炎调方通过Fas/FasL通路抑制ATⅡ细胞凋亡的研究
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摘要
目的体外观察炎调方通过Fas/FasL通路延缓LPS诱导II型肺泡上皮细胞(ATII)凋亡的作用。方法原代分离与培养ATII细胞,运用脂多糖(LPS)干预ATII进行体外ALI细胞造模,结合药物血清技术,观察炎调方对ATII细胞凋亡的影响。Hoechst 33258荧光染色观察细胞凋亡形态,流式细胞仪检测ATII细胞凋亡率,Western blot方法检测Fas、FasL、caspase-8、caspase-3表达。结果 LPS降低了ATII细胞存活率,炎调方对LPS诱导的细胞凋亡有显著的抑制作用;LPS能明显激活caspase-8和caspase-3活性,提高Fas和FasL的水平;炎调方能减弱caspase-8和caspase-3活性,降低Fas和FasL表达。结论炎调方能延缓ATII凋亡,其机制可能与抑制Fas/FasL通路活性有关。
Objective To observe the effect of Yantiao Fang on ATII apoptosis through Fas/FasL pathway in vitro. Methods II type alveolar epithelial cells(ATII) cells are primary isolated and cultured. ATII cells are treated with LPS for the modeling of ALI cells in vitro. With the application of drug serum technique, the effect of Yantiao Fang on apoptosis of ATII cells is observed. Hoechst 33258 fluorescence staining is used to observe the morphology of cell apoptosis. Flow cytometry is used to detect apoptosis rate of apoptotic cells in ATII cells. The expressions of Fas, FasL, caspase-8 and caspase-3 are observed by Western blot analysis. Results The results show that Yantiao Fang could improve cell death induced by LPS and significantly inhibit the apoptosis induced by LPS. The activity of caspase-8 and caspase-3 are activated and the levels of Fas and FasL are improved significantly by LPS. Cell survival of ATII is inhibited by LPS. Yantiao Fang decrease the apoptosis of AT II cells induced by LPS. It inhibited the activity of caspase-8 and caspase-3 and reduced the level of Fas and FasL. Conclusion The mechanisms of Yantiao Fang decreased ATII apoptosis lunduced by LPS may be related to Fas/FasL pathway.
Objective To observe the effect of Yantiao Fang on ATII apoptosis through Fas/FasL pathway in vitro. Methods II type alveolar epithelial cells(ATII) cells are primary isolated and cultured. ATII cells are treated with LPS for the modeling of ALI cells in vitro. With the application of drug serum technique, the effect of Yantiao Fang on apoptosis of ATII cells is observed. Hoechst 33258 fluorescence staining is used to observe the morphology of cell apoptosis. Flow cytometry is used to detect apoptosis rate of apoptotic cells in ATII cells. The expressions of Fas, FasL, caspase-8 and caspase-3 are observed by Western blot analysis. Results The results show that Yantiao Fang could improve cell death induced by LPS and significantly inhibit the apoptosis induced by LPS. The activity of caspase-8 and caspase-3 are activated and the levels of Fas and FasL are improved significantly by LPS. Cell survival of ATII is inhibited by LPS. Yantiao Fang decrease the apoptosis of AT II cells induced by LPS. It inhibited the activity of caspase-8 and caspase-3 and reduced the level of Fas and FasL. Conclusion The mechanisms of Yantiao Fang decreased ATII apoptosis lunduced by LPS may be related to Fas/FasL pathway.
引文
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[10]沈晓红,何淼,熊旭东,等.炎调方对脓毒症大鼠肺热休克蛋白70和核因子-κB的调控作用[J].上海中医药大学学报, 2013, 27(3):69-72.
[11]施荣,熊旭东,李淑芳,等.炎调方对脓毒症大鼠血清炎症因子的调控作用[J].中国中医急症, 2012, 21(3):397-398.
[12]Takano K, Oishi H, Hattori Y. Protection from pulmonary apoptosis:a new therapeutic choice for septic acute lung injury[J]. Nihon Yakurigaku Zasshi,2011, 138(4):146-50.
[13] Song L, Zhou F, Cheng L, et al. MicroRNA-34a Suppresses Autophagy in Alveolar Type II Epithelial Cells in Acute Lung Injury by Inhibiting FoxO3Expression[J]. Inflammation, 2017, 40(3):927-936.
[14]White L E, Cui Y, Shelak C M, et al. Lung endothelial cell apoptosis during ischemic acute kidney injury[J]. Shock, 2012, 38(3):320-327.
[15]施荣,王倩,韩丹,等.炎调方抑制LPS诱导人肺泡上皮细胞炎症因子释放机制研究[J].中国中医急症, 2016, 25(4):572-574.
[2]Bai J, Tang L, Loma-Neira J, et al. TAT-SNAP-23 treatment inhibits the priming of neutrophil functions contributing to shock and/or sepsis-induced extra-pulmonary acute lung injury[J]. Innate Immun, 2015, 21(1):42-54.
[3]Messer M P, Kellermann P, Weber S J, et al. Silencing of fas, fas-associated via death domain, or caspase 3 differentially affects lung inflammation,apoptosis, and development of trauma-induced septic acute lung injury[J].Shock, 2013, 39(1):19-27.
[4]Galani V, Tatsaki E, Bai M, et al. The role of apoptosis in the pathophysiology of Acute Respiratory Distress Syndrome(ARDS):an up-to-date cell-specific review[J]. Pathol Res Pract, 2010, 206(3):145-150.
[5]Gong Y, Lan H, Yu Z, et al. Blockage of glycolysis by targeting PFKFB3alleviates sepsis-related acute lung injury via suppressing inflammation and apoptosis of alveolar epithelial cells[J]. Biochem Biophys Res Commun, 2017,491(2):522-529.
[6]Shao L, Meng D, Yang F, et al. Irisin-mediated protective effect on LPSinduced acute lung injury via suppressing inflammation and apoptosis of alveolar epithelial cells[J]. Biochem Biophys Res Commun, 2017, 487(2):194-200.
[7]施荣,熊旭东.通腑清营法治疗脓毒症热毒内盛证临床观察[J].中国中医急症, 2011, 20(4):521-522.
[8]张涛,熊旭东,李淑芳.通腑泻肺方治疗肺系脓毒症(痰热壅肺证)的临床研究[J].中国中医急症, 2014, 23(10):1788-1790, 1805.
[9]何淼,熊旭东,沈晓红.炎调方对脓毒症急性呼吸窘迫综合征(热炽营血证)患者血细胞因子的影响[J].中国中医急症, 2013, 22(7):1097-1099.
[10]沈晓红,何淼,熊旭东,等.炎调方对脓毒症大鼠肺热休克蛋白70和核因子-κB的调控作用[J].上海中医药大学学报, 2013, 27(3):69-72.
[11]施荣,熊旭东,李淑芳,等.炎调方对脓毒症大鼠血清炎症因子的调控作用[J].中国中医急症, 2012, 21(3):397-398.
[12]Takano K, Oishi H, Hattori Y. Protection from pulmonary apoptosis:a new therapeutic choice for septic acute lung injury[J]. Nihon Yakurigaku Zasshi,2011, 138(4):146-50.
[13] Song L, Zhou F, Cheng L, et al. MicroRNA-34a Suppresses Autophagy in Alveolar Type II Epithelial Cells in Acute Lung Injury by Inhibiting FoxO3Expression[J]. Inflammation, 2017, 40(3):927-936.
[14]White L E, Cui Y, Shelak C M, et al. Lung endothelial cell apoptosis during ischemic acute kidney injury[J]. Shock, 2012, 38(3):320-327.
[15]施荣,王倩,韩丹,等.炎调方抑制LPS诱导人肺泡上皮细胞炎症因子释放机制研究[J].中国中医急症, 2016, 25(4):572-574.