Objective To investigate the mechanism of naringin (NAR) on retinal microvascular endothelial cells (HRMECs) injury based on adenosine-monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway.
Methods HRMECs were randomly divided into control group, high glucose (HG) group, HG+NAR group (3 mg/L NAR), HG+activator (AICAR) group (1 mmol/L AICAR), and HG+NAR+AICAR group (3 mg/L NAR+1 mmol/L AICAR); the control group was treated with 5 mmol/L D-glucose added to the culture medium, while the other groups were treated with 30 mmol/L D-glucose added to the culture medium. CCK-8 and Transwell were used to detect cell proliferation and migration respectively; enzyme-linked immunosorbent assay (ELISA) was applied to detect the levels of interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) in the supernatant; quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was applied to detect the expression levels of autophagy factors LC3 mRNA and p62 mRNA; Western blot was applied to detect the AMPK/mTOR pathway and expression levels of autophagy-related proteins.
Results Compared with the control group, the cell viability, the number of migrating cells, levels of IL-1, IL-6 and TNF-α, and p-AMPK/AMPK, LC3Ⅱ/LC3Ⅰ as well as LC3 mRNA expression increased in the HG group, while the p-mTOR/mTOR, p62 protein and p62 mRNA expression decreased (P < 0.05); compared with the HG group, the cell viability, the number of migrating cells, levels of IL-1, IL-6 and TNF-α, and p-AMPK/AMPK, LC3Ⅱ/LC3Ⅰ as well as LC3 mRNA expression decreased in the HG+NAR group, while the p-mTOR/mTOR, p62 protein and p62 mRNA expression increased, but the cell viability, the number of migrating cells, levels of IL-1, IL-6 and TNF-α, p-AMPK/AMPK and LC3Ⅱ/LC3Ⅰ expression increased in the HG+AICAR group, while the p-mTOR/mTOR, p62 protein and p62 mRNA expression decreased (P < 0.05); compared with the HG+NAR group, the cell viability, the number of migrating cells, levels of IL-1, IL-6 and TNF-α, and p-AMPK/AMPK, LC3Ⅱ/LC3Ⅰ as well as LC3 mRNA expression increased in the HG+NAR+AICAR group, while the p-mTOR/mTOR, p62 protein and p62 mRNA expression decreased (P < 0.05); compared with the HG+AICAR group, the cell viability, the number of migrating cells, levels of IL-1, IL-6 and TNF-α, and p-AMPK/AMPK, LC3Ⅱ/LC3Ⅰ as well as LC3 mRNA expression significantly decreased in the HG+NAR+AICAR group, while the p-mTOR/mTOR, p62 protein and p62 mRNA expression increased (P < 0.05).
Conclusion NAR can alleviate HG induced injury to HRMECs, and its mechanism may be related to the inhibition of AMPK/mTOR pathway mediated autophagy.
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