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

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. Western 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/μl vs. 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/μl vs. 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. Key words: AMP-Activated Protein Kinases; HMGB1 Protein; Cells, Cultured; Neurons; Microglia; Oxygen; Glucose; Inflammation