Effects of AMP-activated protein kinase on HMGB1 release from PC12 cells after oxygen-glucose deprivation and reoxygenation and its mediated inflammatory response in BV2 cells / 国际脑血管病杂志

ABSTRACT

Objective To investigate the effects of adenosine monophosphate-activated protein kinase (AMPK) on high-mobility group box 1 (HMGB1) release from PC12 cells after oxygen-glucose deprivation and reoxygenation (OGD/R) and its mediated inflammatory response in BV2 cells.Methods PC12 and BV2 cells were cultured,respectively.The PC12 cells were used to induce a model of oxygen glucose deprivation for 12 h and reoxygenation for 24 h.After giving 5-aminoimidazole-4-carboxamide (AICAR) 5,50 and 100 μmol/L as well as Compound C 0.1,1 and 10 μmol/L activation or inhibition of AMPK phosphorylation,respectively,methyl thiazolyl tetrazolium (MTT) was used to detect the PC12 cell activity.Enzyme-linked immunosorbent assay was used to detect the HMGB1 release level in the PC12 cell culture media.After OGD/R in each group,the PC12 culture media were acted on normal cultured BV2 cells for 24 h respectively.Westem blotting and Enzyme-linked immunosorbent assay were used to detect the NFκB inhibitory protein (inhibitor of NFκB,IκB) phosphorylation level and TNF-α release level in BV2 cells,respectively.Results After OGD/R,the PC12 cell activity was decreased significantly (68.84％±6.60％ vs.100.04％ ± 8.82％;P ＜ 0.01);the AMPK phosphorylation level was increased significantly (1.95 ±0.39 vs.1.00 ±0.20;P＜0.05),and the extracellular HMGB1 release was increased significantly (287.66 ± 26.42 pg/μl vs.53.05 ± 9.11 pg/μl;P ＜ 0.01).Compared with the OGD/R group,AICAR 100 μmol/L significantly increased the survival rate of PC12 cell after OGD/R (78.6％ ± 3.75％ vs.68.84％ ± 6.60％;P ＜ 0.05),promoted AMPK phosphorylation (3.32 ± 0.66 vs.1.95 ± 0.39;P ＜ 0.01),and reduce the release of extracellular HMGB1 (164.06 ± 12.77 pg/μl vs.287.66 ± 26.42 pg/μl;P ＜0.01).In contrast,Compound C 10 μmol/L significantly reduced the cell survival rate of PC12 (40.44％ ±3.79％ vs.68.84％ ±6.60％;P ＜0.01),inhibited AMPK phosphorylation (1.07 ± 0.21 vs.1.95 ± 0.39;P＜0.05),and increased the release of HMGB1 (337.97 ± 18.9 pg/μlvs.287.66 ± 26.42 pg/μl;P＜0.01).The conditioned medium from the AICAR 100 μmol/L group significantly inhibited IκB phosphorylation (1.68 ±0.51 vs.3.09 ± 0.10;P ＜ 0.05) and reduced the release of TNF-α (669.53 ±38.58 pg/μlvs.841.76 ± 45.82 pg/μl;P＜ 0.05) in BV2 cells.The conditioned medium from the compound C 10 μmol/L group significantly promoted IκB phosphorylation (4.98 ± 1.24 vs.3.09 ± 0.10;P ＜0.01) and increased the release of TNF-α (1 035.32 ± 128.06 pg/μl vs.841.76 ± 45.82 pg/μl;P ＜0.05) in BV2 cells.Conclusions Promoting AMPK phosphorylation activation may reduce the release of HMGB1 from PC12 cells after OGD/R,and inhibit its mediated NF-κB inflammatory pathway and reduce the release of TNF-αin BV2 cells,and thus reducing neuroinflammatory injury.On the contrary,inhibiting AMPK phosphorylation may promote the release of HMGB1 from PC12 cells after OGD/R and aggravate its mediated inflammatory reaction in BV2 cells.