五子衍宗丸对神经管畸形细胞模型凋亡途径的影响
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摘要
目的探讨五子衍宗丸防治神经管畸形(NTDs)的可能作用机制。方法 PC12细胞随机分为空白组、模型组及五子衍宗丸低、中、高剂量组。除空白组外,其余各组采用全反式维甲酸(atRA)干预PC12细胞构建NTDs模型。五子衍宗丸低、中、高剂量组分别给予含五子衍宗丸的RPMI-1640培养基并使其最终浓度分别为125、250、500μg/ml进行预处理,模型组和空白组加入RPMI-1640培养基。测定各组PC12细胞活力及细胞凋亡率,并提取空白组、模型组及经细胞活力、细胞凋亡率实验确定的最佳剂量五子衍宗丸组细胞总RNA测序,筛选差异表达基因,运用维恩图得到目的差异表达基因集,并进行GO功能和KEGG通路显著性富集分析,对与细胞凋亡相关的差异基因进行共表达网络分析。结果模型组PC12细胞活力显著下降,细胞凋亡率显著增加,五子衍宗丸各剂量组细胞活力增强、细胞凋亡率降低,且以五子衍宗丸低剂量组效果最佳(P<0.05或P<0.01)。目的差异表达基因集GO功能分析显著富集到内质网应激介导细胞凋亡相关和细胞增殖生长相关的GO条目,KEGG通路分析显著富集到细胞生长和死亡相关的信号通路。与模型组比较,五子衍宗丸低剂量组内质网应激相关基因Ern1、Casp12、Creb3l1下调,抗凋亡基因Birc5上调,与模型组较空白组这些基因上下调方向正好相反。结论五子衍宗丸可抑制细胞凋亡,保护神经元样细胞,其机制与调控内质网应激相关的基因表达,与染色体分离、细胞核分裂、细胞周期等细胞增殖、生长和死亡等生物过程或信号通路相关。
Objective To explore the possible mechanism of Wuzi Yanzong Pills(五子衍宗丸) on apoptotic pathway of neural tube defects(NTDs) cell model.Methods PC12 cells were randomly divided into a blank group,a model group and Wuzi Yanzong Pills low,medium,high dose group.All groups but the blank group used all-trans retinoic acid(atRA) to intervene PC12 cells to construct NTDs model.The low,medium and high doses of Wuzi Yanzong Pills were given RPMI-1640 medium containing Wuzi Yanzong Pills and the final concentration was 125,250,500 μg/m L,respectively for pre-treatment.The model group and the blank group were added with RPMI-1640 medium.The viability and apoptosis rate of PC12 cells in each group were determined,and the total RNA sequencing of the blank group,the model group and the optimal dose of the Wuzi Yanzong Pills group that determined by cell viability and apoptosis rate was extracted.The differentially expressed genes were screened and the target differentially expressed gene set was obtained by Venn diagram,and performed significant enrichment analysis of GO function and KEGG pathway,and also performed co-expressed network analysis of differential genes related to apoptosis.Results The cell viability of PC12 cells in the model group was significantly decreased,and the apoptosis rate was significantly increased.The cell viability of Wuzi Yanzong Pills groups were increased,apoptosis rate decreased,and the best result appeared in the Wuzi Yanzong Pills low dose group(P<0.05 or P<0.01).Target differentially expressed gene set GO functional analysis was significantly enriched into endoplasmic reticulum stress-mediated apoptosis-associated and cell proliferation-related GO entries,and the KEGG pathway analysis significantly enriched the cell growth and death-related signaling pathways.Compared with the model group,the endoplasmic reticulum stress-related genes Ern1,Casp12 and Creb3l1 were down-regulated in the Wuzi Yanzong Pills low-dose group,and the anti-apoptotic gene Birc5 was up-regulated,which was in the opposite direction to the genes in the model group compared with the blank group.Conclusion Wuzi Yanzong Pills can inhibit apoptosis,protect neuron-like cells,and its mechanism might be related to the regulation of gene expression related to endoplasmic reticulum stress,and biological processes or signal pathways such as chromosome segregation,cell division,cell cycle and other cell proliferation,growth and death.
Objective To explore the possible mechanism of Wuzi Yanzong Pills(五子衍宗丸) on apoptotic pathway of neural tube defects(NTDs) cell model.Methods PC12 cells were randomly divided into a blank group,a model group and Wuzi Yanzong Pills low,medium,high dose group.All groups but the blank group used all-trans retinoic acid(atRA) to intervene PC12 cells to construct NTDs model.The low,medium and high doses of Wuzi Yanzong Pills were given RPMI-1640 medium containing Wuzi Yanzong Pills and the final concentration was 125,250,500 μg/m L,respectively for pre-treatment.The model group and the blank group were added with RPMI-1640 medium.The viability and apoptosis rate of PC12 cells in each group were determined,and the total RNA sequencing of the blank group,the model group and the optimal dose of the Wuzi Yanzong Pills group that determined by cell viability and apoptosis rate was extracted.The differentially expressed genes were screened and the target differentially expressed gene set was obtained by Venn diagram,and performed significant enrichment analysis of GO function and KEGG pathway,and also performed co-expressed network analysis of differential genes related to apoptosis.Results The cell viability of PC12 cells in the model group was significantly decreased,and the apoptosis rate was significantly increased.The cell viability of Wuzi Yanzong Pills groups were increased,apoptosis rate decreased,and the best result appeared in the Wuzi Yanzong Pills low dose group(P<0.05 or P<0.01).Target differentially expressed gene set GO functional analysis was significantly enriched into endoplasmic reticulum stress-mediated apoptosis-associated and cell proliferation-related GO entries,and the KEGG pathway analysis significantly enriched the cell growth and death-related signaling pathways.Compared with the model group,the endoplasmic reticulum stress-related genes Ern1,Casp12 and Creb3l1 were down-regulated in the Wuzi Yanzong Pills low-dose group,and the anti-apoptotic gene Birc5 was up-regulated,which was in the opposite direction to the genes in the model group compared with the blank group.Conclusion Wuzi Yanzong Pills can inhibit apoptosis,protect neuron-like cells,and its mechanism might be related to the regulation of gene expression related to endoplasmic reticulum stress,and biological processes or signal pathways such as chromosome segregation,cell division,cell cycle and other cell proliferation,growth and death.
引文
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[12]王莉,吕爱平,王梅,等.“肾藏精”与生长壮老关系的现代研究[J].现代中西医结合杂志,2018,27(9):1021-1023.
[13]单海军,曹彩红,张英英,等.补肾开窍祛痰方对缺氧缺血性脑损伤新生大鼠海马神经元凋亡基因Bcl-2、Bax及Bcl-2/Bax比例变化的影响[J].时珍国医国药,2018,29(6):1336-1338.
[14]靳贺超,于文涛,刘晓,等.补肾活血方对血管性痴呆大鼠脑海马细胞凋亡及ERK2、CREB表达的影响[J].中国实验方剂学杂志,2018,24(12):129-135.
[15]李莉,戴宁,那莎,等.五子衍宗丸对实验性少弱精子症大鼠的保护作用与机制研究[J].中华男科学杂志,2016,22(9):827-833.
[16]谭为,马柯,周卫东,等.五子衍宗丸含药血清对紫杉醇诱导的小肠隐窝细胞IEC-6化疗损伤的保护机制[J].中华中医药杂志,2017,32(12):5551-5554.
[17]QI B,JI Q,WEN Y,et al. Lyciumbarbarum polysaccharides protect human lens epithelial cells against oxidative stress-induced apoptosis and senescence[J]. PLo S One,2014,9(10):e110275.
[18]SUN SL,GUO L,REN YC,et al. Anti-apoptosis effect of polysaccharide isolated from the seeds of Cuscutachinensis Lam on cardiomyocytes in aging rats[J]. Mol Biol Rep,2014,41(9):6117-6124.
[19]赵梦岚,燕波,刘超,等.覆盆子乙酸乙酯部位不同单体对H2O2诱导PC12细胞损伤的保护作用[J].中药药理与临床,2018,34(3):58-62.
[20]ELMORE S. Apoptosis:a review of programmed cell death[J]. Toxicol Pathol,2007,35(4):495-516.
[21]李敏,林俊.细胞凋亡途径及其机制[J].国际妇产科学杂志,2014,41(2):103-107.
[22]李欲轲,熊孟连,徐僡,等.内质网应激与凋亡研究进展[J].分子植物育种,2018,16(23):7856-7862.
[23]夏元平,王立花,樊燕蓉.细胞凋亡与内质网应激机制[J].药学与临床研究,2010,18(3):291-293,298.
[24]张利平,孟燕,元小冬,等.内质网应激及介导细胞凋亡信号转导研究进展[J].生物医学工程与临床,2018,22(2):214-220.
[25]GREENWOOD M,GREENWOOD MP,PATON JF,et al. Transcription factor CREB3L1 regulates endoplasmic reticulum stress response genes in the osmotically challenged rat hypothalamus[J]. PLo S One,2015,10(4):e0124956.
[26]SCHoNTHAL AH. Endoplasmic reticulum stress:its role in disease and novel prospects for therapy[J]. Scientifica(Cairo),2012,2012:857516. doi:10. 6064/2012/857516.
[27]DUBREZ-DALOZ L,DUPOUX A,CARTIER J. IAPs:more than just inhibitors of apoptosis proteins[J]. Cell Cycle,2008,7(8):1036-1046.
[2]YAMAGUCHI Y,SHINOTSUKA N,NONOMURA K,et al. Live imaging of apoptosis in a novel transgenic mouse highlights its role in neural tube closure[J]. J Cell Bio,2011,195(6):1047-1060.
[3]GREENE ND,COPPA J. Neural tube defects[J]. Annu Rev Neurosci,2014,37:221-242. doi:10. 1146/annurevneuro-062012-170354.
[4]CECCONI F,PIACENTINI M,FIMIA GM. The involvement of cell death and survival in neural tube defects:a distinct role for apoptosis and autophagy?[J]. Cell Death Differ,2008,15(7):1170-1177.
[5]FUKUDA T,TOKUNAGA A,SAKAMOTO R,et al.Fbxl10/Kdm2b deficiency accelerates neural progenitor cell death and leads to exencephaly[J]. Mol Cell Neurosci,2011,46(3):614-624.
[6]PARKER SE,YAZDY MM,TINKER SC,et al. The impact of folic acid intake on the association among diabetes mellitus,obesity,and spina bifida[J]. Am J Obstet Gynecol,2013,209(3):239. e1-e8.
[7]樊慧杰,柴智,解军,等.五子衍宗丸对ATRA诱导的神经管畸形防治作用的初探[J].中成药,2014,36(5):1054-1056.
[8]樊慧杰,李艳彦,柴智,等.五子衍宗丸对NTDs胎鼠神经管细胞凋亡的影响[J].中国实验方剂学杂志,2014,20(9):143-146.
[9]CHAI Z,YANG LH,YU BF,et al. p38 Mitogenactivated protein kinase-dependent regulation of SRC-3 and involvement in retinoic acid receptor a signaling in embryonic cortical neurons[J]. IUBMB Life,2009,61(6):670-678.
[10]LIU JG,ZHOU R,HE QR,et al. Calmodulin kinaseⅡactivation of mitogen-activated protein kinase in PC12 cell following all-trans retinoic acid treatment[J].Neurotoxicology,2009,30(4):599-604.
[11]刘珊,代晓南,崔毓桂,等.早期胚胎发育与细胞凋亡[J].国际生育健康/计划生育杂志,2014,33(1):32-35.
[12]王莉,吕爱平,王梅,等.“肾藏精”与生长壮老关系的现代研究[J].现代中西医结合杂志,2018,27(9):1021-1023.
[13]单海军,曹彩红,张英英,等.补肾开窍祛痰方对缺氧缺血性脑损伤新生大鼠海马神经元凋亡基因Bcl-2、Bax及Bcl-2/Bax比例变化的影响[J].时珍国医国药,2018,29(6):1336-1338.
[14]靳贺超,于文涛,刘晓,等.补肾活血方对血管性痴呆大鼠脑海马细胞凋亡及ERK2、CREB表达的影响[J].中国实验方剂学杂志,2018,24(12):129-135.
[15]李莉,戴宁,那莎,等.五子衍宗丸对实验性少弱精子症大鼠的保护作用与机制研究[J].中华男科学杂志,2016,22(9):827-833.
[16]谭为,马柯,周卫东,等.五子衍宗丸含药血清对紫杉醇诱导的小肠隐窝细胞IEC-6化疗损伤的保护机制[J].中华中医药杂志,2017,32(12):5551-5554.
[17]QI B,JI Q,WEN Y,et al. Lyciumbarbarum polysaccharides protect human lens epithelial cells against oxidative stress-induced apoptosis and senescence[J]. PLo S One,2014,9(10):e110275.
[18]SUN SL,GUO L,REN YC,et al. Anti-apoptosis effect of polysaccharide isolated from the seeds of Cuscutachinensis Lam on cardiomyocytes in aging rats[J]. Mol Biol Rep,2014,41(9):6117-6124.
[19]赵梦岚,燕波,刘超,等.覆盆子乙酸乙酯部位不同单体对H2O2诱导PC12细胞损伤的保护作用[J].中药药理与临床,2018,34(3):58-62.
[20]ELMORE S. Apoptosis:a review of programmed cell death[J]. Toxicol Pathol,2007,35(4):495-516.
[21]李敏,林俊.细胞凋亡途径及其机制[J].国际妇产科学杂志,2014,41(2):103-107.
[22]李欲轲,熊孟连,徐僡,等.内质网应激与凋亡研究进展[J].分子植物育种,2018,16(23):7856-7862.
[23]夏元平,王立花,樊燕蓉.细胞凋亡与内质网应激机制[J].药学与临床研究,2010,18(3):291-293,298.
[24]张利平,孟燕,元小冬,等.内质网应激及介导细胞凋亡信号转导研究进展[J].生物医学工程与临床,2018,22(2):214-220.
[25]GREENWOOD M,GREENWOOD MP,PATON JF,et al. Transcription factor CREB3L1 regulates endoplasmic reticulum stress response genes in the osmotically challenged rat hypothalamus[J]. PLo S One,2015,10(4):e0124956.
[26]SCHoNTHAL AH. Endoplasmic reticulum stress:its role in disease and novel prospects for therapy[J]. Scientifica(Cairo),2012,2012:857516. doi:10. 6064/2012/857516.
[27]DUBREZ-DALOZ L,DUPOUX A,CARTIER J. IAPs:more than just inhibitors of apoptosis proteins[J]. Cell Cycle,2008,7(8):1036-1046.
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