大鼠脑长期慢性低灌注诱导的内质网应激损伤及雌激素干预

STUDY ON ENDOPLASMIC RETICULUM STRESS INJURE INDUCED BY LONG TERM CEREBRAL CHRONIC HYPOPERFUSION AND INTERVENTION EFFECT OF ESTRADIOL

目的观察大鼠长期脑慢性低灌注后海马神经元内质网应激相关因子的蛋白表达,以及持续给予低剂量雌激素的干预作用,旨在为临床治疗血管性痴呆提供理论依据。方法 3月龄SD大鼠行双侧卵巢切除,并随机分成假手术组(sham 3m)、BCCAO3m组、及17β-雌二醇(E2)组。通过永久性结扎双侧颈总动脉建立大鼠慢性脑低灌注及血管性痴呆模型;采用Western blot技术检测海马CA1区内质网应激蛋白GRP78、ATF4、促凋亡转录因子CHOP和促死亡激酶JNK的磷酸化水平;用免疫荧光染色观察海马CA1区CHOP及JNK磷酸化免疫表达的变化。结果与sham 3m组相比,BCCAO后3m海马神经元内GRP78的表达无明显差异,但ATF4蛋白表达显著增加;CHOP和p-JNK蛋白水平较sham对照组显著增加;长期给予低剂量E2可显著逆转此变化;免疫荧光染色验证了以上CHOP和p-JNK的Western blot结果。结论BCCAO可导致海马CA1区神经元长期内质网应激,从而激活促凋亡的CHOP-JNK信号通路;持续低剂量E2替代治疗可有效降低内质网应激损伤。雌激素替代治疗可能成为降低或阻断慢性脑低灌注和血管性痴呆的潜在治疗方法。

Objective To observe protein expression of endoplasmie reticulum stress related factors in hippocampal CA1 neurons of the rats after the long term cerebral chronic hypoperfusion,and the effect of continuously administration of low dose 17β-estradiol （E2）, which aims to explore clinical therapeutic strategies of vascular dementia （VD）. Methods Three - month - old adult Sprague Dawley rats were bilaterally ovariectomized under isoflurane anesthesia and separated into sham 3m group, BCCAO 3m group and E2 - treatment group. Chronic cerebral hypoperfusion and VD was induced by bilateral common carotid artery occlusion （BC- CAO） at 1 week interval. Western Blot analysis was used to investigate the protein expression of endoplasmic reticulum stress （ERS） markers GRP78,ATF4,and apoptosis - inducing factor CHOP, as well as JNK phosphorylation （p- JNK） in hippocampal CA1 region. Immunofluorescence staining was used to observe the immune expression of CHOP, p - JNK in hippocampal CA1 region. Results Compared to sham group, there was no statistic difference in GRP78 protein expression, while the protein expression of ATF4 markedly increased in BCCAO 3m animals. Importantly the protein levels of CHOP and p - JNK robust were increasd at BCCAO 3m compared to sham 3m animals. Longterm low dose of E2 could reverse the changes induced by BCCAO. Immunofluorescence staining for CHOP and p- JNK further mirrored well the Western Blot result, showing stronger fluorescent intensity of CHOP and p- JNK in BCCAO 3m than that in sham 3m and E2- administrated groups. Conclusion Chronic hypoperfusion induced by BCCAO can cause long term ERS in hippocampal CA1 neurons,which may involve JNK death signaling pathway. Continuously low- dose E2 replacement can significantly prevent the ERS damage and may be a potentially therapeutic modality to attenuate or block neurological consequences of chronic cerebral hypoperfusion and VD.