目的 评价7，8‑二羟基黄酮（7，8‑dihydroxyflavone, 7，8‑DHF）对过氧化氢（hydrogen peroxide, H2O2）引起PC12细胞损伤的影响，并探究其机制。 方法 采用随机数字表法将PC12细胞分为4组（每组4皿）：对照组（C组）、7，8‑DHF组（D组）、H2O2组（H组）、7，8‑DHF+H2O2组（D+H组）。C组加入PBS缓冲液，D组加入25 μmol/L 7，8‑DHF，H组和D+H组均加入200 μmol/L H2O2，D+H组在加入H2O2前采用25 μmol/L 7，8‑DHF预处理1 h。各组细胞孵育24 h后采用细胞计数试剂盒（cell counting kit‑8，CCK‑8）法检测细胞活力，2，4‑二硝基苯肼显色法检测乳酸脱氢酶（lactate dehydrogenase, LDH）释放率，Hoechst染色法和膜联蛋白V‑异硫氰酸荧光素（Annexin V‑fluorescein isothiocyanate, FITC）/碘化丙啶（propidium iodide, PI）双染法结合流式细胞仪观察并分析细胞凋亡情况，实时荧光定量PCR（real‐time quantitative PCR, RT‐qPCR）法测定酸敏感离子通道3（acid‑sensing ion channel 3, ASIC3）、B淋巴细胞瘤‑2（B‑cell lymphoma‑2, Bcl‑2）/B淋巴细胞瘤‑2相关X蛋白（B‑cell lymphoma‑2 related X protein, Bax）的mRNA表达，Western blot法测定ASIC3、Bcl‑2/Bax、活化的含半胱氨酸的天冬氨酸蛋白酶‑3（cleaved cysteinyl aspartate specific proteinase‑3, cleaved caspase‑3）蛋白水平。 结果 与C组比较：H组PC12细胞数量和细胞活力明显降低，LDH释放率及细胞凋亡率明显升高，ASIC3 mRNA表达和蛋白水平均上调，cleaved caspase‑3蛋白水平上调，Bcl‑2/Bax mRNA比值和蛋白比值均降低（P<0.05）；D组和D+H组上述指标差异无统计学意义（P>0.05）。与H组比较，D+H组细胞形态接近正常，细胞数量和细胞活力明显升高，LDH释放率及细胞凋亡率明显降低，ASIC3 mRNA表达和蛋白水平均下调，cleaved caspase‑3蛋白表达下调，Bcl‑2/Bax mRNA比值和蛋白比值均升高（P<0.05）。 结论 7，8‑DHF可以减轻H2O2引起的PC12细胞损伤，其机制可能与抑制ASIC3信号从而减轻细胞凋亡有关。
Objective To evaluate the effects of 7, 8‑dihydroxyflavone (7, 8‑DHF) on PC12 cell damage induced by hydrogen peroxide (H2O2) and explore its related mechanisms. Methods According to the random number table method, PC12 cells were divided into four groups (n=4): a control group (group C), a 7，8‑DHF group (group D), a H2O2 group (group H), and a 7，8‑DHF+H2O2 group (group D+H). PBS solution was added in group C, 25 μmol/L 7，8‑DHF was added in group D and 200 μmol/L H2O2 was added in groups H and D+H. Furthermore, group D+H was treated with 25 μmol/L 7，8‑DHF for 1 h before the addition of H2O2. After 24 h of incubation, cell viability was detected by cell counting kit‑8 (CCK‑8) assay in each group. The release rate of lactate dehydrogenase (LDH) was determined by 2,4‑dinitrophenylhydrazine colorimetry. Apoptosis was observed and analyzed by Hoechst staining and Annexin V‑fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining combined with flow cytometry. The expression of acid‑sensing ion channel 3 (ASIC3) and B‑cell lymphoma‑2 (Bcl‑2)/B‑cell lymphoma‑2 related X protein (Bax) mRNA were detected by real‐time quantitative PCR (RT‑qPCR). The expression of ASIC3, Bcl‑2/Bax and cleaved cysteinyl aspartate specific proteinase‑3 (cleaved caspase‑3) were detected by Western blot. Results Compared with group C, group H presented remarkably decreases in the number of PC12 cells and cell viability; significant increases in LDH release rate and apoptosis rate, ASIC3 mRNA and protein expression, the expression of cleaved caspase‑3 protein; and remarkably decreases in Bcl‑2/Bax mRNA ratio and protein ratio (P<0.05). There was no statistical difference in the above indexes in group D and group D+H (P>0.05). Compared with group H, group D+H showed cell morphology that was close to the normal; significant increases in the number of cells and cell viability; remarkable decreases in LDH release rate and apoptosis rate, ASIC3 mRNA and protein expression, and cleaved caspase‑3 protein expression; and significant increases in Bcl‑2/Bax mRNA ratio and protein ratio (P<0.05). Conclusions 7，8‑DHF can relieve PC12 cell damage induced by H2O2 which may be related to reduced apoptosis due to inhibition of ASIC3 signals.
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