吉林大学学报(医学版) ›› 2024, Vol. 50 ›› Issue (5): 1297-1304.doi: 10.13481/j.1671-587X.20240513

• 基础研究 • 上一篇    

肌肽通过AMPK/mTOR信号通路抑制自噬对氧糖剥夺/复糖复氧诱导星形胶质细胞损伤的保护作用

王宇彤,冉睿黎,边疆,姜晓涵,宋俊秋,王德威,杨菁()   

  1. 锦州医科大学基础医学院生物化学与分子生物学教研室,辽宁 锦州 121001
  • 收稿日期:2023-09-23 出版日期:2024-09-28 发布日期:2024-10-28
  • 通讯作者: 杨菁 E-mail:yangjing@jzmu.edu.cn
  • 作者简介:王宇彤(1996-),女,辽宁省沈阳市人,在读硕士研究生,主要从事生化药物方面的研究。
  • 基金资助:
    辽宁省教育厅面上项目(2021LJKZ0823)

Protective effect of carnosine against oxygen-glucose deprivation/reoxygenation-induced astrocyte injury through inhibition of autophagy by AMPK/mTOR signaling pathway

Yutong WANG,Ruili RAN,Jiang BIAN,Xiaohan JIANG,Junqiu SONG,Dewei WANG,Jing YANG()   

  1. Department of Biochemistry and Molecular Biology,School of Basic Medical Sciences,Jinzhou Medical University,Jinzhou 121001,China
  • Received:2023-09-23 Online:2024-09-28 Published:2024-10-28
  • Contact: Jing YANG E-mail:yangjing@jzmu.edu.cn

摘要:

目的 探讨肌肽(CAR)对氧糖剥夺/再灌注(OGD/R)诱导的星形胶质细胞(AS)损伤的改善作用,并阐明其可能的作用机制。 方法 将AS分为对照组、模型组(OGD/R组)、OGD/R+CAR组(CAR组)和OGD/R+CAR+腺苷酸活化蛋白激酶(AMPK)激活剂AICAR组(CAR+AICAR组)。MTT法和绿菁染色检测各组AS存活率和绿菁染色阳性细胞率,Annexin Ⅴ-FITC/PI法流式细胞术检测各组AS凋亡率,Western blotting法检测各组AS中AMPK、磷酸化AMPK(p-AMPK)、哺乳动物雷帕霉素靶蛋白(mTOR)、磷酸化mTOR(p-mTOR)、微管相关蛋白轻链3B(LC3B)、Beclin-1和P62蛋白表达水平,免疫荧光染色法观察各组AS中LC3B阳性荧光强度。 结果 与对照组比较,OGD/R组AS存活率和绿菁染色阳性细胞率明显降低(P<0.01),AS凋亡率明显升高(P<0.01),AS中p-AMPK/AMPK和LC3BⅡ/LC3BⅠ比值及Beclin-1蛋白表达水平明显升高(P<0.01),p-mTOR/mTOR比值及P62蛋白表达水平明显降低(P<0.01)。与OGD/R组比较,CAR组AS存活率和绿菁染色阳性细胞率明显升高(P<0.01),AS凋亡率明显降低(P<0.01),AS中p-AMPK/AMPK和LC3B Ⅱ/LC3BⅠ比值及Beclin-1蛋白表达水平明显降低(P<0.01),p-mTOR/mTOR比值和p62蛋白表达水平明显升高(P<0.01)。与CAR组比较,CAR+AICAR组AS存活率和绿菁染色阳性细胞率明显降低(P<0.01),AS凋亡率升高(P<0.01)、AS中p-AMPK/AMPK和LC3BⅡ/LC3BⅠ比值及Beclin-1蛋白表达水平明显升高(P<0.01),p-mTOR/mTOR比值和P62蛋白表达水平明显降低(P<0.01)。LC3B免疫荧光染色结果与Western blotting法检测结果趋势一致。 结论 CAR对OGD/R所致AS损伤具有保护作用,其作用机制可能与抑制AMPK/mTOR信号途径,进而抑制细胞自噬有关。

关键词: 肌肽, 自噬, 星形胶质细胞, 腺苷酸活化蛋白激酶, 哺乳动物雷帕霉素靶蛋白

Abstract:

Objective To discuss the protective effect of carnosine(CAR) against oxygen-glucose deprivation/reoxygenation(OGD/R)-induced astrocyte(AS) injury,and to clarify its possible mechanism. Methods The AS were divided into control group, model group (OGD/R group), OGD/R+CAR group (CAR group), and OGD/R+CAR+AMP-activated protein kinase (AMPK) activator AICAR group(CAR+AICAR group). MTT assay and green cyanine staining method were used to detect the survival rates and green cyanine staining positive rates of the AS in various groups; Annexin Ⅴ-FITC/PI method and flow cytometry were used to detect the apoptotic rates of the AS in various groups; Western blotting method was used to detect the expression levels of AMPK, phosphorylated AMPK (p-AMPK), mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), microtubule-associated protein light chain 3B(LC3B), Beclin-1, and P62 proteins in the AS in various groups; immunofluorescence staining was used to observe the LC3B positive fluorescence intensities in the AS in various groups. Results Compared with control group, the survival rate and green cyanine staining positive rate of the AS in OGD/R group were decreased (P<0.01), the apoptotic rate of the AS was increased (P<0.01), the ratios of p-AMPK/AMPK and LC3BⅡ/LC3BⅠ and the expression level of Beclin-1 protein were increased (P<0.01), and the ratio of p-mTOR/mTOR and the expression level of P62 protein were decreased (P<0.01). Compared with OGD/R group, the survival rate and green cyanine staining positive rate of the AS in CAR group were increased (P<0.01), the apoptotic rate of the AS was decreased (P<0.01), the ratios of p-AMPK/AMPK and LC3B Ⅱ/LC3BⅠ and the expression level of Beclin-1 protein were decreased (P<0.01), and the ratio of p-mTOR/mTOR and the expression level of P62 protein were increased (P<0.01). Compared with CAR group, the survival rate and green cyanine staining positive rate of the AS in CAR+AICAR group were decreased (P<0.01), the apoptotic rate of the AS was increased (P<0.01), the ratios of p-AMPK/AMPK and LC3BⅡ/LC3BⅠ and the expression level of Beclin-1 protein were increased (P<0.01), and the ratio of p-mTOR/mTOR and the expression level of P62 protein were decreased (P<0.01). The LC3B immunofluorescence staining results were consistent with the Western blotting results. Conclusion CAR has the protective effect on injury of the AS induced by OGD/R, and its molecular mechanism may be related to the inhibition of the AMPK/mTOR signaling pathway, thereby inhibiting autophagy.

Key words: Carnosine, Autophagy, Astrocyte, Adenosine monophosphate activated protein kinase, Mammalian target of rapamycin

中图分类号: 

  • R963