加味脑泰方对去势脑缺血大鼠海马ATF4/CHOP/Puma通路的影响
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摘要
目的:研究加味脑泰方对去卵巢脑缺血大鼠海马活化转录因子4 (ATF4)/C/EBP同源蛋白(CHOP)/p53上调凋亡调控因子(Puma)通路的影响。方法:将雌性大鼠随机分为假手术组、模型组、加味脑泰方组和阳性对照组,除假手术组外,其它大鼠均行去卵巢术;术后11 d,阳性对照组和加味脑泰方组给予相应药物灌胃,连续灌胃3 d后行脑缺血术。术后24 h进行行为学评价,取海马组织行RT-qPCR检测Bax、Bcl-2和caspase-3的mRNA表达,Western blot检测各组大鼠海马组织中Bax、Bcl-2、caspase-3、ATF4、CHOP和Puma蛋白表达。结果:与假手术组比较,各组的海马神经功能评分明显增高;与模型组比较,加味脑泰方组与阳性对照组的神经功能评分明显降低(P <0.05)。与假手术组比较,模型组大鼠海马Bax、caspase-3、ATF4、CHOP和Puma的蛋白表达及Bax和caspase-3的mRNA表达明显升高,而Bcl-2的mRNA和蛋白表达明显降低(P <0.05);与模型组比较,加味脑泰方明显降低Bax、caspase-3、ATF4、CHOP和Puma表达,提高Bcl-2表达(P <0.05)。结论:加味脑泰方可能通过抑制ATF4/CHOP/Puma通路相关mRNA及蛋白的表达而减轻脑缺血损伤。
AIM:To investigate the effects of Jiawei-Naotai formula(JWNTF)on ATF4/CHOP/Puma pathway in hippocampal neurons of ovariectomized female rats with cerebral ischemia.METHODS:The female rats were randomly divided into sham group,model group,JWNTF group and positive control group.The rats,expect in the sham group,were ovariectomized.The rats in each group were intragastric administration 11 days after ovariectomy.The rats in sham group and model group were given a gavage of 0.9%Na Cl,while the rats in other groups were administrated by corresponding therapy intragastrically for 3 d.The regional cerebral ischemia model was established by middle cerebral artery occlusion(MCAO)suture method 14 days after ovariectomy.The behaviors of the rats were evaluated 24 h after cerebral ischemia.The mRNA levels of Bax,Bcl-2 and caspase-3 were detected by RT-qPCR,and the protein expression of Bax,Bcl-2,caspase-3,ATF4,CHOP and Puma was determined by Western blot.RESULTS:Compared with sham group,the neurobehavioral scores significantly increased in other groups(P<0.05).Compared with model group,the neurobehavioral scores were significantly decreased in positive control group and JWNTF group(P<0.05).The protein expression of Bax,caspase-3,ATF4,CHOP and Puma,and the mRNA expression of Bax and caspase-3 in the hippocampus were much higher,and Bcl-2 was lower in model group than those in sham group(P<0.05).JWNTF significantly reduced the protein expression of Bax,caspase-3,ATF4 and CHOP,and the mRNA expression of Puma,Bax and caspase-3,and markedly increased the expression of Bcl-2 at mRNA and protein levels compared with model group.CONCLUSION:The JWNTF protects against brain damage induced by cerebral ischemia,which may be related to inhibitiing the expression of ATF4/CHOP/Puma pathway-related molecules at mRNA and protein levels.
AIM:To investigate the effects of Jiawei-Naotai formula(JWNTF)on ATF4/CHOP/Puma pathway in hippocampal neurons of ovariectomized female rats with cerebral ischemia.METHODS:The female rats were randomly divided into sham group,model group,JWNTF group and positive control group.The rats,expect in the sham group,were ovariectomized.The rats in each group were intragastric administration 11 days after ovariectomy.The rats in sham group and model group were given a gavage of 0.9%Na Cl,while the rats in other groups were administrated by corresponding therapy intragastrically for 3 d.The regional cerebral ischemia model was established by middle cerebral artery occlusion(MCAO)suture method 14 days after ovariectomy.The behaviors of the rats were evaluated 24 h after cerebral ischemia.The mRNA levels of Bax,Bcl-2 and caspase-3 were detected by RT-qPCR,and the protein expression of Bax,Bcl-2,caspase-3,ATF4,CHOP and Puma was determined by Western blot.RESULTS:Compared with sham group,the neurobehavioral scores significantly increased in other groups(P<0.05).Compared with model group,the neurobehavioral scores were significantly decreased in positive control group and JWNTF group(P<0.05).The protein expression of Bax,caspase-3,ATF4,CHOP and Puma,and the mRNA expression of Bax and caspase-3 in the hippocampus were much higher,and Bcl-2 was lower in model group than those in sham group(P<0.05).JWNTF significantly reduced the protein expression of Bax,caspase-3,ATF4 and CHOP,and the mRNA expression of Puma,Bax and caspase-3,and markedly increased the expression of Bcl-2 at mRNA and protein levels compared with model group.CONCLUSION:The JWNTF protects against brain damage induced by cerebral ischemia,which may be related to inhibitiing the expression of ATF4/CHOP/Puma pathway-related molecules at mRNA and protein levels.
引文
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[18]俞益火,田治标,谢嫚花.滋阴补肾对绝经后膝骨关节炎雌激素和C-反应蛋白表达的影响[J].中国中医骨伤科杂志,2014,22(3):34-35.
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[20]熊倩,阮修艳,方向东.SP1/Krüppel样因子的研究进展[J].遗传,2010,32(6):531-538.
[2]秦莉花,刘洋,黄娟,等.加味脑泰方对去势脑缺血大鼠脑损伤的影响与雌激素水平相关性研究[J].中国药理学通报,2018,34(3):428-432.
[3]Cai M,Ma YL,Qin P,et al.The loss of estrogen efficacy against cerebral ischemia in aged postmenopausal female mice[J].Neurosci Lett,2014,558:115-119.
[4]李天,关世奎,武桂铃,等.脑缺血的分子损伤机制及治疗研究进展[J].实用医学杂志,2015,31(17):2775-2777.
[5]Tabas I,Ron D.Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress[J].Nat Cell Biol,2011,13(3):184-190.
[6]贺运河,郝晓元,葛金文.脑泰方治疗气虚血瘀证脑梗塞临床研究[J].中国中医急症,2001,10(6):319-320.
[7]秦莉花,李晟,成邵武,等.加味脑泰方对卵巢摘除脑缺血大鼠海马细胞外信号调节蛋白激酶1/2和c-Jun氨基末端蛋白激酶蛋白活化的影响[J].中国康复理论与实践,2018,24(3):277-281.
[8]Jang Y,Kim J,Ko JW,et al.Homocysteine induces PUMA-mediated mitochondrial apoptosis in SH-SY5Y cells[J].Amino Acids,2016,48(11):2559-2569.
[9]仇健,武菲,王春梅,等.大鼠脑缺血再灌注后GRP78、CHOP表达变化[J].安徽医科大学学报,2018,53(4):497-501.
[10]Liu D,Zhang M,Yin H.Signaling pathways involved in endoplasmic reticulum stress-induced neuronal apoptosis[J].Int J Neurosci,2013,123(3):155-162.
[11]董雅洁,高维娟.Bcl-2、Bax、caspase-3在细胞凋亡中的作用及其关系[J].中国老年学杂志,2012,11(32):4828-4830.
[12]Bickler PE,Clark JP,Gabatto P,et al.Hypoxic preconditioning and cell death from oxygen/glucose deprivation co-opt a subset of the unfolded protein response in hippocampal neurons[J].Neuroscience,2015,310:306-321.
[13]Ding L,Ba XH.Role of ornithine decarboxylase/polyamine pathway in focal cerebral ischemia-reperfusion injury and its mechanism in rats[J].Jnt J Clin Exp Med,2015,8(11):20624-20630.
[14]Yaidikar L,Thaku S.Punicalagin attenuated cerebral ischemia-reperfusion insult via inhibition of proinflammatory cytokines,up-regulation of Bcl-2,down-regulation of Bax,and caspase-3[J].Mol Cell Biochem,2015,402(1-2):141-148.
[15]赵士弟,陈耀,姜丽娜,等.大豆异黄酮对脑缺血/再灌注诱导的线粒体损伤和脑细胞凋亡的影响[J].中国病理生理杂志,2014,30(12):2172-2178.
[16]陈丹丹,彭成,万峰,等.氢溴酸樟柳碱对抗大鼠急性脑缺血/再灌注损伤的作用机制研究[J].中国药理学通报,2017,33(8):1096-1102.
[17]张冬青,王海宝,王淑芳,等.毛蕊异黄酮生物活性的研究进展[J].中国中药杂志,2015,40(22):4339-4343.
[18]俞益火,田治标,谢嫚花.滋阴补肾对绝经后膝骨关节炎雌激素和C-反应蛋白表达的影响[J].中国中医骨伤科杂志,2014,22(3):34-35.
[19]Shah A,Vaidya NK,Bhat HK,et al.HIV-1 gp120 induces type-1 programmed cell death through ER stress employing IRE1α,JNK and AP-1 pathway[J].Sci Rep,2016,6:18929.
[20]熊倩,阮修艳,方向东.SP1/Krüppel样因子的研究进展[J].遗传,2010,32(6):531-538.