脂连蛋白降低氯化钴诱导的巨噬细胞凋亡及机制
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摘要
目的研究氯化钴(CoCl_2)诱导的低氧条件下,脂连蛋白(APN)对RAW264. 7巨噬细胞凋亡的保护作用及其机制。方法巨噬细胞分为CoCl_2处理组和APN预处理组,前者给予100μmol/L CoCl_2,后者先用(0. 5、1、2)μg/m L APN预处理2 h,再给予100μmol/L的CoCl_2处理。CCK-8法检测各组细胞活力;免疫荧光法观察细胞胱天蛋白酶3(caspase-3)的分布及CoCl_2和APN对其表达的影响;流式细胞术检测各组细胞的凋亡比例;分光光度计法检测各组细胞内超氧化物歧化酶(SOD)及丙二醛(MDA)含量; Western blot法检测各组细胞B细胞淋巴瘤因子2(Bcl2)、BAX、裂解型caspase-3(c-caspase-3)及低氧诱导因子1α(HIF-1α)蛋白水平。结果与CoCl_2组相比,APN预处理能提高细胞活力,减少细胞凋亡并升高SOD含量、降低MDA含量;Caspase-3主要分布在细胞膜上,与CoCl_2组相比,APN预处理能上调Bcl2的表达,下调BAX、c-caspase-3及HIF-1α的表达。结论 APN可减少CoCl_2诱导的巨噬细胞凋亡,可能是通过增强细胞内抗氧化活性,以及上调Bcl2、下调BAX、c-caspase-3及HIF-1α的表达来实现的。
Objective To investigate the protective effects and mechanism of adiponectin( APN) on RAW264. 7macrophages under cell model of hypoxia established by CoCl_2. Methods Cells were treated with CoCl_2( 100 μmol/L) anddifferent concentrations of APN( 0. 5,1,2 μg/m L APN,pretreated for 2 hours firstly,and treated with CoCl_2 in a dose of100 μmol/L). The cells' vitality in each group was detected by CCK-8 assay. The distribution of caspase-3,as well as theinfluences of CoCl_2 and APN on the expression of caspase-3 was detected by immunofluorescence technique. Apoptotic ratesof cells in all groups were analyzed by flow cytometry. The levels of SOD and MDA in each group were measured byspectrophotometer. The expressions of Bcl2,BAX,cleaved caspase-3 and HIF-1α of cells in each group were observed byWestern blot analysis. Results Compared with CoCl_2 group,pretreatment with APN could increase cell vitality,decreaseapoptosis,increase the level of SOD,and decrease the level of MDA. Immunofluorescence results revealed that caspase-3in each group was mainly distributed in the cel membranes,and compared with CoCl_2 group,the expression of Bcl2 was evidentlyincreased,BAX,cleaved caspase-3 and HIF-1α were decreased after being pretreated with APN. Conclusion APN coulddecrease CoCl_2-induced apoptosis in RAW264. 7 cells,which may occur via enhancing the intracellular antioxidant activity,and by upregulation of Bcl2,downregulation of BAX,cleaved caspase-3 and HIF-1α.
Objective To investigate the protective effects and mechanism of adiponectin( APN) on RAW264. 7macrophages under cell model of hypoxia established by CoCl_2. Methods Cells were treated with CoCl_2( 100 μmol/L) anddifferent concentrations of APN( 0. 5,1,2 μg/m L APN,pretreated for 2 hours firstly,and treated with CoCl_2 in a dose of100 μmol/L). The cells' vitality in each group was detected by CCK-8 assay. The distribution of caspase-3,as well as theinfluences of CoCl_2 and APN on the expression of caspase-3 was detected by immunofluorescence technique. Apoptotic ratesof cells in all groups were analyzed by flow cytometry. The levels of SOD and MDA in each group were measured byspectrophotometer. The expressions of Bcl2,BAX,cleaved caspase-3 and HIF-1α of cells in each group were observed byWestern blot analysis. Results Compared with CoCl_2 group,pretreatment with APN could increase cell vitality,decreaseapoptosis,increase the level of SOD,and decrease the level of MDA. Immunofluorescence results revealed that caspase-3in each group was mainly distributed in the cel membranes,and compared with CoCl_2 group,the expression of Bcl2 was evidentlyincreased,BAX,cleaved caspase-3 and HIF-1α were decreased after being pretreated with APN. Conclusion APN coulddecrease CoCl_2-induced apoptosis in RAW264. 7 cells,which may occur via enhancing the intracellular antioxidant activity,and by upregulation of Bcl2,downregulation of BAX,cleaved caspase-3 and HIF-1α.
引文
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[24]Chen Z,Zhao L,Zhao F,et al. Tetrandrine suppresses lung cancergrowth and induces apoptosis,potentially via the VEGF/HIF-1α/ICAM-1 signaling pathway[J]. Oncol Lett, 2018, 15(5):7433-7437.
[25]Zhang J,Xia Y,Xu Z,et al. Propofol suppressed hypoxia/reoxygenation-induced apoptosis in HBVSMC by regulation of the expression ofBcl-2,Bax,Caspase3,Kir6. 1,and p-JNK[J/OL]. Oxid Med CellLongev,2016,2016:1518738. DOI:10. 1155/2016/1518738.Epub 2016 Jan 5.
[26] Ying X,Peng Y,Zhang J,et al. Endogenousα-crystallin inhibitsexpression of caspase-3 induced by hypoxia in retinal neurons[J].Life Sci,2014,111(1/2):42-46.
[2]Harwani S C. Macrophages under pressure:the role of macrophagepolarization in hypertension[J]. Transl Res,2018,191:45-63.
[3] Saha S,Shalova I N,Biswas S K. Metabolic regulation of macrophagephenotype and function[J]. Immunol Rev, 2017, 280(1):102-111.
[4]王国红,毕凌云,李超堃,等.氯化钴诱导化学缺氧对N9小胶质细胞的损伤作用及机制[J].中国药理学通报,2013,29(4):557-562.
[5]刘梅,李俊峰.脂联素对棕榈酸诱导H9c2细胞凋亡的抵抗效应以及相关分子机制[J].广东医学,2016,37(12):1811-1814.
[6] Xu N,Zhang Y,Doycheva D M,et al. Adiponectin attenuatesneuronal apoptosis induced by hypoxia-ischemia via the activation ofAdipoR1/APPL1/LKB1/AMPK pathway in neonatal rats[J].Neuropharmacology,2018,133:415-428.
[7]Wang X,Buechler N L,Yoza B K,et al. Adiponectin treatmentattenuates inflammatory response during early sepsis in obese mice[J]. J Inflamm Res,2016,9:167-174.
[8] Hand L E,Usan P,Cooper G J,et al. Adiponectin induces A20expression in adipose tissue to confer metabolic benefit[J].Diabetes,2015,64(1):128-136.
[9] Park M,Sabetski A,Kwan Chan Y,et al. Palmitate induces ERstress and autophagy in H9c2 cells:implications for apoptosis andadiponectin resistance[J]. J Cell Physiol, 2015, 230(3):630-639.
[10]Han X,Wu Y,Liu X,et al. Adiponectin improves coronary no-reflowinjury by protecting the endothelium in rats with type 2 diabetesmellitus[J/OL]. Biosci Rep, 2017, 37(4):BSR20170282.DOI:10. 1042/BSR20170282.
[11]Osathanon T,Vivatbutsiri P,Sukarawan W,et al. Cobalt chloridesupplementation induces stem cell marker expression and inhibitsosteoblastic differentiation in human periodontal ligament cells[J].Arch Oral Biol,2015,60(1):29-36.
[12] Lu Y,Chen W,Lin C,et al. The protective effects of propofolagainst CoCl2-induced HT22 cell hypoxia injury via PP2A/CAMKIIα/nNOS pathway[J/OL]. BMC Anesthesiol, 2017,17(1):32. DOI:10. 1186/s12871-017-0327-1.
[13]Zhang Y B,Wang X,Meister E A,et al. The effects of CoCl2onHIF-1αprotein under experimental conditions of autoprogressivehypoxia using mouse models[J]. Int J Mol Sci,2014,15(6):10999-11012.
[14]Dadson K,Chasiotis H,Wannaiampikul S,et al. Adiponectin mediatedAPPL1-AMPK signaling induces cell migration,MMP activation,andcollagen remodeling in cardiac fibroblasts[J]. J Cell Biochem,2014,115(4):785-793.
[15]Ramezani-Moghadam M,Wang J,Ho V,et al. Adiponectin reduceshepatic stellate cell migration by promoting tissue inhibitor ofmetalloproteinase-1(TIMP-1)secretion[J]. J Biol Chem,2015,290(9):5533-5542.
[16]刘梅,李俊峰.脂联素对棕榈酸诱导H9c2细胞凋亡的抵抗效应以及相关分子机制[J].广东医学,2016,37(12):1811-1814.
[17]Shibata R,Sato K,Kumada M,et al. Adiponectin accumulates inmyocardial tissue that has been damaged by ischemia-reperfusioninjury via leakage from the vascular compartment[J]. CardiovascRes,2007,74(3):471-479.
[18]卢晓梅,马玲,于艳秋.醛固酮对H9C2细胞的诱导作用[J].广东医学,2012,33(10):1388-1389.
[19] Nanasato M,Murohara T. Role of adiponectin in cardiovascularprotection[J]. Circ J,2010,74(3):432-433.
[20] He X,Wang J,Wei W,et al. Hypoxia regulates ABCG2 activitythrough the activivation of ERK1/2/HIF-1αand contributes tochemoresistance in pancreatic cancer cells[J]. Cancer Biol Ther,2016,17(2):188-198.
[21]Zhou L,Feng Y,Jin Y,et al. Verbascoside promotes apoptosis byregulating HIPK2-p53 signaling in human colorectal cancer[J/OL].BMC Cancer,2014,14:747-758. DOI:10. 1186/1471-2407-14-747.
[22]Friedrich D,Fecher R A,Rupp J,et al. Impact of HIF-1αandhypoxia on fungal growth characteristics and fungal immunity[J].Microbes Infect,2017,19(3):204-209.
[23] Nauta T D,van den Broek M,Gibbs S,et al. Identification ofHIF-2α-regulated genes that play a role in human microvascularendothelial sprouting during prolonged hypoxia in vitro[J].Angiogenesis,2017,20(1):39-54.
[24]Chen Z,Zhao L,Zhao F,et al. Tetrandrine suppresses lung cancergrowth and induces apoptosis,potentially via the VEGF/HIF-1α/ICAM-1 signaling pathway[J]. Oncol Lett, 2018, 15(5):7433-7437.
[25]Zhang J,Xia Y,Xu Z,et al. Propofol suppressed hypoxia/reoxygenation-induced apoptosis in HBVSMC by regulation of the expression ofBcl-2,Bax,Caspase3,Kir6. 1,and p-JNK[J/OL]. Oxid Med CellLongev,2016,2016:1518738. DOI:10. 1155/2016/1518738.Epub 2016 Jan 5.
[26] Ying X,Peng Y,Zhang J,et al. Endogenousα-crystallin inhibitsexpression of caspase-3 induced by hypoxia in retinal neurons[J].Life Sci,2014,111(1/2):42-46.