铁皮石斛多糖对缺氧/复氧诱导星形胶质细胞AMPK/ULK1通路相关自噬的影响

Effect of Dendrobium Officinale Polysaccharides on Autophagy Related to AMPK/ULK1 Pathway Induced by Hypoxia/Reoxygenation in Astrocytes

关键词:  铁皮石斛多糖  缺氧/复氧  星形胶质细胞  AMPK/ULK1通路  自噬

Key words:  Dendrobium officinale polysaccharides  hypoxia/reoxygenation  astrocytes  AMPK/ULK1 pathway  autophagy

摘要:

目的 探讨铁皮石斛多糖对缺氧/复氧（hypoxia/reoxygenation，H/R）诱导星形胶质细胞腺苷酸激活蛋白激酶（adenosine monophosphate-activated protein kinase，AMPK）/UNC-51类似自噬激活激酶1（UNC-51 like autophagy activating kinase 1，ULK1）通路相关自噬的影响。方法 体外培养人星形胶质细胞，分为对照组、H/R组（H/R建模）、低浓度铁皮石斛多糖组（H/R建模+100 μg·mL^-1铁皮石斛多糖）、中浓度铁皮石斛多糖组（H/R建模+200 μg·mL^-1铁皮石斛多糖）和高浓度铁皮石斛多糖组（H/R建模+400 μg·mL^-1铁皮石斛多糖）。MTT法检测细胞增殖情况；流式细胞仪检测细胞凋亡情况；试剂盒测定细胞中丙二醛（malondialdehyde，MDA）、超氧化物歧化酶（superoxide dismutase，SOD）水平；Western blotting检测细胞中p-AMPK、AMPK、p-ULK1、ULK1、Beclin1、微管相关蛋白1轻链3（light chain 3，LC3） I、LC3 Ⅱ蛋白表达情况。结果 与对照组相比，H/R组星形胶质细胞存活率、细胞中SOD水平及LC3 I/LC3 Ⅱ蛋白表达水平显著降低（P<0.05），细胞凋亡率、细胞中MDA水平及p-AMPK/AMPK、p-ULK1/ULK1、Beclin1蛋白表达水平显著升高（P<0.05）；随着铁皮石斛多糖的处理及处理浓度的升高，星形胶质细胞存活率、细胞中SOD水平及LC3 I/LC3 Ⅱ蛋白表达水平显著升高（P<0.05），细胞凋亡率、细胞中MDA水平及p-AMPK/AMPK、p-ULK1/ULK1、Beclin1蛋白表达水平显著降低（P<0.05）。结论 铁皮石斛多糖对H/R诱导的星形胶质细胞AMPK/ULK1通路激活及自噬具有抑制作用，可促进细胞存活并减少细胞凋亡。

Abstract:

OBJECTIVE To explore the effect of Dendrobium officinale polysaccharides on autophagy related to adenosine monophosphate-activated protein kinase(AMPK)/UNC-51 like autophagy activating kinase 1 pathway(ULK1) pathway induced by hypoxia/reoxygenation(H/R) in astrocytes. METHODS Human astrocytes were cultured in vitro and divided into control group, H/R group(H/R modeling), low concentration Dendrobium officinale polysaccharide group(H/R modeling+100 μg·mL^-1 Dendrobium officinale polysaccharide), medium concentration Dendrobium officinale polysaccharide group(H/R modeling+ 200 μg·mL^-1 Dendrobium officinale polysaccharide) and high concentration Dendrobium officinale polysaccharide group(H/R modeling+400 μg·mL^-1 Dendrobium officinale polysaccharide). MTT assay was used to detect cell proliferation. Flow cytometry was used to detect apoptosis. The kit was used to determine the levels of malondialdehyde(MDA) and superoxide dismutase(SOD) in cells. Western blotting was used to detect the expression of p-AMPK, AMPK, p-ULK1, ULK1, Beclin1, microtubule-associated protein 1 light chain 3(LC3) I and LC3 Ⅱ proteins. RESULTS Compared with control group, the survival rate of astrocytes, the level of SOD and the protein expression level of LC3 I/LC3 Ⅱ were significantly lower in H/R group(P<0.05), the apoptosis rate, the level of MDA and the protein expression levels of p-AMPK/AMPK, p-ULK1/ULK1 and Beclin1 were significantly higher(P<0.05); with the treatment of Dendrobium officinale polysaccharide and the increase of its concentration, the survival rate of astrocytes, the level of SOD and the protein expression level of LC3 I/LC3 Ⅱ were significantly increased(P<0.05), the apoptosis rate, the level of MDA and the protein expression levels of p-AMPK/AMPK, p-ULK1/ULK1 and Beclin1 were significantly decreased(P<0.05). CONCLUSION Dendrobium officinale polysaccharides can inhibit the activation of AMPK/ULK1 pathway and autophagy of astrocytes induced by H/R, which can promote cell survival and reduce cell apoptosis.