Effect of hydrogen on lipopolysaccharide-caused inflammatory responses in BV-2 microglia and the role of autophagy / 中华麻醉学杂志

ABSTRACT

Objective:To evaluate the effect of hydrogen on lipopolysaccharide (LPS)-caused inflammatory responses in BV-2 microglia and the role of autophagy.Methods:The BV-2 microglial cells cultured in vitro were seeded in 6- or 96-well plates and were divided into 4 groups ( n=24 each) using a random number table method: control group (group C), group LPS, hydrogen-rich medium group (group H) and autophagy inhibitor 3-methylpurine group (group 3-MA). In group C, cells were cultured in MEM culture medium supplemented with 15% fetal bovine serum for 24 h. In group LPS, LPS was added at a final concentration of 1 μg/ml, and cells were incubated for 24 h. In group H, LPS was added at a final concentration of 1 μg/ml, the culture medium was replaced with a hydrogen-rich medium at a final concentration of 0.6 mmol/L, and cells were incubated for 24 h. In group 3-MA, 3-methylpurine was added at a final concentration of 2 mmol/L, and the subsequent treatment was similar to those previously described in group H. The cell survival rate was detected by CCK-8 assay.The concentrations of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-10 and transforming growth factor-β (TGF-β) in supernatant were detected by enzyme-linked immunosorbent assay.The percentage of ionized calcium binding adaptor molecule-1 (Iba-1) +, Iba-1 + CD86 + and Iba-1 + CD206 + cells was detected by flow cytometry.The expression of microtubule-associated protein 1 light chain 3 Ⅰ (LC3 Ⅰ), LC3Ⅱ, Beclin-1 and p62 was detected by Western blot, and the ratio of LC3Ⅱ/LC3Ⅰ was calculated. Results:There was no significant difference in the cell survival rate among the four groups ( P>0.05). Compared with group C, the concentrations of TNF-α, IL-6, IL-10 and TGF-β and percentage of Iba-1 +, Iba-1 + CD86 + and Iba-1 + CD206 + cells were significantly increased in LPS, H and 3-MA groups, the LC3Ⅱ/LC3Ⅰ ratio and Beclin-1 expression was significantly down-regulated, and p62 expression was up-regulated in LPS and 3-MA groups, and the ratio of LC3LC3Ⅱ/LC3Ⅰ and Beclin-1 expression was significantly up-regulated, and p62 expression was down-regulated in group H ( P<0.05). Compared with group LPS, the concentrations of TNF-α and IL-6 were significantly decreased, the concentrations of IL-10 and TGF-β were increased, the percentage of Iba-1 + and Iba-1 + CD86 + cells were decreased, the percentage of Iba-1 + CD206 + cells was increased, the LC3Ⅱ/LC3Ⅰ ratio and Beclin-1 expression was up-regulated, and p62 expression was down-regulated in group H ( P<0.05), and no significant change was found in the above indexes in group 3-MA ( P>0.05). Compared with group H, the concentrations of TNF-α and IL-6 were significantly increased, the concentrations of IL-10 and TGF-β were decreased, the percentage of Iba-1 + and Iba-1 + CD86 + cells was increased, the percentage of Iba-1 + CD206 + cells was decreased, the LC3Ⅱ/LC3Ⅰ ratio and Beclin-1 expression was down-regulated, and p62 expression was up-regulated in group 3-MA ( P<0.05). Conclusion:The mechanism by which hydrogen reduces LPS-caused inflammatory responses in BV-2 microglia is related to enhancing autophagy and inhibiting microglial activation.