人参皂苷Rg_1对LPS诱导的肺上皮细胞凋亡和自噬的影响
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摘要
为了研究人参皂苷Rg_1对内毒素(LPS)导致的肺上皮细胞凋亡的保护作用及其作用机制。首先用LPS处理小鼠肺上皮细胞(MLE-12),观察其自噬变化和凋亡情况与LPS作用的时间、作用浓度关系。然后选择在特定LPS浓度和作用时间下调控肺上皮细胞自噬水平,观察肺上皮细胞凋亡的变化。其次选择相同的LPS浓度和作用时间,同时分别加入人参皂苷Rg_1,自噬抑制剂3-MA,将肺上皮细胞分组,观察人参皂苷Rg_1对LPS导致的肺上皮细胞凋亡的影响及其作用机制。动物实验中将小鼠分组,分别通过检测凋亡蛋白,肺损伤评分及HE染色切片,在体内验证人参皂苷Rg_1对LPS导致的肺损伤是否具有保护作用。结果显示,LPS作用12 h,随着浓度增加,凋亡增加,自噬也逐渐增加。25 g·L-1LPS处理不同时间组,凋亡逐渐增加,自噬先增加后降低。LPS组的凋亡较对照组增加,LPS+3-MA组凋亡进一步增加,而LPS+RAM(雷帕霉素,自噬促进剂)组凋亡明显下降。LPS组自噬增加,LPS+3-MA组自噬下降,LPS+RAM组自噬增加。LPS组凋亡较对照组升高,LPS+Rg_1组凋亡下降,而LPS+Rg_1+3-MA组凋亡再次升高,自噬在LPS组增加,给予人参皂苷Rg_1后进一步增加,而给予3-MA后下降。小鼠体内实验显示,LPS组凋亡较对照组显著增加,LPS+人参皂苷Rg_1组下降,肺损伤评分及HE染色也符合上述趋势。LPS能够导致肺上皮细胞的凋亡具有一定的时间浓度依赖性,肺上皮细胞的自噬随LPS浓度的增加而增加,在LPS特定浓度下,随时间的延长,自噬先增加,在12～16 h后开始降低。特定时间内适当提高肺上皮细胞的自噬可以减少LPS导致的凋亡,而抑制其自噬,凋亡增加。人参皂苷Rg_1通过提高自噬对LPS导致的肺上皮细胞的凋亡具有保护作用。
This paper aimed to study the protective effect of ginsenoside Rg_1 on endotoxin(LPS)-induced apoptosis of lung epithelial cells and its mechanism of action. Mouse lung epithelial cells(MLE-12) were first treated with LPS. The autophagy changes and apoptosis and the relationship with concentration and time of LPS were observed. Then,the level of autophagy in MLE-12 was regulated at a specific concentration and action time of LPS,and the changes of apoptosis were observed. Secondly,ginsenoside Rg_1 and autophagy inhibitor 3-MA were added respectively at the same concentration and action time of LPS. The lung epithelial cells were grouped to observe the effect of ginsenoside Rg_1 on LPS-induced apoptosis of lung epithelial cells and its mechanism. In the animal experiment,the mice were grouped and tested by apoptosis protein,lung injury score and HE staining section to verify whether ginsenoside Rg_1 has a protective effect on LPS-induced lung injury. The results showed that apoptosis and autophagy increased as the rise of concentration after treatment with LPS for 12 h. The apoptosis increased gradually,and the autophagy increased first and then decreased over time at the LPS concentration of 25 g·L-1. The apoptosis of LPS group was higher than that of control group,and LPS+3-MA group increased further,while apoptosis decreased significantly in LPS+RAM(rapamycin,autophagy promoter) group. The autophagy increased in LPS group,decreased in LPS+3-MA group and increased in LPS+RAM group. The apoptosis of LPS group was higher than that of control group,and the apoptosis of LPS+Rg_1 group decreased. The apoptosis of LPS+Rg_1+3-MA group increased again. The autophagy of LPS group further increased after administration of ginsenoside Rg_1,but decreased after administration of 3-MA. In the in vivo experiments in mice,the apoptosis of LPS group increased significantly compared with the control group,while LPS + ginsenoside Rg_1 group decreased. Lung injury score and HE staining also conformed to the above trend. LPS can induce the apoptosis of lung epithelial cells in a time-dependent and concentration-dependent manner. The autophagy of lung epithelial cells increases with the rise of LPS concentration. At the specific concentration of LPS,autophagy increases first and then decreases after 12-16 hours. Proper increase of autophagy in lung epithelial cells within a certain period of time can reduce the apoptosis induced by LPS,while inhibition of autophagy can increase apoptosis. Ginsenoside Rg_1 has a protective effect on lung cancer epithelial cell apoptosis induced by autophagy.
This paper aimed to study the protective effect of ginsenoside Rg_1 on endotoxin(LPS)-induced apoptosis of lung epithelial cells and its mechanism of action. Mouse lung epithelial cells(MLE-12) were first treated with LPS. The autophagy changes and apoptosis and the relationship with concentration and time of LPS were observed. Then,the level of autophagy in MLE-12 was regulated at a specific concentration and action time of LPS,and the changes of apoptosis were observed. Secondly,ginsenoside Rg_1 and autophagy inhibitor 3-MA were added respectively at the same concentration and action time of LPS. The lung epithelial cells were grouped to observe the effect of ginsenoside Rg_1 on LPS-induced apoptosis of lung epithelial cells and its mechanism. In the animal experiment,the mice were grouped and tested by apoptosis protein,lung injury score and HE staining section to verify whether ginsenoside Rg_1 has a protective effect on LPS-induced lung injury. The results showed that apoptosis and autophagy increased as the rise of concentration after treatment with LPS for 12 h. The apoptosis increased gradually,and the autophagy increased first and then decreased over time at the LPS concentration of 25 g·L-1. The apoptosis of LPS group was higher than that of control group,and LPS+3-MA group increased further,while apoptosis decreased significantly in LPS+RAM(rapamycin,autophagy promoter) group. The autophagy increased in LPS group,decreased in LPS+3-MA group and increased in LPS+RAM group. The apoptosis of LPS group was higher than that of control group,and the apoptosis of LPS+Rg_1 group decreased. The apoptosis of LPS+Rg_1+3-MA group increased again. The autophagy of LPS group further increased after administration of ginsenoside Rg_1,but decreased after administration of 3-MA. In the in vivo experiments in mice,the apoptosis of LPS group increased significantly compared with the control group,while LPS + ginsenoside Rg_1 group decreased. Lung injury score and HE staining also conformed to the above trend. LPS can induce the apoptosis of lung epithelial cells in a time-dependent and concentration-dependent manner. The autophagy of lung epithelial cells increases with the rise of LPS concentration. At the specific concentration of LPS,autophagy increases first and then decreases after 12-16 hours. Proper increase of autophagy in lung epithelial cells within a certain period of time can reduce the apoptosis induced by LPS,while inhibition of autophagy can increase apoptosis. Ginsenoside Rg_1 has a protective effect on lung cancer epithelial cell apoptosis induced by autophagy.
引文
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[24] Hou Y C,Chiu W C,Yeh C L,et al. Glutamine modulates lipopolysaccharide-induced activation of NF-κB via the Akt/m TOR pathway in lung epithelial cells[J]. Am J Physiol Lung Cell Mol Physiol,2012,302(1):L174.
[2] Sandrock C E,Albertson T E. Controversies in the treatment of sepsis[J]. Semin Respir Crit Care Med,2010,31:66.
[3] Dellinger R P,Levy M M,Rhodes A,et al. Surviving sepsis campaign:international guidelines for management of severe sepsis and septic shock:2012[J]. Crit Care Med,2013,41:580.
[4] Ware L B,Matthay M A. Alveolar fluid clearance is impaired in the majority of patients with acute lung injury and the acute respiratory distress syndrome[J]. Am J Resp Crit Care Med,2001,163(6):1376.
[5] King E G,BauzáG J,Mella J R,et al. Pathophysiologic mechanisms in septic shock[J]. Lab Invest,2014,94:4.
[6] Deutschman C S,Tracey K J. Sepsis:current dogma and new perspectives[J]. Immunity,2014,40:463.
[7] Perl M,Lomas-Neira J,Chung C S. Epithelial cell apoptosis and neutrophil recruitment in acute lung injury--a unifying hypothesis? What we have learned from small interfering RNAs[J]. Mol Med,2008,14(7/8):465.
[8] Tao W,Su Q,Wang H,et al. Platycodin D attenuates acute lung injury by suppressing apoptosis and inflammation in vivo and in vitro[J]. Int Immunopharmacol,2015,27(1):138.
[9] Hale A N,Ledbetter D J,Gawriluk T R,et al. Autophagy:regulation and role in development[J]. Autophagy,2013,9:951.
[10] Ryter S W,Choi A M K. Autophagy in lung disease pathogenesis and therapeutics[J]. Redox Biol,2015,4(147):215.
[11] Liu F,Nie C,Zhao N,et al. MiR-155 alleviates septic lung injury by inducing autophagy via inhibition of transforming growth factor-β-activated binding protein 2[J]. Shock,2017,48(1):61.
[12] Li S,Guo L,Qian P,et al. Lipopolysaccharide induces autophagic cell death through the PERK-dependent branch of the unfolded protein response in human alveolar epithelial A549 cells[J].Cell Physiol Biochem,2015,36(6):2403.
[13] Ni X J,Xu Z Q,Jin H,et al. Ginsenoside Rg1protects human renal tubular epithelial cells from lipopolysaccharide-induced apoptosis and inflammation damage[J]. Braz J Med Biol Res,2017,51(2):e6611.
[14] Bao S,Zou Y,Wang B,et al. Ginsenoside Rg1improves lipopolysaccharide-induced acute lung injury by inhibiting inflammatory responses and modulating infiltration of M2 macrophages[J]. Int Immunopharmacol,2015,28(1):429.
[15] Kim T W,Joh E H,Kim B,et al. Ginsenoside Rg5ameliorates lung inflammation in mice by inhibiting the binding of LPS to toll-like receptor-4 on macrophages[J]. Int Immunopharmacol,2012,12(1):110.
[16] Cao Y,Zhang D,Moon H G,et al. MiR-15a/16 regulates apoptosis of lung epithelial cells after oxidative stress[J]. Mol Med,2016,22:233.
[17] Yang L,Li D,Zhuo Y,et al. Protective role of liriodendrin in sepsis-induced acute lung injury[J]. Inflammation,2016,39(5):1805.
[18] Jones S A,Mills K H,Harris J. Autophagy and inflammatory diseases[J]. Immunol Cell Biol,2013,91(3):250.
[19] Tatsuya Saitoh,Naonobu Fujita,Myoung Ho Jang,et al. Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1b production[J]. Nature,2008,456(7219):264.
[20]赵国桢,郭玉红,李博,等.中医药防治脓毒症的研究进展[J].中国中药杂志,2017,42(8):1423.
[21]赵滋苗,蔡宛如,陆军,等.大黄素对脓毒症大鼠血糖和胰岛素抵抗的影响[J].中国现代应用药学,2016,33(9):1115.
[22]徐煜彬,张洪伟,陈赛贞,等.巴马汀抗脓毒症潜在靶点的筛选与鉴定[J].中国现代应用药学,2018,35(11):1602.
[23] Sun R,Li Y,Chen W,et al. Total ginsenosides synergize with ulinastatin against septic acute lung injury and acute respiratory distress syndrome[J]. Int J Clin Exp Pathol,2015,8(6):7385.
[24] Hou Y C,Chiu W C,Yeh C L,et al. Glutamine modulates lipopolysaccharide-induced activation of NF-κB via the Akt/m TOR pathway in lung epithelial cells[J]. Am J Physiol Lung Cell Mol Physiol,2012,302(1):L174.