龙蛭汤对衣霉素诱导人脐静脉内皮细胞内GRP78和CHOP的影响
|
摘要
目的探讨龙蛭汤对衣霉素(TM)诱导的人脐静脉内皮细胞(HUVEC)内质网应激及其标志蛋白葡萄糖调节蛋白78(GRP78)和特异性蛋白C增强子结合蛋白同源蛋白(CHOP)表达的影响。方法制备龙蛭汤含药血清,体外培养HUVEC细胞株,分为对照组(10%空白血清),TM组(10%空白血清+3μg/mL TM),TM+龙蛭汤组(10%含药血清+3μg/mL TM),龙蛭汤组(10%含药血清),继续培养6 h。使用CCK-8法比较各组细胞活力,细胞免疫荧光法和Western Blot比较各组GRP78和CHOP蛋白表达,RT-PCR法比较各组GRP78和CHOP mRNA表达。结果与对照组比较,TM组HUVEC细胞活力下降,GRP78、CHOP蛋白及mRNA表达增加(均P<0.05);与TM组比较,TM+龙蛭汤组、龙蛭汤组HUVEC细胞活力升高,GRP78、CHOP蛋白及mRNA表达降低(均P<0.05)。结论龙蛭汤可通过下调GRP78、CHOP表达,减轻细胞过度的内质网应激反应,提高HUVEC细胞活力。
Objective To investigate the effect of Longzhi Decoction(LZD) on endoplasmic reticulum stress of human umbilical vein endothelial cells(HUVEC) induced by Tunicamycin(TM) and the expression of corresponding marker proteins, glucose regulated protein 78(GRP78) and C/EBP homologous protein(CHOP). Methods LZD containing Serum was prepared. HUVEC were cultured in vitro and divided into four groups: control group(10% blank serum), TM group(10% blank serum+3 μg/mL TM), TM+LZD group(10% LZD serum+3 μg/mL TM) and LZD group(10% LZD serum). Cells were cultured for 6 hours. The cell viability was assessed by CCK-8 method. The protein expressions of GRP78 and CHOP were evaluated by immunofluorescence and Western Blot. The mRNA expressions of GRP78 and CHOP were detected by RT-PCR. Results Compared with the control group, the cell viability was decreased, while the protein and mRNA expressions of GRP78 and CHOP were increased in TM group(all P<0.05). Compared with the TM group, the cell viability was increased, while the protein and mRNA expressions of GRP78 and CHOP were decreased in TM+LZD and LZD groups(P<0.05). Conclusion LZD can improve HUVEC cell activity by reducing excessive endoplasmic reticulum stress via down-regulating GRP78 and CHOP expression.
Objective To investigate the effect of Longzhi Decoction(LZD) on endoplasmic reticulum stress of human umbilical vein endothelial cells(HUVEC) induced by Tunicamycin(TM) and the expression of corresponding marker proteins, glucose regulated protein 78(GRP78) and C/EBP homologous protein(CHOP). Methods LZD containing Serum was prepared. HUVEC were cultured in vitro and divided into four groups: control group(10% blank serum), TM group(10% blank serum+3 μg/mL TM), TM+LZD group(10% LZD serum+3 μg/mL TM) and LZD group(10% LZD serum). Cells were cultured for 6 hours. The cell viability was assessed by CCK-8 method. The protein expressions of GRP78 and CHOP were evaluated by immunofluorescence and Western Blot. The mRNA expressions of GRP78 and CHOP were detected by RT-PCR. Results Compared with the control group, the cell viability was decreased, while the protein and mRNA expressions of GRP78 and CHOP were increased in TM group(all P<0.05). Compared with the TM group, the cell viability was increased, while the protein and mRNA expressions of GRP78 and CHOP were decreased in TM+LZD and LZD groups(P<0.05). Conclusion LZD can improve HUVEC cell activity by reducing excessive endoplasmic reticulum stress via down-regulating GRP78 and CHOP expression.
引文
[1] Hemphill JC 3rd,Greenberg SM,Anderson CS,et al.Guidelines for the management of spontaneous intracerebral hemorrhage:a guideline for healthcare professionals from the American Heart Association/American Stroke Association[J].Stroke,2015,46(7):2032-2060.
[2] Fann DY,Lee SY,Manzanero S,et al.Pathogenesis of acute stroke and the role of inflammasomes[J].Ageing Res Rev,2013,12(4):941-966.
[3] Hotamisligil GS.Endoplasmic reticulum stress and the inflammatory basis of metabolic disease[J].Cell,2010,140(6):900-917.
[4] Sano R,Reed JC.ER stress-induced cell death mechanisms[J].Biochim Biophys Acta,2013,1833(12):3460-3470.
[5] 冯容,张欣,陈永斌.龙蛭汤对气虚血瘀证急性脑梗死患者的临床疗效及作用机制研究[J].中国中西医结合急救杂志,2016,23(3):225-227.
[6] 姜薇,李若林,陈永斌.龙蛭汤对大鼠脑梗死模型梗死灶周围组织VEGF及Flk-1 mRNA表达的影响[J].广西医科大学学报,2014,31(2):192-196.
[7] 张宏,王旭昀,刘美奇,等.中药含药血清实验动物灌胃给药剂量探讨[J].吉林中医药,2015,35(6):623-625.
[8] Ron D,Walter P.Signal integration in the endoplasmic reticulum unfolded protein response[J].Nat Rev Mol Cell Biol,2007,8(7):519-529.
[9] Heusschen R,Schulkens IA,van Beijnum J,et al.Endothelial LGALS9 splice variant expression in endothelial cell biology and angiogenesis[J].Biochim Biophys Acta,2014,1842(2):284-292.
[10] Tabas I,Ron D.Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress[J].Nat Cell Biol,2011,13(3):184-190.
[11] Dalkara T,Alarcon-Martinez L.Cerebral microvascular pericytes and neurogliovascular signaling in health and disease[J].Brain Res,2015,1623:3-17.
[12] Christoph M,Ibrahim K,Hesse K,et al.Local inhibition of hypoxia-inducible factor reduces neointima formation after arterial injury in ApoE-/- mice[J].Atherosclerosis,2014,233(2):641-647.
[13] An C,Shi Y,Li P,et al.Molecular dialogs between the ischemic brain and the peripheral immune system:dualistic roles in injury and repair[J].Prog Neurobiol,2014,115:6-24.
[14] Liu J,Wang Y,Akamatsu Y,et al.Vascular remodeling after ischemic stroke:mechanisms and therapeutic potentials[J].Prog Neurobiol,2014,115:138-156.
[15] Yang Y,Thompson J F,Taheri S,et al.Early inhibition of MMP activity in ischemic rat brain promotes expression of tight junction proteins and angiogenesis during recovery[J].J Cereb Blood Flow Metab,2013,33(7):1104-1114.
[16] Zeng L,He X,Wang Y,et al.MicroRNA-210 overexpression induces angiogenesis and neurogenesis in the normal adult mouse brain[J].Gene Ther,2014,21(1):37-43.
[17] 蒙家泉,陈永斌,刘启华.龙蛭汤方治疗缺血性中风的理论探源[J].中国中西医结合急救杂志,2017,24(1):104-106.
[18] Wang J,Li G,Wang Y,et al.Suppression of tumor angiogenesis by metformin treatment via a mechanism linked to targeting of HER2/HIF-1alpha/VEGF secretion axis[J].Oncotarget,2015,6(42):44579-44592.
[19] Cheang WS,Tian XY,Wong WT,et al.Metformin protects endothelial function in diet-induced obese mice by inhibition of endoplasmic reticulum stress through 5' adenosine monophosphate-activated protein kinase-peroxisome proliferator-activated receptor delta pathway[J].Arterioscler Thromb Vasc Biol,2014,34(4):830-836.
[20] Luo B,Lee A S.The critical roles of endoplasmic reticulum chaperones and unfolded protein response in tumorigenesis and anticancer therapies[J].Oncogene,2013,32(7):805-818.
[21] Han J,Back SH,Hur J,et al.ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death[J].Nat Cell Biol,2013,15(5):481-490.
[22] Cao SS,Kaufman RJ.Endoplasmic reticulum stress and oxidative stress in cell fate decision and human disease[J].Antioxid Redox Signal,2014,21(3):396-413.
[23] Atkins C,Liu Q,Minthorn E,et al.Characterization of a novel PERK kinase inhibitor with antitumor and antiangiogenic activity[J].Cancer Res,2013,73(6):1993-2002.
[24] Binet F,Mawambo G,Sitaras N,et al.Neuronal ER stress impedes myeloid-cell-induced vascular regeneration through IRE1alpha degradation of netrin-1[J].Cell Metab,2013,17(3):353-571.
[25] Yin J,Gu L,Wang Y,et al.Rapamycin improves palmitate-induced ER stress/NF kappa B pathways associated with stimulating autophagy in adipocytes[J].Mediators Inflamm,2015,2015:272313.
[26] Yao C,Zhang J,Liu G,et al.Neuroprotection by (-)-epigallocatechin-3-gallate in a rat model of stroke is mediated through inhibition of endoplasmic reticulum stress[J].Mol Med Rep,2014,9(1):69-76.
[27] Liu D,Zhang M,Yin H.Signaling pathways involved in endoplasmic reticulum stress-induced neuronal apoptosis[J].Int J Neurosci,2013,123(3):155-162.
[2] Fann DY,Lee SY,Manzanero S,et al.Pathogenesis of acute stroke and the role of inflammasomes[J].Ageing Res Rev,2013,12(4):941-966.
[3] Hotamisligil GS.Endoplasmic reticulum stress and the inflammatory basis of metabolic disease[J].Cell,2010,140(6):900-917.
[4] Sano R,Reed JC.ER stress-induced cell death mechanisms[J].Biochim Biophys Acta,2013,1833(12):3460-3470.
[5] 冯容,张欣,陈永斌.龙蛭汤对气虚血瘀证急性脑梗死患者的临床疗效及作用机制研究[J].中国中西医结合急救杂志,2016,23(3):225-227.
[6] 姜薇,李若林,陈永斌.龙蛭汤对大鼠脑梗死模型梗死灶周围组织VEGF及Flk-1 mRNA表达的影响[J].广西医科大学学报,2014,31(2):192-196.
[7] 张宏,王旭昀,刘美奇,等.中药含药血清实验动物灌胃给药剂量探讨[J].吉林中医药,2015,35(6):623-625.
[8] Ron D,Walter P.Signal integration in the endoplasmic reticulum unfolded protein response[J].Nat Rev Mol Cell Biol,2007,8(7):519-529.
[9] Heusschen R,Schulkens IA,van Beijnum J,et al.Endothelial LGALS9 splice variant expression in endothelial cell biology and angiogenesis[J].Biochim Biophys Acta,2014,1842(2):284-292.
[10] Tabas I,Ron D.Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress[J].Nat Cell Biol,2011,13(3):184-190.
[11] Dalkara T,Alarcon-Martinez L.Cerebral microvascular pericytes and neurogliovascular signaling in health and disease[J].Brain Res,2015,1623:3-17.
[12] Christoph M,Ibrahim K,Hesse K,et al.Local inhibition of hypoxia-inducible factor reduces neointima formation after arterial injury in ApoE-/- mice[J].Atherosclerosis,2014,233(2):641-647.
[13] An C,Shi Y,Li P,et al.Molecular dialogs between the ischemic brain and the peripheral immune system:dualistic roles in injury and repair[J].Prog Neurobiol,2014,115:6-24.
[14] Liu J,Wang Y,Akamatsu Y,et al.Vascular remodeling after ischemic stroke:mechanisms and therapeutic potentials[J].Prog Neurobiol,2014,115:138-156.
[15] Yang Y,Thompson J F,Taheri S,et al.Early inhibition of MMP activity in ischemic rat brain promotes expression of tight junction proteins and angiogenesis during recovery[J].J Cereb Blood Flow Metab,2013,33(7):1104-1114.
[16] Zeng L,He X,Wang Y,et al.MicroRNA-210 overexpression induces angiogenesis and neurogenesis in the normal adult mouse brain[J].Gene Ther,2014,21(1):37-43.
[17] 蒙家泉,陈永斌,刘启华.龙蛭汤方治疗缺血性中风的理论探源[J].中国中西医结合急救杂志,2017,24(1):104-106.
[18] Wang J,Li G,Wang Y,et al.Suppression of tumor angiogenesis by metformin treatment via a mechanism linked to targeting of HER2/HIF-1alpha/VEGF secretion axis[J].Oncotarget,2015,6(42):44579-44592.
[19] Cheang WS,Tian XY,Wong WT,et al.Metformin protects endothelial function in diet-induced obese mice by inhibition of endoplasmic reticulum stress through 5' adenosine monophosphate-activated protein kinase-peroxisome proliferator-activated receptor delta pathway[J].Arterioscler Thromb Vasc Biol,2014,34(4):830-836.
[20] Luo B,Lee A S.The critical roles of endoplasmic reticulum chaperones and unfolded protein response in tumorigenesis and anticancer therapies[J].Oncogene,2013,32(7):805-818.
[21] Han J,Back SH,Hur J,et al.ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death[J].Nat Cell Biol,2013,15(5):481-490.
[22] Cao SS,Kaufman RJ.Endoplasmic reticulum stress and oxidative stress in cell fate decision and human disease[J].Antioxid Redox Signal,2014,21(3):396-413.
[23] Atkins C,Liu Q,Minthorn E,et al.Characterization of a novel PERK kinase inhibitor with antitumor and antiangiogenic activity[J].Cancer Res,2013,73(6):1993-2002.
[24] Binet F,Mawambo G,Sitaras N,et al.Neuronal ER stress impedes myeloid-cell-induced vascular regeneration through IRE1alpha degradation of netrin-1[J].Cell Metab,2013,17(3):353-571.
[25] Yin J,Gu L,Wang Y,et al.Rapamycin improves palmitate-induced ER stress/NF kappa B pathways associated with stimulating autophagy in adipocytes[J].Mediators Inflamm,2015,2015:272313.
[26] Yao C,Zhang J,Liu G,et al.Neuroprotection by (-)-epigallocatechin-3-gallate in a rat model of stroke is mediated through inhibition of endoplasmic reticulum stress[J].Mol Med Rep,2014,9(1):69-76.
[27] Liu D,Zhang M,Yin H.Signaling pathways involved in endoplasmic reticulum stress-induced neuronal apoptosis[J].Int J Neurosci,2013,123(3):155-162.