AMP-活化蛋白激酶对氧葡萄糖剥夺复氧PC12细胞HMGB1释放及其介导的BV2细胞炎性反应的影响

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 celts

目的探讨调节AMP-活化蛋白激酶（AMP-activated protein kinase, AMPK）对PC12细胞氧葡萄糖剥夺复氧（oxygen glucose deprivation and reoxygenation, OGD/R）后高迁移率族蛋白1（high mobility group box 1, HMGB1）释放及其介导的BV2细胞炎性反应的影响。方法分别培养PC12和BV2细胞，应用PC12细胞建立氧葡萄糖剥夺12 h复氧24 h模型，分别给予5-氨基-4-甲酰胺咪唑核糖核苷酸（5-aminoimidazole-4-carboxamide, AICAR）5、50和100 μmol/L以及Compound C 0.1、1和10 μmol/L激活或抑制AMPK磷酸化后，应用四甲基偶氮唑蓝（methyl thiazolyl tetrazolium, MTT）法检测PC12细胞活性，酶联免疫吸附法检测PC12细胞培养基中HMGB1释放水平。将各组OGD/R后PC12培养基分别作用于BV2细胞正常培养24 h。分别采用免疫印迹法和酶联免疫吸附法检测BV2细胞中NF-κB抑制蛋白（inhibitor of NF-κB, IκB）磷酸化水平和TNF-α释放水平。结果OGD/R后，PC12细胞活性显著降低[（68.84±6.60）%对（100.04±8.82）%；P〈0.01]，AMPK磷酸化水平显著增高（1.95±0.39对1.00±0.20；P〈0.05），细胞外HMGB1释放显著增多[（287.66±26.42）pg/μl对（53.05±9.11）pg/μl；P〈0.01]。与OGD/R组比较，AICAR 100 μmol/L能显著增高OGD/R后PC12细胞存活率[（78.60±3.75）%对（68.84±6.60）%；P〈0.05]、促进AMPK磷酸化（3.32±0.66对1.95±0.39；P〈0.01）和减少细胞外HMGB1的释放[（164.06±12.77）pg/μl对（287.66±26.42）pg/μl；P〈0.01]。相比之下，Compound C 10 μmol/L则会显著降低PC12细胞存活率[（40.44±3.79）%对（68.84±6.60）%；P〈0.01]、抑制AMPK磷酸化（1.07±0.21对1.95±0.39；P〈0.05）和增加HMGB1的释放[（337.97±18.90）pg/μl对（287.66±26.42）pg/μl；P〈0.01]。AICAR 100 μmol/L组条件培养基能显著抑制BV2细胞的IκB磷酸化（1.68±0.51对3.09±0.10；P〈0.05）和减少TNF-α释放[（669.53±38.58）pg/μl对（841.76±45.82）pg/μl；P〈0.05]；Compound C 10 μmol/L组条件培养基则能显著促进BV2细胞IκB磷酸化（4.98±1.24对3.09±0.10；P〈0.01）和增加TNF-α释放[（1 035.32±128.06）pg/μl对（841.76±45.82）pg/μl；P〈0.05]。结论促进AMPK磷酸化激活能减少PC12细胞OGD/R后HMGB1的释放、抑制其介导的BV2细胞NF-κB炎症通路激活并且减少TNF-α释放，从而减轻神经炎性损伤；相反，抑制AMPK磷酸化则会促进PC12细胞OGD/R后HMGB1释放和加重其介导的BV2细胞炎性反应。

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 ixmol/L as well as Compound C 0. 1, 1 and 10 μmol/L activation or inhibition of AMPK phosphoryiation, 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 （inhlbitor 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/p,l vs. 53.05± 9. 11 pg/μl; P〈 0. 01）. Compared with the OGD/R group, AICAR 100 txmol/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 phosphoryiation （3.32 ± 0. 66 vs. 1.95 ± 0.39; P 〈 0. 01）, and reduce the release of extracellular HMGB1 （164. 06± 12.77 pg/txlvs. 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/pul vs. 287.66 ± 26.42 pg/μl; P 〈 0.01）. The conditioned medium from the AICAR 100 μmol/L group significantly inhibited IKB 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/pAvs. 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 IKB phosphoryiation （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/μ1; 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-αxin BV2 cells, and thus reducing neurointlammatory 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.