Timosaponin B-II reduces necroptosis of VSC4.1 cells induced by oxygen-glucose deprivation

doi:10.3969/j.issn.2095-4344.1777

Abstract

Abstract:

BACKGROUND: Timosaponin B-II has neuroprotective effects and reduces necroptosis of cells, but its mechanism is still unknown and needs further study.
OBJECTIVE: To study the mechanism by which the extract of timosaponin B-II of Anemarrhena asphodeloides on necroptosis of VSC4.1 cells induced by oxygen-glucose deprivation.
METHODS: Firstly, VSC4.1 cells in good growth condition were cultured in six groups: group A as normal control; group B with hydrogen peroxide solution for 24 hours; groups C-F with 1, 10, 100, 1 000 μmol/L timosaponin B-II solution for 24 hours, respectively, and then each group was cultured with hydrogen peroxide solution for another 24 hours. MTT assay was used to detect the cell survival rate, and the drug concentration of the group with the highest cell survival rate was selected for the following experiments. Secondly, VSC4.1 cells were cultured in three groups: routine culture in normal control group; anaerobic hypoglycemic culture for 8 hours followed by reoxygenation and hyperglycemic culture for 6 or 12 hours in model group; necrostatin-1, a necroptosis inhibitor, was added in inhibitor group for 24 hours, and anaerobic hypoglycemic culture for 8 hours followed by reoxygenation and hyperglycemic culture for 6 or 12 hours. Flow cytometry was used to detect the number of necrotic cells after 6 hours of reoxygenation, and western blot assay was used to detect the expression of receptor-interacting protein 3 after 6 or 12 hours of reoxygenation. Thirdly, VSC4.1 cells were cultured in three groups: routine culture in normal control group; anaerobic hypoglycemic culture for 8 hours followed by reoxygenation and hyperglycemic culture for 6 hours in model group; drug group was treated with 100 μmol/L timosaponin B-II solution for 24 hours, followed by anaerobic hypoglycemic culture for 8 hours and anaerobic hyperglycemic culture for 6 hours. Flow cytometry was used to detect the number of necrotic cells after 6 hours of reoxygenation and western blot assay was used to detect the expression of receptor-interacting protein 3 after 6 hours of reoxygenation.
RESULTS AND CONCLUSION: (1) The MTT assay showed that compared with group B, group E had the highest cell survival rate. Hence, 100 μmol/L timosaponin B-II was selected for subsequent experiments. (2) The number of necrotic cells in the model group was higher than that in the normal control group (P < 0.05), and the number of necrotic cells in the inhibitor group was lower than that in the model group (P < 0.05). After 6 and 12 hours of reoxygenation culture the expression of receptor-interacting protein 3 in the inhibitor group was higher than that in the normal control group (P < 0.05), and the highest expression was observed at 6 hours after reoxygenation (P < 0.05). (3) The number of necrotic cells and the expression of receptor-interacting protein 3 in the model group were higher than those in the normal control group (P < 0.05), and the number of necrotic cells and the expression of receptor-interacting protein 3 in the drug group were lower than those in the model group (P < 0.05). All these results indicate that timosaponin B-II can effectively reduce the necroptosis of VSC4.1 cells deprived of oxygen-glucose, which may be related to the reduction of receptor-interacting protein 3.

Key words: VSC4.1 cell, necrostatin-1, necroptosis, oxygen-glucose deprivation, cell damage, receptor-interacting protein 3, necrosome, timosaponin B-II

CLC Number:

Gao Junyan, Cao Yanfei, Zhang Yun, Liu Xuemin, Hou Yanhong, Li Jianbin, Wu Zhibing. Timosaponin B-II reduces necroptosis of VSC4.1 cells induced by oxygen-glucose deprivation [J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 4062-4067.

Figures/Tables 4

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