受体相互作用蛋白1(RIP1)对BCG诱导小鼠巨噬细胞凋亡的调控作用
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摘要
旨在通过构建受体相互作用蛋白1(RIP1)腺病毒干扰载体,研究其对BCG诱导的RAW264.7细胞凋亡相关指标的影响,以探讨其在BCG诱导RAW264.7凋亡过程中的调控作用。笔者构建RIP1腺病毒干扰载体,并转染感染BCG的小鼠RAW264.7细胞系,利用流式细胞仪检测各处理细胞凋亡率、细胞线粒体膜电位、细胞活性氧水平及细胞周期等指标,并用Western blot检测凋亡相关蛋白的表达水平。结果显示:BCG感染显著上调了RIP1的蛋白表达水平并提高了小鼠巨噬细胞RAW264.7的凋亡率,当RIP1被干扰后,BCG感染后的RAW264.7细胞凋亡率和活性氧水平显著降低,而促凋亡蛋白Bax表达量显著下调,线粒体膜电位和抑凋亡蛋白表达量上调。同时,BCG感染后细胞周期滞留于G_1期。BCG感染可有效上调RIP1表达量并诱导RAW264.7细胞凋亡。RIP1通过下调BCG感染后RAW264.7细胞的线粒体膜电位,上调活性氧含量并提高凋亡相关蛋白Bax/Bcl-2比值,使细胞周期阻滞于G_1期从而参与诱导细胞凋亡。
The purpose of this study was to explore the effect of RIP1 on apoptosis of RAW264.7 cell by assessment the apoptosis related indicators of RAW264.7 cell treated with RIP1 RNAi vector or/and BCG infection. The RIP1 adenovirus RNAi vector was constructed and transfected into BCG-infected RAW264.7 cell line. Flow cytometry was used to detect the apoptotic rate, mitochondrial membrane potential, cell reactive oxygen species and cell cycle of RAW264.7 cell. The expression of RIP1 and apoptosis associated proteins were examined by Western blot. The results showed that BCG-infection can significantly induce RAW264.7 cells apoptosis, which was accompanied with up-regulation of PIP1 protein. In addition, a RIP1 adenovirus RNAi vector showed an ability to reduce the BCG-induced macrophage apoptosis along with an increased mitochondrial membrane potential and reduced reactive oxygen species(ROS). It also down-regulated Bax expression and up-regulated Bcl-2 expression of RAW264.7 cell infected with BCG. Intriguingly, cell cycle of RAW264.7 cell was arrested in G_1 phase. In conclusion, RIP1 involves in BCG-induced apoptosis by down-regulating mitochondrial membrane potential, increasing the production of ROS and the ratio of apoptosis-related proteins(Bax/Bcl-2) and arresting the cell cycle in G_1 phase in BCG-infectied RAW264.7.
The purpose of this study was to explore the effect of RIP1 on apoptosis of RAW264.7 cell by assessment the apoptosis related indicators of RAW264.7 cell treated with RIP1 RNAi vector or/and BCG infection. The RIP1 adenovirus RNAi vector was constructed and transfected into BCG-infected RAW264.7 cell line. Flow cytometry was used to detect the apoptotic rate, mitochondrial membrane potential, cell reactive oxygen species and cell cycle of RAW264.7 cell. The expression of RIP1 and apoptosis associated proteins were examined by Western blot. The results showed that BCG-infection can significantly induce RAW264.7 cells apoptosis, which was accompanied with up-regulation of PIP1 protein. In addition, a RIP1 adenovirus RNAi vector showed an ability to reduce the BCG-induced macrophage apoptosis along with an increased mitochondrial membrane potential and reduced reactive oxygen species(ROS). It also down-regulated Bax expression and up-regulated Bcl-2 expression of RAW264.7 cell infected with BCG. Intriguingly, cell cycle of RAW264.7 cell was arrested in G_1 phase. In conclusion, RIP1 involves in BCG-induced apoptosis by down-regulating mitochondrial membrane potential, increasing the production of ROS and the ratio of apoptosis-related proteins(Bax/Bcl-2) and arresting the cell cycle in G_1 phase in BCG-infectied RAW264.7.
引文
[1] 司壮丽,周远忠. 耐药结核病发生和传播的分子流行病学研究进展[J]. 牡丹江医学院学报, 2015, 36(5):92-96.SI Z L, ZHOU Y Z. Advances in molecular epidemiology of the occurrence and transmission of drug-resistant tuberculosis[J]. Journal of Mudanjiang Medical University, 2015, 36(5):92-96. (in Chinese)
[2] World Health Organization. Global tuberculosis report[R]. Global Tuberculosis Report. France: WHO, 2017.
[3] VARIAVA E, MARTINSON N. Drug-resistant tuberculosis:the rise of the monos[J]. Lancet Infect Dis, 2018, 18(7):705-706.
[4] OFENGEIM D, YUAN J Y. Regulation of RIP1 kinase signalling at the crossroads of inflammation and cell death[J]. Nat Rev Mol Cell Biol, 2013, 14(11):727-736.
[5] XU Q, JITKAEW S, CHOKSI S, et al. The cytoplasmic nuclear receptor RARγ controls RIP1 initiated cell death when cIAP activity is inhibited[J]. Nat Commun, 2017, 8(1):425.
[6] ROCA F J, RAMAKRISHNAN L. TNF dually mediates resistance and susceptibility to mycobacteria via mitochondrial reactive oxygen species[J]. Cell, 2013, 153(3):521-534.
[7] XU Y, JAGANNATH C, LIU X D, et al. Toll-like receptor 4 is a sensor for autophagy associated with innate immunity[J]. Immunity, 2007, 27(1):135-144.
[8] BERTRAND M J M, MILUTINOVIC S, DICKSON K M, et al. cIAP1 and cIAP2 facilitate cancer cell survival by functioning as E3 ligases that promote RIP1 ubiquitination[J]. Mol Cell, 2008, 30(6):689-700.
[9] CHEN C, XIAO W, HUANG L, et al. Shikonin induces apoptosis and necroptosis in pancreatic cancer via regulating the expression of RIP1/RIP3 and synergizes the activity of gemcitabine[J]. Am J Transl Res, 2017, 9(12):5507-5517.
[10] LUAN Q, JIN L, JIANG C C, et al. RIPK1 regulates survival of human melanoma cells upon endoplasmic reticulum stress through autophagy[J]. Autophagy, 2015, 11(7):975-994.
[11] FESTJENS N, VANDEN BERGHE T, CORNELIS S, et al. RIP1, a kinase on the crossroads of a cell’s decision to live or die[J]. Cell Death Differ, 2007, 14(3):400-410.
[12] 张嘉美,马臣杰,赵宁,等. Necrostatin-1对BCG感染后小鼠巨噬细胞RAW264. 7凋亡的调控[J]. 农业生物技术学报, 2016, 24(10):1552-1559.ZHANG J M,MA C J, ZHAO N, et al. Necrostatin-1 modulate the apoptosis induced by BCG in a macrophage cell line RAW264. 7[J]. Journal of Agricultural Biotechnology, 2016, 24(10):1552-1559. (in Chinese)
[13] ASHKENAZI A, FAIRBROTHER W J, LEVERSON J D, et al. From basic apoptosis discoveries to advanced selective BCL-2 family inhibitors[J]. Nat Rev Drug Discov, 2017, 16(4):273-284.
[14] ROOS W P, THOMAS A D, KAINA B. DNA damage and the balance between survival and death in cancer biology[J]. Nat Rev Cancer, 2016, 16(1):20-33.
[15] COLSTON M J. The molecular basis of mycobacterial infection[J]. Mol Aspects Med, 1996, 17(4):385-454.
[16] AWUH J A, FLO T H. Molecular basis of mycobacterial survival in macrophages[J]. Cell Mol Life Sci, 2017, 74(9):1625-1648.
[17] BEHAR S M, MARTIN C J, BOOTY M G, et al. Apoptosis is an innate defense function of macrophages against Mycobacterium tuberculosis[J]. Mucosal Immunol, 2011, 4(3):279-287.
[18] WU X L, DENG G C, HAO X J, et al. A caspase-dependent pathway is involved in Wnt/β-catenin signaling promoted apoptosis in bacillus calmette-guerin infected RAW264. 7 macrophages[J]. Int J Mol Sci, 2014, 15(3):5045-5062.
[19] HOLLER N, ZARU R, MICHEAU O, et al. Fas triggers an alternative, caspase-8-independent cell death pathway usingthe kinase RIP as effector molecule[J]. Nat Immunol, 2000, 1(6):489-495.
[20] ZWERLING A, BEHR M A, VERMA A, et al. The BCG World Atlas:a database of global BCG vaccination policies and practices[J]. PLoS Med, 2011, 8(3):e1001012.
[21] ORME I M. The Achilles heel of BCG[J]. Tuberculosis (Edinb), 2010, 90(6):329-332.
[22] WINAU F, WEBER S, SAD S, et al. Apoptotic vesicles crossprime CD8 T cells and protect against tuberculosis[J]. Immunity, 2006, 24(1):105-117.
[23] FARINACCI M, WEBER S, KAUFMANN S H E. The recombinant tuberculosis vaccine rBCG ΔureC::hly+ induces apoptotic vesicles for improved priming of CD4+ and CD8+ T cells[J]. Vaccine, 2012, 30(52):7608-7614.
[24] CRISTOFANON S, ABHARI B A, KRUEGER M, et al. Identification of RIP1 as a critical mediator of Smac mimetic-mediated sensitization of glioblastoma cells for Drozitumab-induced apoptosis[J]. Cell Death Dis, 2015, 6:e1724.
[25] WANG J S, WU D, HUANG D Y, et al. TAK1 inhibition-induced RIP1-dependent apoptosis in murine macrophages relies on constitutive TNF-α signaling and ROS production[J]. J Biomed Sci, 2015, 22(1):76.
[26] DONDELINGER Y, AGUILETA M A, GOOSSENS V, et al. RIPK3 contributes to TNFR1-mediated RIPK1 kinase-dependent apoptosis in conditions of cIAP1/2 depletion or TAK1 kinase inhibition[J]. Cell Death Differ, 2013, 20(10):1381-1392.
[27] FAYNGERTS S A, WANG Z J, ZAMANI A, et al. Direction of leukocyte polarization and migration by the phosphoinositide-transfer protein TIPE2[J]. Nat Immunol, 2017, 18(12):1353-1360.
[28] MIHALY S R, NINOMIYA-TSUJI J, MORIOKA S. TAK1 control of cell death[J]. Cell Death Differ, 2014, 21(11):1667-1676.
[29] YU X F, PAN Y H, MA H S, et al. Simvastatin inhibits proliferation and induces apoptosis in human lung cancer cells[J]. Oncol Res, 2013, 20(8):351-357.
[30] DíAZ-GARCíA A, MORIER-DíAZ L, FRIóN-HERRERA Y, et al. In vitro anticancer effect of venom from Cuban scorpion Rhopalurus junceus against a panel of human cancer cell lines[J]. J Venom Res, 2013, 4:5-12.
[31] PARK S, RAMNARAIN D B, HATANPAA K J, et al. The death domain-containing kinase RIP1 regulates p27Kip1 levels through the PI3K-Akt-forkhead pathway[J]. EMBO Rep, 2008, 9(8):766-773.
[2] World Health Organization. Global tuberculosis report[R]. Global Tuberculosis Report. France: WHO, 2017.
[3] VARIAVA E, MARTINSON N. Drug-resistant tuberculosis:the rise of the monos[J]. Lancet Infect Dis, 2018, 18(7):705-706.
[4] OFENGEIM D, YUAN J Y. Regulation of RIP1 kinase signalling at the crossroads of inflammation and cell death[J]. Nat Rev Mol Cell Biol, 2013, 14(11):727-736.
[5] XU Q, JITKAEW S, CHOKSI S, et al. The cytoplasmic nuclear receptor RARγ controls RIP1 initiated cell death when cIAP activity is inhibited[J]. Nat Commun, 2017, 8(1):425.
[6] ROCA F J, RAMAKRISHNAN L. TNF dually mediates resistance and susceptibility to mycobacteria via mitochondrial reactive oxygen species[J]. Cell, 2013, 153(3):521-534.
[7] XU Y, JAGANNATH C, LIU X D, et al. Toll-like receptor 4 is a sensor for autophagy associated with innate immunity[J]. Immunity, 2007, 27(1):135-144.
[8] BERTRAND M J M, MILUTINOVIC S, DICKSON K M, et al. cIAP1 and cIAP2 facilitate cancer cell survival by functioning as E3 ligases that promote RIP1 ubiquitination[J]. Mol Cell, 2008, 30(6):689-700.
[9] CHEN C, XIAO W, HUANG L, et al. Shikonin induces apoptosis and necroptosis in pancreatic cancer via regulating the expression of RIP1/RIP3 and synergizes the activity of gemcitabine[J]. Am J Transl Res, 2017, 9(12):5507-5517.
[10] LUAN Q, JIN L, JIANG C C, et al. RIPK1 regulates survival of human melanoma cells upon endoplasmic reticulum stress through autophagy[J]. Autophagy, 2015, 11(7):975-994.
[11] FESTJENS N, VANDEN BERGHE T, CORNELIS S, et al. RIP1, a kinase on the crossroads of a cell’s decision to live or die[J]. Cell Death Differ, 2007, 14(3):400-410.
[12] 张嘉美,马臣杰,赵宁,等. Necrostatin-1对BCG感染后小鼠巨噬细胞RAW264. 7凋亡的调控[J]. 农业生物技术学报, 2016, 24(10):1552-1559.ZHANG J M,MA C J, ZHAO N, et al. Necrostatin-1 modulate the apoptosis induced by BCG in a macrophage cell line RAW264. 7[J]. Journal of Agricultural Biotechnology, 2016, 24(10):1552-1559. (in Chinese)
[13] ASHKENAZI A, FAIRBROTHER W J, LEVERSON J D, et al. From basic apoptosis discoveries to advanced selective BCL-2 family inhibitors[J]. Nat Rev Drug Discov, 2017, 16(4):273-284.
[14] ROOS W P, THOMAS A D, KAINA B. DNA damage and the balance between survival and death in cancer biology[J]. Nat Rev Cancer, 2016, 16(1):20-33.
[15] COLSTON M J. The molecular basis of mycobacterial infection[J]. Mol Aspects Med, 1996, 17(4):385-454.
[16] AWUH J A, FLO T H. Molecular basis of mycobacterial survival in macrophages[J]. Cell Mol Life Sci, 2017, 74(9):1625-1648.
[17] BEHAR S M, MARTIN C J, BOOTY M G, et al. Apoptosis is an innate defense function of macrophages against Mycobacterium tuberculosis[J]. Mucosal Immunol, 2011, 4(3):279-287.
[18] WU X L, DENG G C, HAO X J, et al. A caspase-dependent pathway is involved in Wnt/β-catenin signaling promoted apoptosis in bacillus calmette-guerin infected RAW264. 7 macrophages[J]. Int J Mol Sci, 2014, 15(3):5045-5062.
[19] HOLLER N, ZARU R, MICHEAU O, et al. Fas triggers an alternative, caspase-8-independent cell death pathway usingthe kinase RIP as effector molecule[J]. Nat Immunol, 2000, 1(6):489-495.
[20] ZWERLING A, BEHR M A, VERMA A, et al. The BCG World Atlas:a database of global BCG vaccination policies and practices[J]. PLoS Med, 2011, 8(3):e1001012.
[21] ORME I M. The Achilles heel of BCG[J]. Tuberculosis (Edinb), 2010, 90(6):329-332.
[22] WINAU F, WEBER S, SAD S, et al. Apoptotic vesicles crossprime CD8 T cells and protect against tuberculosis[J]. Immunity, 2006, 24(1):105-117.
[23] FARINACCI M, WEBER S, KAUFMANN S H E. The recombinant tuberculosis vaccine rBCG ΔureC::hly+ induces apoptotic vesicles for improved priming of CD4+ and CD8+ T cells[J]. Vaccine, 2012, 30(52):7608-7614.
[24] CRISTOFANON S, ABHARI B A, KRUEGER M, et al. Identification of RIP1 as a critical mediator of Smac mimetic-mediated sensitization of glioblastoma cells for Drozitumab-induced apoptosis[J]. Cell Death Dis, 2015, 6:e1724.
[25] WANG J S, WU D, HUANG D Y, et al. TAK1 inhibition-induced RIP1-dependent apoptosis in murine macrophages relies on constitutive TNF-α signaling and ROS production[J]. J Biomed Sci, 2015, 22(1):76.
[26] DONDELINGER Y, AGUILETA M A, GOOSSENS V, et al. RIPK3 contributes to TNFR1-mediated RIPK1 kinase-dependent apoptosis in conditions of cIAP1/2 depletion or TAK1 kinase inhibition[J]. Cell Death Differ, 2013, 20(10):1381-1392.
[27] FAYNGERTS S A, WANG Z J, ZAMANI A, et al. Direction of leukocyte polarization and migration by the phosphoinositide-transfer protein TIPE2[J]. Nat Immunol, 2017, 18(12):1353-1360.
[28] MIHALY S R, NINOMIYA-TSUJI J, MORIOKA S. TAK1 control of cell death[J]. Cell Death Differ, 2014, 21(11):1667-1676.
[29] YU X F, PAN Y H, MA H S, et al. Simvastatin inhibits proliferation and induces apoptosis in human lung cancer cells[J]. Oncol Res, 2013, 20(8):351-357.
[30] DíAZ-GARCíA A, MORIER-DíAZ L, FRIóN-HERRERA Y, et al. In vitro anticancer effect of venom from Cuban scorpion Rhopalurus junceus against a panel of human cancer cell lines[J]. J Venom Res, 2013, 4:5-12.
[31] PARK S, RAMNARAIN D B, HATANPAA K J, et al. The death domain-containing kinase RIP1 regulates p27Kip1 levels through the PI3K-Akt-forkhead pathway[J]. EMBO Rep, 2008, 9(8):766-773.