Autophagy has an early protective effect against fibroblast injury induced by advanced glycation end products

doi:10.3969/j.issn.2095-4344.2921

Abstract

Abstract:

BACKGROUND: Advanced glycation end products (AGEs) are closely related to diabetic wound healing. AGEs can affect the proliferation and migration of fibroblasts, but the early protective effect of autophagy in AGE-induced fibroblast injury has not been explored.

OBJECTIVE: To confirm the effect of AGEs on fibroblast cell viability and autophagy, and to explore the early protective effect of autophagy on cell injury.

METHODS: Human fibroblasts were cultured in vitro. Normal control group (DMEM medium), AGE group (DMEM medium+100 mg/L AGEs), AGE+PTB (AGE inhibitor) group (DMEM medium+100 mg/L AGEs+100 mg/L PTB), and AGE+chloroquine (autophagy inhibitor) group (DMEM medium+100 mg/L AGEs+10 μmol/L chloroquine) were established. After 48 hours of culture, trypan blue staining was used to detect the cell viability of each group, western blot was used to detect the expression of LC3 and P62 protein, cell immunofluorescence assay was used to detect the formation of autophagy using, and transmission electron microscopy was used to observe the ultrastructure of autophagy vesicles.

RESULTS AND CONCLUSION: Compared with the control group, cell death rate in the AGE group increased significantly (P < 0.01). Compared with the AGE group, the cell death rate in the AGE+PTB group decreased significantly (P < 0.05), while that in the AGE+chloroquine group increased significantly (P < 0.05). Compared with the control group, the ratio of LC3 II/LC3 I in the AGE group increased significantly, and the expression of P 62 decreased significantly (P < 0.01); compared with the AGE group, the ratio of LC3 II/LC3 I in the AGE+PTB group decreased significantly, the expression of P62 increased significantly (P < 0.05), whereas the content of LC3 II/LC3 I in the AGE+chloroquine group did not change significantly (P > 0.05) and the expression of P62 increased significantly (P < 0.05). Both in the AGE group and AGE+chloroquine group, the green fluorescent dot-like aggregation of LC3 was increased. By comparing these two groups, there were more autophagy bodies and autophagy vesicles in the AGE group, whereas the number of autophagy structures and vacuole like structures were less in the AGE+chloroquine group. These findings indicate that AGEs could damage fibroblasts in vitro, reduce cell survival rate, and activate autophagy activity in an early stage, and moreover induced autophagy could certainly protect fibroblasts.

Key words: cell, fibroblast, advanced glycation end products, autophagy, PTB, chloroquine, cell viability, immune, wound, experiment

CLC Number:

Han Yanfu, Tao Ran, Sun Tianjun. Autophagy has an early protective effect against fibroblast injury induced by advanced glycation end products[J]. Chinese Journal of Tissue Engineering Research, 2020, 24(35): 5619-5624.

Figures/Tables 10

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