OBJECTIVE To investigate icariside(ICS)Ⅱ protects against PC12 cel damage induced by oxygen-glucose deprivation and reoxygenation and explore its mechanism.METHODS The oxidative stress injury model was induced by oxy...OBJECTIVE To investigate icariside(ICS)Ⅱ protects against PC12 cel damage induced by oxygen-glucose deprivation and reoxygenation and explore its mechanism.METHODS The oxidative stress injury model was induced by oxygen-glucose deprivation/reoxygenation(OGD/R) 2 h/24 h in PC12 cells.N-acetyl-lcysteine(NAC),a classical anti-oxidant,was used as positive control.Pharmacodynamic experimental study groups as follows:control,control+ICS Ⅱ50 μmol·L^(-1),OGD/R,OGD/R+ICSⅡ 12.5 μmol·L^(-1),OGD/R + ICS Ⅱ 25 μmol·L^(-1),OGD/R + ICS Ⅱ50 μmol·L^(-1),and OGD/R+NAC 100 μmol·L^(-1) groups.Cell viability and lactate dehydrogenase(LDH) leakage rate were measured by MTT assay and LDH ELISA kit,respectively.Moreover,reactive oxygen species(ROS) ELISA kit was used for detection of intracellular ROS generation,Mito-SOX fluorescence staining was used for detecting production of ROS in mitochondria and mitochondrial membrane potential(MMP)was detected by rhodamine 123 dye.In addition,PC12 cells apoptosis was detected by one-step TUNEL assay.Furthermore,the expressions of nuclear factor erythroid 2-related factors(Nrf2),Keap1,HO^(-1),NQO^(-1),silent information regulator 3(SIRT3),IDH2,Bax,Bcl-2 and caspase 3 were detected by Western blotting analysis.RESULTS The results of MTT and LDH assay showed that OGD/R reduced the cell viability and improved LDH release compared with the control or ICSⅡ 50 μmol·L^(-1) alone(P<0.01).Meanwhile,OGD/R not only increased intracellular and mitochondrial ROS generation,but also elevated the fluorescence intensity of TUNEL staining,at the same time,the MMP was declined when challenged by OGD/R.Furthermore,the Western blotting results showed that OGD/R induced the increase in the expression of cytoplasm-Nrf2,Keap1,Bax and cleaved-caspase 3 level,while the decrease in the expression of nucleus-Nrf2,HO^(-1),NQO^(-1),SIRT3,IDH2 and Bcl-2(P<0.05).However,ICS Ⅱ significantly increased the viability of PC12 cells and reduced LDH leakage(P<0.01).Notably,ICS Ⅱ also suppressed ROS generation both in the intracellular and mitochondria,as well as restored MMP.It was also worthy to note that ICS Ⅱ decreased the expressions of cytoplasmNrf2,Keap1,Bax and the level of cleaved-caspase3,whereas,it increased the expressions of nucleus-Nrf2,HO^(-1),NQO^(-1),SIRT3,IDH2 and Bcl-2(P<0.05).CONCLUSION ICSⅡ reduced OGD/Rinduced oxidative damage in PC12 cells under the laboratory conditions,and its underlying mechanism may be related to the regulation of Nrf2/SIRT3 signaling pathway.展开更多
Sirtuin家族是酵母沉默信息调节因子(silent information regulator factor)在哺乳动物中的同源物,属于Ⅲ类组蛋白去乙酰化酶。沉默信息调节因子2相关酶1(silent information regulator factor 2-related enzyme 1,SIRT1)是Sirtuin家族...Sirtuin家族是酵母沉默信息调节因子(silent information regulator factor)在哺乳动物中的同源物,属于Ⅲ类组蛋白去乙酰化酶。沉默信息调节因子2相关酶1(silent information regulator factor 2-related enzyme 1,SIRT1)是Sirtuin家族目前研究最为充分的成员之一。主要分布于细胞核及细胞质,通过行使其NAD+依赖的去乙酰化活性,参与调节DNA损伤修复、基因转录、能量代谢、应激及凋亡等细胞生理过程。细胞凋亡是在精确的内在遗传调控下发生的一种程序性死亡,旨在维持机体内环境稳态。异常的细胞凋亡参与多种疾病的发生,例如肿瘤、神经退行性病变、自身免疫病和缺血性疾病等,了解细胞凋亡的机制对于这些疾病的治疗有着极为重要的意义。SIRT1除靶向组蛋白外,还作用于多种非组蛋白质例如转录因子和蛋白激酶等,其中有很多非组蛋白分子都与细胞凋亡息息相关。本文总结了SIRT1通过调控相关下游非组蛋白分子p53、叉头转录因子FOXO3a、AMP依赖的蛋白激酶(AMPK)、核转录因子NF-κB、ku70蛋白、转录因子E2F1和缺氧诱导因子-1α的乙酰化修饰水平,进而影响基因转录、DNA损伤修复、炎症、氧化应激等过程,直接或间接触发细胞凋亡的分子机制。希望通过调控SIRT1表达来影响其下游非组蛋白分子的乙酰化修饰水平,进而对细胞凋亡进行干预,为多种相关疾病的治疗提供新的靶点。展开更多
基金National Natural Science Foundation of China(81560666)Program for Excellent Young Talents of Zunyi Medical Uiverstity(15zy-002)+1 种基金Science and Technology Innovation Talent Team of Guizhou Province(20154023)the ″Hundred″Level of High-level Innovative Talents in Guizhou Province(QKHRCPT 20165684)；and Program forChangjiang Scholars and Innovative ResearchTeam in University of China(IRT一17R113).
文摘OBJECTIVE To investigate icariside(ICS)Ⅱ protects against PC12 cel damage induced by oxygen-glucose deprivation and reoxygenation and explore its mechanism.METHODS The oxidative stress injury model was induced by oxygen-glucose deprivation/reoxygenation(OGD/R) 2 h/24 h in PC12 cells.N-acetyl-lcysteine(NAC),a classical anti-oxidant,was used as positive control.Pharmacodynamic experimental study groups as follows:control,control+ICS Ⅱ50 μmol·L^(-1),OGD/R,OGD/R+ICSⅡ 12.5 μmol·L^(-1),OGD/R + ICS Ⅱ 25 μmol·L^(-1),OGD/R + ICS Ⅱ50 μmol·L^(-1),and OGD/R+NAC 100 μmol·L^(-1) groups.Cell viability and lactate dehydrogenase(LDH) leakage rate were measured by MTT assay and LDH ELISA kit,respectively.Moreover,reactive oxygen species(ROS) ELISA kit was used for detection of intracellular ROS generation,Mito-SOX fluorescence staining was used for detecting production of ROS in mitochondria and mitochondrial membrane potential(MMP)was detected by rhodamine 123 dye.In addition,PC12 cells apoptosis was detected by one-step TUNEL assay.Furthermore,the expressions of nuclear factor erythroid 2-related factors(Nrf2),Keap1,HO^(-1),NQO^(-1),silent information regulator 3(SIRT3),IDH2,Bax,Bcl-2 and caspase 3 were detected by Western blotting analysis.RESULTS The results of MTT and LDH assay showed that OGD/R reduced the cell viability and improved LDH release compared with the control or ICSⅡ 50 μmol·L^(-1) alone(P<0.01).Meanwhile,OGD/R not only increased intracellular and mitochondrial ROS generation,but also elevated the fluorescence intensity of TUNEL staining,at the same time,the MMP was declined when challenged by OGD/R.Furthermore,the Western blotting results showed that OGD/R induced the increase in the expression of cytoplasm-Nrf2,Keap1,Bax and cleaved-caspase 3 level,while the decrease in the expression of nucleus-Nrf2,HO^(-1),NQO^(-1),SIRT3,IDH2 and Bcl-2(P<0.05).However,ICS Ⅱ significantly increased the viability of PC12 cells and reduced LDH leakage(P<0.01).Notably,ICS Ⅱ also suppressed ROS generation both in the intracellular and mitochondria,as well as restored MMP.It was also worthy to note that ICS Ⅱ decreased the expressions of cytoplasmNrf2,Keap1,Bax and the level of cleaved-caspase3,whereas,it increased the expressions of nucleus-Nrf2,HO^(-1),NQO^(-1),SIRT3,IDH2 and Bcl-2(P<0.05).CONCLUSION ICSⅡ reduced OGD/Rinduced oxidative damage in PC12 cells under the laboratory conditions,and its underlying mechanism may be related to the regulation of Nrf2/SIRT3 signaling pathway.
文摘Sirtuin家族是酵母沉默信息调节因子(silent information regulator factor)在哺乳动物中的同源物,属于Ⅲ类组蛋白去乙酰化酶。沉默信息调节因子2相关酶1(silent information regulator factor 2-related enzyme 1,SIRT1)是Sirtuin家族目前研究最为充分的成员之一。主要分布于细胞核及细胞质,通过行使其NAD+依赖的去乙酰化活性,参与调节DNA损伤修复、基因转录、能量代谢、应激及凋亡等细胞生理过程。细胞凋亡是在精确的内在遗传调控下发生的一种程序性死亡,旨在维持机体内环境稳态。异常的细胞凋亡参与多种疾病的发生,例如肿瘤、神经退行性病变、自身免疫病和缺血性疾病等,了解细胞凋亡的机制对于这些疾病的治疗有着极为重要的意义。SIRT1除靶向组蛋白外,还作用于多种非组蛋白质例如转录因子和蛋白激酶等,其中有很多非组蛋白分子都与细胞凋亡息息相关。本文总结了SIRT1通过调控相关下游非组蛋白分子p53、叉头转录因子FOXO3a、AMP依赖的蛋白激酶(AMPK)、核转录因子NF-κB、ku70蛋白、转录因子E2F1和缺氧诱导因子-1α的乙酰化修饰水平,进而影响基因转录、DNA损伤修复、炎症、氧化应激等过程,直接或间接触发细胞凋亡的分子机制。希望通过调控SIRT1表达来影响其下游非组蛋白分子的乙酰化修饰水平,进而对细胞凋亡进行干预,为多种相关疾病的治疗提供新的靶点。
