Overexpression of TRPV1 activates autophagy in human lens epithelial cells under hyperosmotic stress through Ca2+-dependent AMPK/mTOR pathway
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Pei-Quan Zhao. Department of Ophthalmology, Shanghai Xinhua Hospital Affiliated with Shanghai Jiao Tong University School of Medicine, Shanghai 200120, China. zhaopeiquan@xinhuamed.com.cn; Hai-Ying Jin. Department of Ophthalmology, Shanghai East Hospital Affiliated with Shanghai Tongji University School of Medicine, Shanghai 200120, China. eagle_jin@163.com

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Supported by the National Natural Science Foundation of China (No.82201199 to Huang LH, No.82171069 to Zhao PQ; No.81770964 to Zhao PQ), Melbourne Research Scholarship (Huang LH), the Shanghai Clinical Research Plan of Shenkang Hospital Development Center (No.SHDC2020CR4081 to Jin HY); the Shanghai Science and Technology Innovation Fund (No.20Y11910900 to Jin HY).

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    Abstract:

    AIM: To explore whether autophagy functions as a cellular adaptation mechanism in lens epithelial cells (LECs) under hyperosmotic stress. METHODS: LECs were treated with hyperosmotic stress at the concentration of 270, 300, 400, 500, or 600 mOsm for 6, 12, 18, 24h in vitro. Polymerase chain reaction (PCR) was employed for the mRNA expression of autophagy-related genes, while Western blotting detected the targeted protein expression. The transfection of stub-RFP-sens-GFP-LC3 autophagy-related double fluorescence lentivirus was conducted to detect the level of autophagy flux. Scanning electron microscopy was used to detect the existence of autolysosome. Short interfering RNA of autophagy-related gene (ATG) 7, transient receptor potential vanilloid (TRPV) 1 overexpression plasmid, related agonists and inhibitors were employed to their influence on autophagy related pathway. Flow cytometry was employed to test the apoptosis and intracellular Ca2+ level. Mitochondrial membrane potential was measured by JC-1 staining. The cell counting kit-8 assay was used to calculate the cellular viability. The wound healing assay was used to evaluate the wound closure rate. GraphPad 6.0 software was utilized to evaluate the data. RESULTS: The hyperosmotic stress activated autophagy in a pressure- and time-dependent manner in LECs. Beclin 1 protein expression and conversion of LC3B II to LC3B I increased, whereas sequestosome-1 (SQSTM1) protein expression decreased. Transient Ca2+ influx was stimulated caused by hyperosmotic stress, levels of mammalian target of rapamycin (mTOR) phosphorylation decreased, and the level of AMP-activated protein kinase (AMPK) phosphorylation increased in the early stage. Based on this evidence, autophagy activation through the Ca2+-dependent AMPK/mTOR pathway might represent an adaptation process in LECs under hyperosmotic stress. Hyperosmotic stress decreased cellular viability and accelerated apoptosis in LECs and cellular migration decreased. Inhibition of autophagy by ATG7 knockdown had similar results. TRPV1 overexpression increased autophagy and might be crucial in the occurrence of autophagy promoted by hyperosmotic stress. CONCLUSION: A combination of hyperosmotic stress and autophagy inhibition may be a promising approach to decrease the number of LECs in the capsular bag and pave the way for improving prevention of posterior capsular opacification and capsular fibrosis.

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Liu-Hui Huang, Jiao Lyu, Sheng Chen, et al. Overexpression of TRPV1 activates autophagy in human lens epithelial cells under hyperosmotic stress through Ca2+-dependent AMPK/mTOR pathway. Int J Ophthalmol, 2024,17(3):420-434

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Publication History
  • Received:August 14,2023
  • Revised:December 20,2023
  • Adopted:
  • Online: February 27,2024
  • Published: