褪黑素对缺血再灌注损伤的人肝细胞的自噬影响及功能研究

Effect of Melatonin on Autophagy of Ischemia-reperfusion-injured Human Normal Liver Cells and the Function Research

目的:探讨褪黑素对缺血再灌注的人正常肝细胞LO_2自噬的影响以及褪黑素的功能机制。方法:采用MTT法检测细胞的活性;采用Western blot检测LO_2细胞中自噬相关蛋白LC-3Ⅱ、Beclin1的表达情况。结果:MTT结果显示:褪黑素对缺血再灌注损伤的肝细胞有一定的保护作用,促进细胞的存活。Western blot结果显示:缺血再灌注能够上调LO_2细胞的自噬活性,增强细胞内的自噬,缺血再灌注6 h后LC-3Ⅱ和Beclin1的表达迅速上升,并且维持在高自噬水平状态;褪黑素能够在缺血再灌注3 h后显著下调LO_2细胞的自噬活性,降低细胞内的自噬水平,细胞内LC-3Ⅱ和Beclin1在3 h后逐渐下降表达,从而有效降低过度自噬的发生;褪黑素预处理后的缺血再灌注组的自噬活性在24 h较褪黑素治疗组低,预处理对缺血再灌注细胞后期能够起到更好的保护作用。结论:褪黑素能够抑制因缺血再灌注导致的肝细胞内自噬的发生,并且可以防止细胞发生过度自噬,提示褪黑素可以对各种因缺血再灌注导致损伤的肝细胞起到保护作用,以维持细胞内环境的稳态。

To explore the effect of melatonin on autophagy of ischemia-reperfusion-injured LO2cells and its function. Methods: MTT was used to detect the cell activity, and Western bloting was used to detect the expression of autophagy related proteins LC-3Ⅱ and Beclin1 in LO2cells. Results: The MTT results showed that melatonin had a certain protective effect on ischemia -reperfusion injured livercells, and could also promote cell survival. The results of western blot showed that: Ischemia reperfusion could raise the autophagy activity in LO2cells, enhance cell autophagy, 6 h after ischemia-reperfusion, LC-3Ⅱ and Beclin1 were rapidly expressed and autophagy state was maintained at a high level; Melatonincould significantly lower the autophagy activity 3 h after schemia -reperfusion and reduce the level ofautophagy, the expression of LC-3Ⅱ and Beclin1 gradually declined after 3 h, so as to effectively reducedthe excessive autophagy; Pretreatment of melatonin lowered the autophagy activity within 24 h, showed abetter protection effect in the later stage. Conclusion: Melatonin can inhibit ischemia-reperfusion caused autophagy in liver cells and prevent excessive autophagy, which hints that melatonin can protect liver cells from ischemia-reperfusion injury and maintain steady cell environment.