大麻素受体1通过PI3K/AKT信号通路介导单核巨噬细胞向M1型极化
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摘要
该文采用实时荧光定量聚合酶链反应(RT-qPCR)、蛋白印记实验(Western blot)、流式细胞分析(flurescence-activated cell sorting, FACS)技术检测骨髓单核巨噬细胞(bone marrow monocytes/macrophages, BMMs)中大麻素受体1(cannabinoid receptor 1, CB1)对BMMs极化的作用。结果表明,使用1μmol/L ACEA(CB1激动剂)处理细胞发现, BMMs中M1型的标志物CD86、IL-1、MIP-1β、NOS2、IL-6、TNF-α的m RNA水平均上调;用流式细胞分析技术检测发现, BMMs中M1型的标志物CD86蛋白质水平上调;使用PI3K/AKT信号通路的特异性抑制剂(LY294002)预处理BMMs,再加入1μmol/L ACEA刺激细胞,与未加入LY294002的对照组相比,这些M1型BMMs标志物的mRNA表达均被抑制;通过Western blot法证实ACEA使p-AKT增加,而使用1μmol/L AM281(CB1药理学拮抗剂)阻断CB1功能,则抑制了ACEA导致的p-AKT增加。上述结果说明, CB1通过PI3K/AKT信号通路介导BMMs向M1型极化。
RT-qPCR, Western blot, flurescence-activated cell sorting(FACS) were employed to detect whether cannabinoid receptor 1(CB1) was involved in bone marrow monocytes/macrophage(BMMs) M1 polarization. The results showed that ACEA(CB1 agonist, 1 μmol/L) promoted the mRNA levels of M1 type macrophage gene signatures(CD86, IL-1, MIP-1β, NOS2, IL-6, TNF-α) in BMMs. The protein level of CD86 deteced by FACS were increased in BMMs induced by ACEA. When BMMs were pretreated with LY294002(specific PI3 K/AKT signal pathway inhibitor), ACEA-induced(1 μmol/L) increases of M1 gene signatures mRNA levels was suppressed. Furthermore, Western blot analysis showed the protein level of phosphorylated AKT(p-AKT) was increased in ACEA-treated BMMs. When BMMs were pretreated with AM281(CB1 antagonist, 1 μmol/L), p-AKT protein level was inhibited. The result showed that CB1 mediated monocyte/macrophage M1 polarization via PI3 K/AKT signaling pathway.
RT-qPCR, Western blot, flurescence-activated cell sorting(FACS) were employed to detect whether cannabinoid receptor 1(CB1) was involved in bone marrow monocytes/macrophage(BMMs) M1 polarization. The results showed that ACEA(CB1 agonist, 1 μmol/L) promoted the mRNA levels of M1 type macrophage gene signatures(CD86, IL-1, MIP-1β, NOS2, IL-6, TNF-α) in BMMs. The protein level of CD86 deteced by FACS were increased in BMMs induced by ACEA. When BMMs were pretreated with LY294002(specific PI3 K/AKT signal pathway inhibitor), ACEA-induced(1 μmol/L) increases of M1 gene signatures mRNA levels was suppressed. Furthermore, Western blot analysis showed the protein level of phosphorylated AKT(p-AKT) was increased in ACEA-treated BMMs. When BMMs were pretreated with AM281(CB1 antagonist, 1 μmol/L), p-AKT protein level was inhibited. The result showed that CB1 mediated monocyte/macrophage M1 polarization via PI3 K/AKT signaling pathway.
引文
1Ginhoux F, Jung S. Monocytes and macrophages:developmental pathways and tissue homeostasis. Nat Rev Immunol 2014; 14(6):392-404.
2 BhargavaP,LeeCH.Roleandfunctionofmacrophagesin the metabolic syndrome. Biochem J 2012; 442(2):253-62.
3 ParkEJ,LeeJH, YuGY,HeG, AliSR,HolzerRG,etal.Dietaryandgeneticobesitypromoteliverinflammationand tumorigenesis by enhancing IL-6 and TNF expression. Cell 2010;140(2):197-208.
4 Han Z, Zhu TP, Liu XH, Li CY, Yue S, Liu X, et al. 15-deoxy-Δ12,14-prostaglandin J2 reduces recruitment of bone marrowderived monocyte/macrophages in chronic liver injury in mice.Hepatology 2012; 56(1):350-60.
5 Sica A,InvernizziP,Mantovani A.Macrophageplasticityand polarizationinliverhomeostasisandpathology.Hepatology2014; 59(5):2034-42.
6Sica A, Mantovani A. Macrophage plasticity and polarization:in vivo veritas. J Clin Invest 2012; 122(3):787-95.
7 DiMarzo V. Targetingtheendocannabinoidsystem:toenhance or reduce? Nat Rev Drug Discov 2008; 7(5):438-55.
8 Siegmund SV, Schwabe RF. Endocannabinoids and liver disease.II. Endocannabinoids in the pathogenesis and treatment ofliver fibrosis. Am J Physiol Gastrointest Liver Physio 2008; 294(2):G357-62.
9 LiuLY, AlexaK,CortesM,Schatzman-BoneS,Kim AJ,MukhopadhyayB,etal.Cannabinoidreceptorsignaling regulates liver development and metabolism. Development 2016;143(4):609-22.
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11 Louvet A, Teixeira-Clerc F, Chobert MN, Deveaux V, Pavoine C, Zimmer A, et al. Cannabinoid CB2 receptors protect against alcoholic liver disease by regulating Kupffer cell polarization in mice. Hepatology 2011; 54(4):1217-26.
12 Montalbano R, Honrath B, Wissniowski TT, Elxnat M, Roth S,Ocker M, et al. Exogenous hepatitis B virus envelope proteins induce endoplasmic reticulum stress:involvement of cannabinoid axis in liver cancer cells. Oncotarget 2016; 7(15):20312-23.
13 MaiP,YangL,TianL,WangL,JiaS,ZhangY,etal.Endocannabinoidsystemcontributestoliverinjuryand inflammation by activation of bone marrow-derived monocytes/macrophagesinaCB1-dependentmanner.JImmunol2015;195(7):3390-401.
14 Tian L, Li W, Yang L, Chang N, Fan X, Ji X, et al. Cannabinoid receptor 1 participates in liver inflammation by promoting M1macrophagepolarizationviaRhoA/NF-κBp65andERK1/2pathways,respectively,inmouseliverfibrogenesis.Front Immunol 2017; 28; 8:1214.
15 ChangL,ZhaoD,LiuHB, WangQS,ZhangP,LiCL,etal.Activation of soni hedgehog signaling enhances cell migration and invasion by induction of matrix metalloproteinase-2 and-9viathephosphoinositide-3kinase/AKTsignalingpathwayin glioblastoma. Mol Med Rep 2015; 12(5):6702-10.
16 GreenBD,Jabbour AM,SandowJJ,RiffkinCD,Masouras D, Daunt CP, et al. Akt1 is the principal Akt isoform regulating apoptosis in limiting cytokine concentrations. Cell Death Differ2013; 20(10):1341-9.
17 张媛媛,李伟阳,杨琳,李丽英.磷酸鞘胺醇调节小鼠单核巨噬细胞炎性细胞因子表达的机制研究.首都医科大学学报(Zhang Yuanyuan, Li Weiyang, Yang Lin, Li Liying. Regulation of inflammatory cytokine expression of sphingosine 1-phosphate inmurinemonocyte/macrophageJ774A.1.JournalofCapital Medical University)2015; 5(36):729-33.
18 YangL,HanZ,TianL,MaiP,ZhangY,WangL,etal.Sphingosine 1-phosphate receptor 2 and 3 mediate bone marrowderived monocyte/macrophage motility in cholestatic liver injury in mice. Sci Rep 2015; 5:13423.
19 Ammit AJ,Hastie AT,EdsallLC,HoffmanRK, Amrani Y,Krymskaya VP, et al. Sphingosine 1-phosphate modulates human airway smooth muscle cell functions that promote inflammation and airway remodeling in asthma. FASEB J 2001; 15(7):1212-4.
20 WongBS,CamilleriM,EckertD,CarlsonP,RyksM,Burton D,etal.Randomizedpharmacodynamicandpharmacogenetic trialofdronabinoleffectsoncolontransitinirritablebowel syndrome-diarrhea. Neurogastroenterol Motil 2012; 24(4):358-e169.
21 Storr M, Emmerdinger D, Diegelmann J, Pfennig S, Ochsenkuhn T, Goke B, et al. The cannabinoid 1 receptor(CNR1)1359 G/A polymorphism modulates susceptibility to ulcerative colitis and the phenotype in Crohn’s disease. PLoS One 2010; 5(2):e9453.
22 Turu G, Hunyady L. Signal transduction of the CB1 cannabinoid receptor. J Mol Endocrinol 2010; 44(2):75-85.
23 Bekhite MM, Müller V, Tr?ger SH, Müller JP, Figulla HR, Sauer H,etal.Involvementofphosphoinositide3-kinaseclassIA(PI3K110α)andNADPHoxidase1(NOX1)inregulationof vascular differentiation induced by vascular endothelial growth factor(VEGF)in mouse embryonic stem cells. Cell Tissue Res2016; 364(1):159-74.
24 高广勋,董红娟,顾宏涛,高瑛,潘耀柱,杨洋,等. PI3K调控血小板聚集过程中RhoA活性及其与m Dia1相互作用.中国实验血液学杂志(Gao Guangxun, Dong hongJuan, Gu Hongtao, Gao Ying, Pan Yaozhu, Yang Yang, et al. PI3-kinase mediates activity ofRhoAwithmDia1thrombin-inducedplateletaggregation.Journal of Experimental Hematology)2009; 17(6):1555-9.
25 王清清,刘俊,曹浩强,张浩. LY294002阻断PI3k/Akt信号通路逆转肝癌细胞对索拉菲尼耐药的研究.全科医学临床与教育(Wang Qingqing, Liu Jun, Cao Haoqiang, Zhang Hao. Research about the sorafenib resistance of hepatocellular carcinoma cells reversedbyLY294002throughblockingPI3k/Aktpathway.Clinical Education of General Practice)2016; 14(6):612-6, 721.
26 Le Foll B, Gorelick DA, Goldberg SR. The future of endocannabinoid-orientedclinicalresearchafterCB1antagonists.Psychopharmacology(Berl)2009; 205(1):171-4.
27 Teixeira-Clerc F, Julien B, Grenard P, Tran Van Nhieu J, Deveaux V,LiL,etal.CB1cannabinoidreceptorantagonism:anew strategy for the treatment of liver fibrosis. Nat Med 2006; 12(6):671-6.
2 BhargavaP,LeeCH.Roleandfunctionofmacrophagesin the metabolic syndrome. Biochem J 2012; 442(2):253-62.
3 ParkEJ,LeeJH, YuGY,HeG, AliSR,HolzerRG,etal.Dietaryandgeneticobesitypromoteliverinflammationand tumorigenesis by enhancing IL-6 and TNF expression. Cell 2010;140(2):197-208.
4 Han Z, Zhu TP, Liu XH, Li CY, Yue S, Liu X, et al. 15-deoxy-Δ12,14-prostaglandin J2 reduces recruitment of bone marrowderived monocyte/macrophages in chronic liver injury in mice.Hepatology 2012; 56(1):350-60.
5 Sica A,InvernizziP,Mantovani A.Macrophageplasticityand polarizationinliverhomeostasisandpathology.Hepatology2014; 59(5):2034-42.
6Sica A, Mantovani A. Macrophage plasticity and polarization:in vivo veritas. J Clin Invest 2012; 122(3):787-95.
7 DiMarzo V. Targetingtheendocannabinoidsystem:toenhance or reduce? Nat Rev Drug Discov 2008; 7(5):438-55.
8 Siegmund SV, Schwabe RF. Endocannabinoids and liver disease.II. Endocannabinoids in the pathogenesis and treatment ofliver fibrosis. Am J Physiol Gastrointest Liver Physio 2008; 294(2):G357-62.
9 LiuLY, AlexaK,CortesM,Schatzman-BoneS,Kim AJ,MukhopadhyayB,etal.Cannabinoidreceptorsignaling regulates liver development and metabolism. Development 2016;143(4):609-22.
10 Suk KT, Mederacke I, Gwak GY, Cho SW, Adeyemi A, Friedman R,etal.Oppositerolesofcannabinoidreceptors1and2in hepatocarcinogenesis. Gut 2016; 65(10):1721-32.
11 Louvet A, Teixeira-Clerc F, Chobert MN, Deveaux V, Pavoine C, Zimmer A, et al. Cannabinoid CB2 receptors protect against alcoholic liver disease by regulating Kupffer cell polarization in mice. Hepatology 2011; 54(4):1217-26.
12 Montalbano R, Honrath B, Wissniowski TT, Elxnat M, Roth S,Ocker M, et al. Exogenous hepatitis B virus envelope proteins induce endoplasmic reticulum stress:involvement of cannabinoid axis in liver cancer cells. Oncotarget 2016; 7(15):20312-23.
13 MaiP,YangL,TianL,WangL,JiaS,ZhangY,etal.Endocannabinoidsystemcontributestoliverinjuryand inflammation by activation of bone marrow-derived monocytes/macrophagesinaCB1-dependentmanner.JImmunol2015;195(7):3390-401.
14 Tian L, Li W, Yang L, Chang N, Fan X, Ji X, et al. Cannabinoid receptor 1 participates in liver inflammation by promoting M1macrophagepolarizationviaRhoA/NF-κBp65andERK1/2pathways,respectively,inmouseliverfibrogenesis.Front Immunol 2017; 28; 8:1214.
15 ChangL,ZhaoD,LiuHB, WangQS,ZhangP,LiCL,etal.Activation of soni hedgehog signaling enhances cell migration and invasion by induction of matrix metalloproteinase-2 and-9viathephosphoinositide-3kinase/AKTsignalingpathwayin glioblastoma. Mol Med Rep 2015; 12(5):6702-10.
16 GreenBD,Jabbour AM,SandowJJ,RiffkinCD,Masouras D, Daunt CP, et al. Akt1 is the principal Akt isoform regulating apoptosis in limiting cytokine concentrations. Cell Death Differ2013; 20(10):1341-9.
17 张媛媛,李伟阳,杨琳,李丽英.磷酸鞘胺醇调节小鼠单核巨噬细胞炎性细胞因子表达的机制研究.首都医科大学学报(Zhang Yuanyuan, Li Weiyang, Yang Lin, Li Liying. Regulation of inflammatory cytokine expression of sphingosine 1-phosphate inmurinemonocyte/macrophageJ774A.1.JournalofCapital Medical University)2015; 5(36):729-33.
18 YangL,HanZ,TianL,MaiP,ZhangY,WangL,etal.Sphingosine 1-phosphate receptor 2 and 3 mediate bone marrowderived monocyte/macrophage motility in cholestatic liver injury in mice. Sci Rep 2015; 5:13423.
19 Ammit AJ,Hastie AT,EdsallLC,HoffmanRK, Amrani Y,Krymskaya VP, et al. Sphingosine 1-phosphate modulates human airway smooth muscle cell functions that promote inflammation and airway remodeling in asthma. FASEB J 2001; 15(7):1212-4.
20 WongBS,CamilleriM,EckertD,CarlsonP,RyksM,Burton D,etal.Randomizedpharmacodynamicandpharmacogenetic trialofdronabinoleffectsoncolontransitinirritablebowel syndrome-diarrhea. Neurogastroenterol Motil 2012; 24(4):358-e169.
21 Storr M, Emmerdinger D, Diegelmann J, Pfennig S, Ochsenkuhn T, Goke B, et al. The cannabinoid 1 receptor(CNR1)1359 G/A polymorphism modulates susceptibility to ulcerative colitis and the phenotype in Crohn’s disease. PLoS One 2010; 5(2):e9453.
22 Turu G, Hunyady L. Signal transduction of the CB1 cannabinoid receptor. J Mol Endocrinol 2010; 44(2):75-85.
23 Bekhite MM, Müller V, Tr?ger SH, Müller JP, Figulla HR, Sauer H,etal.Involvementofphosphoinositide3-kinaseclassIA(PI3K110α)andNADPHoxidase1(NOX1)inregulationof vascular differentiation induced by vascular endothelial growth factor(VEGF)in mouse embryonic stem cells. Cell Tissue Res2016; 364(1):159-74.
24 高广勋,董红娟,顾宏涛,高瑛,潘耀柱,杨洋,等. PI3K调控血小板聚集过程中RhoA活性及其与m Dia1相互作用.中国实验血液学杂志(Gao Guangxun, Dong hongJuan, Gu Hongtao, Gao Ying, Pan Yaozhu, Yang Yang, et al. PI3-kinase mediates activity ofRhoAwithmDia1thrombin-inducedplateletaggregation.Journal of Experimental Hematology)2009; 17(6):1555-9.
25 王清清,刘俊,曹浩强,张浩. LY294002阻断PI3k/Akt信号通路逆转肝癌细胞对索拉菲尼耐药的研究.全科医学临床与教育(Wang Qingqing, Liu Jun, Cao Haoqiang, Zhang Hao. Research about the sorafenib resistance of hepatocellular carcinoma cells reversedbyLY294002throughblockingPI3k/Aktpathway.Clinical Education of General Practice)2016; 14(6):612-6, 721.
26 Le Foll B, Gorelick DA, Goldberg SR. The future of endocannabinoid-orientedclinicalresearchafterCB1antagonists.Psychopharmacology(Berl)2009; 205(1):171-4.
27 Teixeira-Clerc F, Julien B, Grenard P, Tran Van Nhieu J, Deveaux V,LiL,etal.CB1cannabinoidreceptorantagonism:anew strategy for the treatment of liver fibrosis. Nat Med 2006; 12(6):671-6.