地黄饮子抑制能量障碍诱导的APP/PS1小鼠内质网应激及神经元凋亡的作用机制

Effect of Dihuang Yinzi on Endoplasmic Reticulum Stress and Neuronal Apoptosis Induced by Energy Disorders in APP/PS1 Mice

目的：研究地黄饮子对能量代谢障碍诱导的APP/PS1转基因小鼠内质网应激（endoplasmic reticulum stress,ERS）活性转录因子4（activating transcription factor4,ATF4）/C/EBP同源蛋白（C/EBP homologous protein,CHOP）信号通路激活及其引发的神经元凋亡的作用机制。方法：4月龄APP/PS1转基因小鼠120只,随机分为正常组、模型组、阳性药（安理申,1 mg·kg-1）组、地黄饮子低、中、高剂量组（1.25,2.5,5 g·kg-1）。除正常组外,其余各组均腹腔注射100 mg·kg-1剂量的3-硝基丙酸（3-NP）,正常组小鼠腹腔注射等体积的无菌生理盐水。经灌胃给予地黄饮子和安理申1周。实时荧光定量聚合酶链式方应（Real-time PCR）检测小鼠脑组织ATF4,CHOP mRNA水平。蛋白免疫印迹法（Western blot）检测小鼠脑组织内质网应激标志蛋白葡萄糖调节蛋白78（GRP78）,ATF4,CHOP,B淋巴细胞瘤-2（B-cell lymphoma-2,Bcl-2）,Bcl-2相关X蛋白（Bcl-2-associated X protein,Bax）蛋白表达水平。末端标记法（TUNEL）染色观察小鼠脑组织神经元凋亡,并计算凋亡率。结果：与正常组比较,3-NP诱导的能量代谢障碍可以显著增加ERS标记性蛋白GRP78表达量（P〈0.01）,提高ATF4,CHOP mRNA水平（P〈0.01）和蛋白表达水平（P〈0.01）,下调抗凋亡蛋白Bcl-2（P〈0.01）和上调促凋亡蛋白Bax（P〈0.01）,增加模型小鼠脑组织神经元凋亡率。与模型组比较,地黄饮子各剂量组能显著降低模型小鼠脑组织GRP78表达水平（P〈0.05,P〈0.01）,ATF4,CHOP基因mRNA（P〈0.05,P〈0.01）及蛋白（P〈0.05,P〈0.01）水平;能够上调抗凋亡蛋白Bcl-2（P〈0.05,P〈0.01）,下调促凋亡蛋白Bax（P〈0.05,P〈0.01）,减少脑组织神经元凋亡。TUNEL结果显示地黄饮子可以显著减少小鼠脑组织神经元凋亡率。结论：地黄饮子可以抑制能量代谢障碍导致的ERS,抑制ATF4/CHOP信号通路激活,调节凋亡相关蛋白,显著减少神经元凋亡。

Objective： To study the mechanism of Dihuang Yinzi on the activation of energy metabolism disorder-induced endoplasmic reticulum stress（ ERS） activating transcription factor4（ ATF4）/C/EBP homologous protein（ CHOP） signaling pathway in APP/PS1 transgenic mice,and investigate its mechanism on neuronal apoptosis. Method： The 120 APP/PS1 transgenic mice at 4-month-old were randomly divided into normal control group,model group,positive drug（ Aricept,1 mg·kg-1） group,and low-dose,middle-dose,and high-dose groups of Dihuang Yinzi（ 1. 25,2. 5,5 g·kg-1）. Except for the normal control group,the other groups were intraperitoneally injected with 3-nitropropionic acid（ 3-NP） at a dose of 100 mg·kg-1 to establish models.The mice in normal control group were injected intraperitoneally with an equal volume of normal saline. Dihuang Yinzi and Aricept were given by oral administration for 1 week. Then Real-time PCR was used to detect ATF4,CHOP mRNA levels in mouse brain. The protein expression levels of glucose-regulated protein 78（ GRP78）,ATF4,CHOP,B-cell lymphoma-2（ Bcl-2）,and Bcl-2-associated X protein（ Bax） in brain tissue of mice were detected by Western blot. Terminal-deoxynucleoitidyl transferase mediated nick end labeling（ TUNEL） staining was used to evaluate the neuronal apoptosis in mouse brain and the apoptosis rate. Result： As compared with the normal group,3-NP-induced energy metabolism disorder significantly increased the expression of ERS marker protein GRP78（ P 〈 0. 01）,increased ATF4 and CHOP mRNA levels（ P 〈 0. 01） and protein expression（ P 〈 0. 01）,down-regulated anti-apoptosis protein Bcl-2（ P 〈 0. 01） and up-regulated of pro-apoptotic protein Bax（ P 〈 0. 01）,and promoted the rate of neuronal apoptosis in the brain of model mice. As compared with the model group,Dihuang Yinzi could significantly reduce the mRNA and protein expression of GRP78（ P 〈 0. 05,P 〈 0. 01）,ATF4 and CHOP（ P 〈 0. 05, P 〈 0. 01）,up-regulate anti-apoptotic protein Bcl-2（ P 〈 0. 05,P 〈 0. 01）,down-regulate pro-apoptotic protein Bax（ P 〈 0. 05,P 〈 0. 01）,and reduce neuronal apoptosis in brain tissue. TUNEL results showed that Dihuang Yinzi can significantly reduce the neuronal apoptosis rate in mouse brain.