二氢杨梅素对间歇低氧大鼠脑组织内质网应激诱导细胞凋亡的影响

Effect of dihydromyricetin on apoptosis induced by endoplasmic reticulum stress in rats with intermittent hypoxia

目的观察二氢杨梅素(DHM)对间歇低氧大鼠脑组织内质网应激诱导细胞凋亡的影响。方法选择96只Wistar雄性大鼠,按随机数字表法分为正常对照组、5%间歇低氧组及DHM组,每组再分为制模后2、4、6、8周4个亚组,每个亚组8只大鼠。5%间歇低氧组向实验箱内依次注入30 s氮气、40 s空气和50 s空气,使箱内氧浓度在5%~21%之间变化,每2 min为1个循环,反复进行,每天7 h;正常对照组大鼠向实验箱内持续注入空气,使箱内氧气浓度为21%。DHM组大鼠于制模前30 min每日晨起07:00灌胃DHM 250 mg·kg^(-1)·d^(-1),正常对照组和5%间歇低氧组不进行任何处理。实验2、4、6、8周取材,于透射电镜下观察皮质神经细胞内质网的超微结构;采用免疫组化法检测各组皮质脑组织细胞C/EBP同源蛋白(CHOP)的蛋白表达水平;采用原位末端缺刻标记试验(TUNEL)检测各组皮质神经细胞的凋亡情况。结果透射电镜下可见:正常对照组神经元形态无异常,内质网丰富,形态完整,无扩张肿胀及脱颗粒改变;5%间歇低氧组内质网出现脱颗粒、扩张及肿胀,甚至出现融合,随低氧时间延长内质网损伤越严重;DHM组神经细胞内质网的损伤较5%间歇低氧组减轻。正常对照组各时间点神经细胞CHOP的蛋白表达及凋亡指数均较低,皮质神经细胞阳性细胞较少。与正常对照组比较,5%间歇低氧组及DHM组神经细胞CHOP蛋白表达和凋亡指数升高,于制模型2周开始出现统计学差异〔CHOP(A值):19.15±0.92、14.99±0.71比10.51±0.28;凋亡指数:0.398±0.026、0.283±0.053比0.058±0.014,均P<0.05〕,持续到制模后8周〔CHOP(A值):58.44±2.45、40.99±1.60比10.42±0.29;凋亡指数:0.826±0.034、0.645±0.039比0.058±0.016,均P<0.05〕;DHM组上述指标均较5%间歇低氧组明显降低(均P<0.05)。结论DHM可通过抑制间歇低氧大鼠脑组织内质网应激诱导的神经细胞凋亡,进而发挥神经保护作用。

Objective To investigate the effect of dihydromyricetin(DHM)on endoplasmic reticulum stress induced apoptosis in rats with intermittent hypoxia.Methods A total of 96 Wistar male rats were randomly divided into normal control group,5%intermittent hypoxia group,and DHM group according to random number table method.Each group was further divided into four subgroups at 2,4,6,and 8 weeks after modeling with 8 rats in each subgroup.In the 5%intermittent hypoxia group,30 seconds nitrogen,40 seconds air and 50 seconds air were injected into the experimental chamber successively,so that the oxygen concentration in the chamber varied between 5%-21%,every 2 minutes was a cycle,repeated for 7 hours a day;normal control group rats continued to inject air into the experimental chamber,the oxygen concentration in the chamber was 21%.DHM group rats were injected with DHM 250 mg·kg^(-1)·d^(-1)from 07:00 every morning before 30 minutes modeling,while normal control group and 5%intermittent hypoxia group were given no treatment.The samples were collected at experiment 2,4,6,and 8 weeks for index detection.The ultrastructure of endoplasmic reticulum of cortical nerve cells was observed under transmission electron microscope;The protein expression level of C/EBP homologous protein(CHOP)in cortical brain cells of each group was detected by immunohistochemistry;The apoptosis of cortical nerve cells in each group was detected by terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling(TUNEL).Results Under transmission electron microscopy:the morphology of neurons in the normal control group was complete,no abnormalities,abundant endoplasmic reticulum,no expansion,swelling,and degranulation changes.In the 5%intermittent hypoxia group,the endoplasmic reticulum appeared degranulation,dilation,swelling,and even fusion and the injury became more serious with the extension of hypoxia time.The damage of endoplasmic reticulum of nerve cells in DHM group was less than that in 5%intermittent hypoxia group.CHOP protein expression and apoptosis index of nerve cells were lower in normal control group at all time points,and there were fewer positive cortical nerve cells.Under light microscope,apoptotic cells are brownish yellow or brown.CHOP protein expression and apoptosis index of nerve cells in 5%intermittent hypoxia group and DHM group were significantly increased compared with normal control group,and statistical differences began to appear after 2 weeks of model building[CHOP(A value):19.15±0.92,14.99±0.71 vs.10.51±0.28,apoptotic index:0.398±0.026,0.283±0.053 vs.0.058±0.014,all P<0.05],and lasted until 8 weeks after modeling[CHOP(A value):58.44±2.45,40.99±1.60 vs.10.42±0.29,apoptotic index:0.826±0.034,0.645±0.039 vs.0.058±0.016,all P<0.05],while above indexes in DHM group were significantly decreased compared with 5%intermittent hypoxia group(all P<0.05).Conclusion DHM can play a neuroprotective role by inhibiting the apoptosis of nerve cells induced by endoplasmic reticulum stress in intermittently hypoxic rats.