher than that in PQ group on the 1st day （0.731 ±0.035 vs.0.618 ±0.029,P＜0.05）,and it was elevated steadily,reaching the peak on the 7th day （0.732 ± 0.037 vs.0.669 ± 0.034,P＜0.05）,but it was lower than that of PQ group on the 14th day （0.708 ± 0.034 vs.0.765 ± 0.041,P＜0.05）.Conclusions Xuebijing reduces lung inflammatory reaction and pulmonary fibrosis as a result of PQ poisoning.The mechanism is that Xnebijing regulates and increases expression of Mfn2 in lung tissue.