氟西汀对蛛网膜下腔出血模型大鼠线粒体自噬及Keap1/Nrf2/PHB2通路的调节作用

Effect of fluoxetine on mitochondrial autophagy and Keap1/Nrf2/PHB2 pathway in rats with subarachnoid hemorrhage

目的探究氟西汀对蛛网膜下腔出血(Subarachnoid hemorrhage,SAH)模型大鼠线粒体自噬及Kelch样环氧氯丙烷相关蛋白1(Kelch-like epichlorohydrin-related protein-1,Keap1)/E2相关因子2(Nuclear factor E2-related factor 2,Nrf2)/抗增殖蛋白2(Prohibitin2,PHB2)通路的影响。方法将Sprague Dawley(SD)大鼠分为假手术(Sham)组、模型(Model)组、氟西汀低、中、高剂量组(Low dose group,middle dose group,high dose group);假手术组不刺破血管壁,其余4组均建立SAH模型;术后1 h假手术组、模型组当天腹腔注射等量生理盐水,氟西汀低、中、高剂量组注射10、20、30 mg/kg的氟西汀,1次/d,连续3 d;观察各组大鼠神经功能、SAH评分、脑组织含水量,原位末端标记(TdT-mediated dUTP nick and labeling,TUNEL)检测脑组织细胞凋亡;蛋白免疫印迹法检测p62、自噬相关基因5(Autophagy related gene 5,Atg5)、酵母Atg6同源物(Yeast Atg6 homolog,Beclin-1)、Keap1/Nrf2/PHB2通路蛋白表达水平;苏木精-伊红(Hematoxylin-eosin,HE)染色观察脑组织病理学变化;放射免疫法检测血清和脑脊液神经肽Y(Neuropetide Y,NPY)水平。结果与Sham组比较,Model组神经功能评分、p62蛋白表达水平降低,SAH评分、脑组织含水量、细胞凋亡率、血清和脑脊液NPY水平增高,Atg5,Beclin-1,Nrf2,Keap1、磷酸酶张力蛋白样同源物诱导激酶1(Tension protein analogue induced putative kinase 1,PINK1)、帕金森病相关基因(Parkin protein,Parkin)、PHB2蛋白表达水平上调(P<0.05);与Model组比较,Low dose group,middle dose group,high dose group神经功能评分、p62蛋白表达水平增高,SAH评分、脑组织含水量、细胞凋亡率、血清和脑脊液NPY水平降低,Atg5,Beclin-1,Nrf2,Keap1,PINK1,Parkin,PHB2蛋白表达水平下调(P<0.05)。结论氟西汀可减轻SAH模型大鼠脑组织线粒体自噬和凋亡,并降低Keap1/Nrf2/PHB2通路蛋白表达水平。

Objective To investigate the effects of fluoxetine on mitochondrial autophagy and Kelch-like epichlorohydrin-associated protein 1(Keap1)/E2-associated factor 2(Nrf2)/antiproliferative protein 2(PHB2)pathways in rats with subarachnoid hemorrhage(SAH).Methods SD rats were divided into Sham group,Model group,fluoxetine Low dose group,Middle dose group,and High dose group(Low dose group,middle dose group,High dose group).The blood vessel wall was not pierced in the sham operation,while SAH model was established in the other 4 groups.1 h after operation,sham operation group and model group were intraperitoneally injected with the same amount of normal saline on the same day,while fluoxetine low,medium and high dose groups were injected with 10,20 and 30 mg/kg fluoxetine once a day for continuous 3 days.Neural function,SAH score and brain tissue water content of rats in each group were observed.TdT-mediated dUTP nick and labeling(TUNEL)was used to detect apoptosis in brain tissue.The protein expression levels of p62,autophagy related gene 5(Atg5),Yeast Atg6 homolog(Beclin-1),Keap1/Nrf2/PHB2 pathway were detected by Western blotting.Hematoxylin-eosin(HE)staining was used to observe the pathological changes of brain tissue.The levels of neuropeptide Y(NPY)in serum and cerebrospinal fluid were detected by radioimmunoassay.Results Compared with sham operation group,neural function score and p62 protein expression were decreased in model group,while SAH score,brain tissue water content,apoptosis rate,serum and cerebrospinal fluid NPY were increased,and protein expressions of Atg5,Beclin-1,Nrf2,Keap1,tension protein analogue induced putative kinase 1(PINK1),Parkin protein(Parkin)and PHB2 were up-regulated(P<0.05).Compared with the model group,the nerve function score and p62 protein expression were increased in fluoxetine low-dose,medium-dose and high-dose groups,while the SAH score,brain tissue water content,apoptosis rate,serum and cerebrospinal fluid NPY were decreased.The protein expressions of Atg5,Beclin-1,Nrf2,Keap1,PINK1,Parkin and PHB2 were down-regulated(P<0.05).Conclusion Fluoxetine can reduce the mitochondrial autophagy and apoptosis in SAH rats and decrease the expression level of Keap1/Nrf2/PHB2 pathway.