TSA对不同糖浓度下小鼠海马神经元HT-22细胞凋亡的影响

Effects of TSA on the apoptosis of HT-22 cells in mouse hippocampal neurons under differentconcentrations of glucose

目的探究曲古抑菌素A(TSA)对不同糖浓度下小鼠海马神经元细胞系HT-22细胞凋亡的影响。方法分别用高糖培养基(葡萄糖浓度55 mmol/L)及普通培养基(葡萄糖浓度25 mmol/L)培养小鼠海马神经元HT-22细胞,高糖培养基及普通培养基各分为对照组(NC组)和抑制剂组(TSA组)。用1 mmol/L的TSA作用细胞1、4、8 h,同时用0.4 mmol/L的TSA作用细胞1、4、8、12、14、16、20、24 h,CCK8法检测细胞存活率,确定抑制剂最佳作用浓度和作用时间;酶联免疫吸附测定(ELISA)法检测组蛋白去乙酰化酶(HDAC)和组蛋白乙酰转移酶(HAT)活性;流式细胞术检测细胞凋亡情况;蛋白免疫印迹法检测B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、半胱氨酸天冬氨酸蛋白酶3(Caspase-3)的表达情况。结果以0.4 mmol/L、20 h为TSA处理HT-22细胞的最适条件;ELISA结果显示,TSA作用后,HDAC显著减少,HAT显著增加(P<0.05);流式细胞术结果显示,在TSA抑制20 h后,细胞凋亡率显著增加,高糖环境下加入TSA,细胞凋亡情况更加严重;TSA作用20 h后,Bcl-2表达显著下调,Caspase-3显著上调(P<0.05)。结论TSA可能通过抑制HDAC增加组蛋白乙酰化水平,导致小鼠神经元HT-22细胞的凋亡。

Objective To explore the effect of trichostatin A(TSA)on the apoptosis of mouse hippocampal neuron cell line HT-22 cells under different sugar concentrations.Methods The mouse hippocampal neuron HT-22 cells in high glucose medium(glucose concentration 55 mmol/L)and normal medium(grape concentration 25 mmol/L)were cultured.The cells in high glucose medium and normal medium were divided into control group(NC group)and inhibitor group(TSA group).Cells were treated with 1 mmol/L TSA for 1,4 and 8 h,or 0.4 mmol/L TSA for 1,4,8,12,14,16,20 and 24 h.The cell viability was detected by the CCK8 method after treatment.Histone deacetylase(HDAC)and histone acetyltransferase(HAT)enzyme activities were detected by ELISA.Cell apoptosis was detected by flow cytometry.The expression levels of B lymphoma-2(Bcl-2),Bcl-2 related X protein(Bax)and cysteine protease 3(Caspase-3)were detected by Western blot assay.Results The 0.4 mmol/L of TSA and 20 hours were the optimal condition for culturing HT-22 cells after testing.ELISA results showed that after treatment with TSA,HDAC significantly decreased and HAT increased significantly(P<0.05).Flow cytometry results showed that after TSA inhibiting for 20 h,the apoptosis rate increased significantly.While TSA was added to cells under high glucose conditions,the apoptosis rate increased more.After 20 hours of TSA treatment,Bcl-2 expression was significantly down-regulated,while Caspase-3 was significantly up-regulated(P<0.05).Conclusion TSA may increase the level of histone acetylation by inhibiting HDAC, which could lead to the apoptosis of mouse neuronal cellsHT-22.