梓醇对TNF-α诱导的HAECs细胞损伤抑制作用的机制研究
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摘要
梓醇是从中药地黄的根中提取出的一种环烯醚萜葡萄糖苷类化合物,据报道有抗氧化应激作用。腺苷5'磷酸腺苷活化蛋白激酶(AMPK)在抑制氧化应激方面起到重要作用。该研究通过对氧化应激相关指标的检测探讨梓醇对TNF-α诱导的人主动脉内皮细胞(human aorta epithelial cells,HAECs)细胞损伤的抑制作用,通过siRNA技术检测其调控氧化应激与AMPK的关系。采用比色法测定细胞中SOD,MDA,GSH和LDH水平,流式细胞仪测定ROS生成水平。免疫印迹(Westernblot)法检测细胞中AMPK,磷酸化AMPK和NOX4的表达。RNA干扰技术敲低AMPK水平,通过再次检测各项指标探讨梓醇抑制氧化应激与其激活AMPK的相关性。结果表明,TNF-α诱导损伤的HAECs磷酸化AMPK表达下降,而梓醇上调AMPK的磷酸化水平。未转染AMPK siRNA前,梓醇能够抑制TNF-α诱导的NOX4蛋白表达上调,并减少ROS生成,AMPK siRNA作用后,梓醇对ROS过度生成、NOX4蛋白表达上调的抑制作用均减弱。以上结果提示,梓醇通过激活AMPK抑制细胞中氧化应激,进而抑制TNF-α诱导的HAECs细胞损伤。
Catalpol is an iridoid glycoside extracted from the root of Rehmannia glutinosa. It has been reported to have antioxidant stress effects. Adenosine 5' monophosphate-activated protein kinase( AMPK) plays an important role in inhibiting oxidative stress. This study was designed to investigate the protective effects of catalpol on TNF-α-exposed human aorta epithelial cells( HAECs) via inhibit oxidative stress,and the relationship between catalpol and AMPK was detected by RNA interference technique. Levels of superoxide dismutase( SOD),malonaldehyde( MDA),glutathione( GSH) and lactate dehydrogenase( LDH) were measured with a colorimetric assay kit. The level of ROS was measured with FACS calibur. Western blot was employed to detect the protein expression of AMPK,phosphorylated-AMPK and NOX4. Finally,RNA interference technique was used to investigate the role of AMPK in catalpol-induced protective effects. TNF-α treatment decreased the expression of phosphorylated-AMPK protein level,however,catalpol could reverse the decreased phosphorylated-AMPK level. Catalpol could inhibit NOX4 protein expression and decrease ROS overproduction. After using AMPK siRNA that effects of catalpol on ROS overproduction and NOX4 protein expression inhibition were attenuated. The above results suggest that catalpol inhibits oxidative stress in TNF-α-exposed HAECs by activating AMPK.
Catalpol is an iridoid glycoside extracted from the root of Rehmannia glutinosa. It has been reported to have antioxidant stress effects. Adenosine 5' monophosphate-activated protein kinase( AMPK) plays an important role in inhibiting oxidative stress. This study was designed to investigate the protective effects of catalpol on TNF-α-exposed human aorta epithelial cells( HAECs) via inhibit oxidative stress,and the relationship between catalpol and AMPK was detected by RNA interference technique. Levels of superoxide dismutase( SOD),malonaldehyde( MDA),glutathione( GSH) and lactate dehydrogenase( LDH) were measured with a colorimetric assay kit. The level of ROS was measured with FACS calibur. Western blot was employed to detect the protein expression of AMPK,phosphorylated-AMPK and NOX4. Finally,RNA interference technique was used to investigate the role of AMPK in catalpol-induced protective effects. TNF-α treatment decreased the expression of phosphorylated-AMPK protein level,however,catalpol could reverse the decreased phosphorylated-AMPK level. Catalpol could inhibit NOX4 protein expression and decrease ROS overproduction. After using AMPK siRNA that effects of catalpol on ROS overproduction and NOX4 protein expression inhibition were attenuated. The above results suggest that catalpol inhibits oxidative stress in TNF-α-exposed HAECs by activating AMPK.
引文
[1]林艾雯,陈竹君.动脉粥样硬化与内皮细胞损伤机制的研究进展[J].岭南心血管病杂志,2015,21(4):580.
[2] Taniyama Y,Griendling K K. Reactive oxygen species in the vas-culature:molecular and cellular mechanisms[J]. Hypertension,2003,42(6):1075.
[3] Jin X,Chen M,Yi L,et al. Delphinidin-3-glucoside protectshuman umbilical vein endothelial cells against oxidized low-densi-ty lipoprotein-induced injury by autophagy upregulation via theAMPK/SIRT1 signaling pathway[J]. Mol Nutr Food Res,2014,58(10):1941.
[4] Chen M L,Yi L,Jin X,et al. Resveratrol attenuates vascularendothelial inflammation by inducing autophagy through thec AMP signaling pathway[J]. Autophagy,2013,9(12):2033.
[5] Inoki K,Kim J,Guan K L. AMPK and m TOR in cellular energyhomeostasis and drug targets[J]. Annu Rev Pharmacol Toxicol,2012,52:381.
[6]李营营,张吟.腺苷酸活化蛋白激酶促胰岛β细胞凋亡机制的研究进展[J].中国现代应用药学,2018,35(1):143.
[7] Han X,Tai H,Wang X,et al. AMPK activation protects cellsfrom oxidative stress-induced senescence via autophagic flux res-toration and intracellular NAD+elevation[J]. Aging Cell,2016,15(3):416.
[8] Mihaylova M M,Shaw R J. The AMPK signalling pathway coor-dinates cell growth,autophagy and metabolism[J]. Nat Cell Bi-ol,2011,13(9):1016.
[9] Zhang X L,Jiang B,Li Z B,et al. Catalpol ameliorates cognitiondeficits and attenuates oxidative damage in the brain of senescentmice induced by D-galactose[J]. Pharmacol Biochem Behav,2007,88(1):64.
[10] Li X,Xu Z,Jiang Z,et al. Hypoglycemic effect of catalpol onhigh-fat diet/streptozotocin-induced diabetic mice by increasingskeletal muscle mitochondrial biogenesis[J]. Acta Biochim Bio-phys Sin,2014,46(9):738.
[11] Vasamsetti S B,Karnewar S,Gopoju R,et al. Resveratrol atten-uates monocyte-to-macrophage differentiation and associated in-flammation via modulation of intracellular GSH homeostasis:Rel-evance in atherosclerosis[J]. Free Radic Biol Med,2016,96:392.
[12] Glass C K,Witztum J L. Atherosclerosis. The road ahead[J].Cell,2001,104(4):503.
[13] Chen W,Li X,Jia L Q,et al. Neuroprotective activities of catal-pol against CaMKⅡ-dependent apoptosis induced by LPS inPC12 cells[J]. Br J Pharmacol,2013,169(5):1140.
[14] Shah S A,Yoon G H,Chung S S,et al. Novel osmotin inhibitsSREBP2 via the AdipoR1/AMPK/SIRT1 pathway to improveAlzheimer's disease neuropathological deficits[J]. Mol Psychia-try,2017,22(3):407.
[15] Chan E Y. Regulation and function of uncoordinated-51 like ki-nase proteins[J]. Antioxid Redox Signal,2012,17(5):775.
[16] Fan X,Wang J,Hou J,et al. Berberine alleviates ox-LDL in-duced inflammatory factors by up-regulation of autophagy viaAMPK/m TOR signaling pathway[J]. J Transl Med,2015,13(1):1.
[17] Crosas-Molist E,Bertran E,Rodriguez-Hernandez I,et al. TheNADPH oxidase NOX4 represses epithelial to amoeboid transitionand efficient tumour dissemination[J]. Oncogene,2017,36(21):3002.
[18] Xu H,Goettsch C,Xia N,et al. Differential roles of PKCalphaand PKCepsilon in controlling the gene expression of Nox4 in hu-man endothelial cells[J]. Free Radic Biol Med,2008,44(8):1656.
[19] Zhang M,Wang C M,Li J,et al. Berberine protects against pal-mitate-induced endothelial dysfunction:involvements of upregu-lation of AMPK and e NOS and downregulation of NOX4[J]. Me-diators Inflamm,2013,2013:260464.
[20] Ugusman A,Zakaria Z,Hui C K,et al. Piper sarmentosum in-hibits ICAM-1 and Nox4 gene expression in oxidative stress-in-duced human umbilical vein endothelial cells[J]. BMC Comple-ment Altern Med,2011,11(1):31.
[21] Xia F,Wang C,Jin Y,et al. Luteolin protects HUVECs fromTNF-alpha-induced oxidative stress and inflammation via itseffects on the Nox4/ROS-NF-kappaB and MAPK pathways[J].J Atheroscler Thromb,2014,21(8):768.
[22] Li G H,Lin X L,Zhang H,et al. Ox-Lp(a)transiently inducesHUVEC autophagy via an ROS-dependent PAPR-1-LKB1-AMPK-mTOR pathway[J]. Atherosclerosis,2015,243(1):223.
[23] Razani B,Feng C,Coleman T,et al. Autophagy links inflamma-somes to atherosclerotic progression[J]. Cell Metab,2012,15(4):534.
[24] Li X,He S,Zhou X,et al. Lyn delivers bacteria to lysosomes foreradication through TLR2-initiated autophagy related phagocyto-sis[J]. PLoS Pathog,2016,12(1):e1005363.
[2] Taniyama Y,Griendling K K. Reactive oxygen species in the vas-culature:molecular and cellular mechanisms[J]. Hypertension,2003,42(6):1075.
[3] Jin X,Chen M,Yi L,et al. Delphinidin-3-glucoside protectshuman umbilical vein endothelial cells against oxidized low-densi-ty lipoprotein-induced injury by autophagy upregulation via theAMPK/SIRT1 signaling pathway[J]. Mol Nutr Food Res,2014,58(10):1941.
[4] Chen M L,Yi L,Jin X,et al. Resveratrol attenuates vascularendothelial inflammation by inducing autophagy through thec AMP signaling pathway[J]. Autophagy,2013,9(12):2033.
[5] Inoki K,Kim J,Guan K L. AMPK and m TOR in cellular energyhomeostasis and drug targets[J]. Annu Rev Pharmacol Toxicol,2012,52:381.
[6]李营营,张吟.腺苷酸活化蛋白激酶促胰岛β细胞凋亡机制的研究进展[J].中国现代应用药学,2018,35(1):143.
[7] Han X,Tai H,Wang X,et al. AMPK activation protects cellsfrom oxidative stress-induced senescence via autophagic flux res-toration and intracellular NAD+elevation[J]. Aging Cell,2016,15(3):416.
[8] Mihaylova M M,Shaw R J. The AMPK signalling pathway coor-dinates cell growth,autophagy and metabolism[J]. Nat Cell Bi-ol,2011,13(9):1016.
[9] Zhang X L,Jiang B,Li Z B,et al. Catalpol ameliorates cognitiondeficits and attenuates oxidative damage in the brain of senescentmice induced by D-galactose[J]. Pharmacol Biochem Behav,2007,88(1):64.
[10] Li X,Xu Z,Jiang Z,et al. Hypoglycemic effect of catalpol onhigh-fat diet/streptozotocin-induced diabetic mice by increasingskeletal muscle mitochondrial biogenesis[J]. Acta Biochim Bio-phys Sin,2014,46(9):738.
[11] Vasamsetti S B,Karnewar S,Gopoju R,et al. Resveratrol atten-uates monocyte-to-macrophage differentiation and associated in-flammation via modulation of intracellular GSH homeostasis:Rel-evance in atherosclerosis[J]. Free Radic Biol Med,2016,96:392.
[12] Glass C K,Witztum J L. Atherosclerosis. The road ahead[J].Cell,2001,104(4):503.
[13] Chen W,Li X,Jia L Q,et al. Neuroprotective activities of catal-pol against CaMKⅡ-dependent apoptosis induced by LPS inPC12 cells[J]. Br J Pharmacol,2013,169(5):1140.
[14] Shah S A,Yoon G H,Chung S S,et al. Novel osmotin inhibitsSREBP2 via the AdipoR1/AMPK/SIRT1 pathway to improveAlzheimer's disease neuropathological deficits[J]. Mol Psychia-try,2017,22(3):407.
[15] Chan E Y. Regulation and function of uncoordinated-51 like ki-nase proteins[J]. Antioxid Redox Signal,2012,17(5):775.
[16] Fan X,Wang J,Hou J,et al. Berberine alleviates ox-LDL in-duced inflammatory factors by up-regulation of autophagy viaAMPK/m TOR signaling pathway[J]. J Transl Med,2015,13(1):1.
[17] Crosas-Molist E,Bertran E,Rodriguez-Hernandez I,et al. TheNADPH oxidase NOX4 represses epithelial to amoeboid transitionand efficient tumour dissemination[J]. Oncogene,2017,36(21):3002.
[18] Xu H,Goettsch C,Xia N,et al. Differential roles of PKCalphaand PKCepsilon in controlling the gene expression of Nox4 in hu-man endothelial cells[J]. Free Radic Biol Med,2008,44(8):1656.
[19] Zhang M,Wang C M,Li J,et al. Berberine protects against pal-mitate-induced endothelial dysfunction:involvements of upregu-lation of AMPK and e NOS and downregulation of NOX4[J]. Me-diators Inflamm,2013,2013:260464.
[20] Ugusman A,Zakaria Z,Hui C K,et al. Piper sarmentosum in-hibits ICAM-1 and Nox4 gene expression in oxidative stress-in-duced human umbilical vein endothelial cells[J]. BMC Comple-ment Altern Med,2011,11(1):31.
[21] Xia F,Wang C,Jin Y,et al. Luteolin protects HUVECs fromTNF-alpha-induced oxidative stress and inflammation via itseffects on the Nox4/ROS-NF-kappaB and MAPK pathways[J].J Atheroscler Thromb,2014,21(8):768.
[22] Li G H,Lin X L,Zhang H,et al. Ox-Lp(a)transiently inducesHUVEC autophagy via an ROS-dependent PAPR-1-LKB1-AMPK-mTOR pathway[J]. Atherosclerosis,2015,243(1):223.
[23] Razani B,Feng C,Coleman T,et al. Autophagy links inflamma-somes to atherosclerotic progression[J]. Cell Metab,2012,15(4):534.
[24] Li X,He S,Zhou X,et al. Lyn delivers bacteria to lysosomes foreradication through TLR2-initiated autophagy related phagocyto-sis[J]. PLoS Pathog,2016,12(1):e1005363.