基于TLR3/TLR9介导巨噬细胞自噬/极化效应探讨血管软化丸抗AS的作用机制
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摘要
目的:观察血管软化丸通过对TLR3的影响,调控PI3K/Akt/mTOR信号通路,进而对巨噬细胞自噬产生影响,以及观察血管软化丸通过对TLR9的影响,调控下游NF-κB和IRF7信号途径,影响巨噬细胞M2/M1平衡,探讨血管软化丸抗动脉粥样硬化(AS)的分子机制。方法:体外实验,将巨噬细胞随机分为6组,即对照组(空白血清),血管软化丸高剂量组、血管软化丸中剂量组、血管软化丸低剂量组、ODN组(TLR9激动剂:ODN1826)、poly组(TLR3激动剂:poly(I:C))。体内实验,将ApoE~(-/-)小鼠分为6组,对照组(生理盐水,灌胃)血管软化丸高剂量组(浓度为3.456 g/mL),血管软化丸中剂量组(浓度为1.728 g/mL)、血管软化丸低剂量组(浓度为0.864 g/mL)、ODN组(ODN1826,腹腔注射)、poly组(poly(I:C),腹腔注射),腹腔注射每周两次,灌胃每日1次,连续给药8周后取材进行检测。观察血管软化丸含药血清和TLR9对巨噬细胞的极化状态的影响,对动脉斑块iNOS和CD206的表达的影响,对巨噬细胞(M1/M2)平衡相关炎症因子的影响和对巨噬细胞(M1/M2)平衡相关炎症因子的影响;采用ELISA法检测细胞分泌炎性因子相关水平,采用Western blot检测和RT-PCR法检测血管软化丸对主动脉斑块TLR9和TLR3及其下游信号分子表达的影响,观测指标:采用Western blot检测各组细胞TLR9的蛋白含量,及主动脉TLR9及其下游的分子MyD88,p-NF-κB和IRF7的蛋白含量;采用流式细胞术和免疫化学荧光检测各组细胞M1型和M2型的比例;采用RT-PCR和ELISA法检测各组细胞TNF-α的表达;采用RT-PCR和免疫荧光检测RAW264.7细胞和动脉斑块的M1型和M2型巨噬细胞的相关标志性因子和炎症因子;病理学检测验证血管软化丸抗动脉粥样硬化的疗效。结果:血管软化丸含药血清可以诱导巨噬细胞高表达M2型巨噬细胞的标志CD206;中药组斑块中iNOS的荧光密度表现为低表达(P<0.05),而斑块中CD206的荧光密度则表现为高表达(P<0.05);血管软化丸含药血清调低IL-1β、iNOS、Ciita表达(P<0.05),调高Ym1、Fizz1表达水平(P<0.05);能够改善细胞分泌炎性因子IFN-γ和IL-10相关水平(P<0.05);血管软化丸含药血清调低TLR9蛋白和TLR3蛋白表达水平(P<0.05);血管软化丸含药血清调低TLR9下游信号MyD88、p-NF-κB、IRF7 mRNA表达水平(P<0.05);血管软化丸含药血清调低TLR3下游信号LC3Ⅱ、Beclin、Akt、mTOR mRNA表达水平(P<0.05)。结论:血管软化丸通过影响TLR9,调控下游NF-κB和IRF7信号途径,进而调节巨噬细胞M2/M1平衡;通过调控TLR3影响PI3K/Akt/mTOR信号通路,进而调节巨噬细胞自噬,抑制动脉粥样硬化的发生发展。
Objective:To observe the effect of Vascular Softening Pills on TLR3,regulating PI3K/Akt/mTOR signaling pathway,and then affecting macrophages autophagy,and observing the effect of vascular softening pills on TLR9,regulating downstream NF-κB and IRF7 signaling pathways.To influence the M2/M1 balance of macrophages and explore the molecular mechanism of Vascular Softening Pills against atherosclerosis(AS).Methods:In vitro,macrophages were randomly divided into 6 groups,namely the control group(blank serum),the high-dose group of Vascular Softening Pills,the middle dose group of Vascular Softening Pills,the low-dose group of Vascular Softening Pills,and the ODN group(TLR9 agonist:ODN1826),poly group(TLR3 agonist:poly(I:C).In vivo experiments,ApoE~(-/-) mice were divided into 6 groups,control group(normal saline,intragastric),high-dose group of Vascular Softening Pills(concentration 3.456 g/mL),Vascular Softening Pills medium dose group(concentration 1.728 g/mL),low-dose group of Vascular Softening Pills(concentration of 0.864 g/mL),ODN group(ODN1826,intraperitoneal injection),poly group[poly(I:C),intraperitoneal injection],intraperitoneal injection twice a week.The stomach was taken out once a day,and after 8 weeks of continuous administration,the material was taken out for testing.We observed the effect of serum containing Vascular Softening Pills and TLR9 on the polarization state of macrophages,the effect on the expressions of iNOS and CD206 in arterial plaques,the influence of macrophage(M1/M2) on inflammatory factors related to macrophages and the effect of phagocyte(M1/M2) balance-related inflammatory factors.The level of inflammatory factors secreted by cells was detected by ELISA,and the Vascular Softening Pills were used to detect TLR9 and TLR3 and downstream of aortic plaque by Western blot and RT-PCR.Western blot was used to detect the protein content of TLR9 in each group,and the protein content of aortic TLR9 and its downstream molecules MyD88,p-NF-κB and IRF7.The ratios of M1 and M2 in each group were detected by chemical fluorescence.The expression of TNF-αwas detected by RT-PCR and ELISA.The M1 type of RAW264.7 cells and arterial plaques were detected by RT-PCR and immunofluorescence.And related markers and inflammatory factors of M2 macrophages were observed.The pathological examination was used to verify the efficacy of vascular softening pills against atherosclerosis.Results:The Vascular Softening Pill-containing serum can induce macrophage high expression of M2 type macrophage marker CD206.The fluorescence density of iNOS in the plaque of Chinese medicine group was low expression(P<0.05),while the fluorescence density of CD206 in plaque was high expression(P<0.05).Vascular Softening Pills containing serum decreased IL-1β,iNOS,Ciita expression(P<0.05),increased Ym1 and Fizz1 expressions(P<0.05).Vascular Softening Pills containing serum can improve cell secretion inflammation.The levels of IFN-γand IL-10 were correlated(P<0.05).The serum containing Vascular Softening Pills lowered the expression of TLR9 protein and TLR3 protein(P<0.05).The serum containing Vascular Softening Pills lowered the downstream signal of TLR9 MyD88,p-NF-κB and IRF7 mRNA expression levels(P<0.05).Vascular Softening Pills containing serum can regulated the expressions of LC3 II,Beclin,Akt,mTOR mRNA downstream of TLR3(P<0.05).Conclusion:Vascular Softening Pills regulate TLR9,downstream NF-κB and IRF7 signaling pathways,and then regulate macrophage M2/M1 balance and TLR3 to affect PI3K/Akt/mTOR signaling pathway,thereby regulating macrophage autophagy and inhibiting atherosclerosis the occurrence and development of hardening.
Objective:To observe the effect of Vascular Softening Pills on TLR3,regulating PI3K/Akt/mTOR signaling pathway,and then affecting macrophages autophagy,and observing the effect of vascular softening pills on TLR9,regulating downstream NF-κB and IRF7 signaling pathways.To influence the M2/M1 balance of macrophages and explore the molecular mechanism of Vascular Softening Pills against atherosclerosis(AS).Methods:In vitro,macrophages were randomly divided into 6 groups,namely the control group(blank serum),the high-dose group of Vascular Softening Pills,the middle dose group of Vascular Softening Pills,the low-dose group of Vascular Softening Pills,and the ODN group(TLR9 agonist:ODN1826),poly group(TLR3 agonist:poly(I:C).In vivo experiments,ApoE~(-/-) mice were divided into 6 groups,control group(normal saline,intragastric),high-dose group of Vascular Softening Pills(concentration 3.456 g/mL),Vascular Softening Pills medium dose group(concentration 1.728 g/mL),low-dose group of Vascular Softening Pills(concentration of 0.864 g/mL),ODN group(ODN1826,intraperitoneal injection),poly group[poly(I:C),intraperitoneal injection],intraperitoneal injection twice a week.The stomach was taken out once a day,and after 8 weeks of continuous administration,the material was taken out for testing.We observed the effect of serum containing Vascular Softening Pills and TLR9 on the polarization state of macrophages,the effect on the expressions of iNOS and CD206 in arterial plaques,the influence of macrophage(M1/M2) on inflammatory factors related to macrophages and the effect of phagocyte(M1/M2) balance-related inflammatory factors.The level of inflammatory factors secreted by cells was detected by ELISA,and the Vascular Softening Pills were used to detect TLR9 and TLR3 and downstream of aortic plaque by Western blot and RT-PCR.Western blot was used to detect the protein content of TLR9 in each group,and the protein content of aortic TLR9 and its downstream molecules MyD88,p-NF-κB and IRF7.The ratios of M1 and M2 in each group were detected by chemical fluorescence.The expression of TNF-αwas detected by RT-PCR and ELISA.The M1 type of RAW264.7 cells and arterial plaques were detected by RT-PCR and immunofluorescence.And related markers and inflammatory factors of M2 macrophages were observed.The pathological examination was used to verify the efficacy of vascular softening pills against atherosclerosis.Results:The Vascular Softening Pill-containing serum can induce macrophage high expression of M2 type macrophage marker CD206.The fluorescence density of iNOS in the plaque of Chinese medicine group was low expression(P<0.05),while the fluorescence density of CD206 in plaque was high expression(P<0.05).Vascular Softening Pills containing serum decreased IL-1β,iNOS,Ciita expression(P<0.05),increased Ym1 and Fizz1 expressions(P<0.05).Vascular Softening Pills containing serum can improve cell secretion inflammation.The levels of IFN-γand IL-10 were correlated(P<0.05).The serum containing Vascular Softening Pills lowered the expression of TLR9 protein and TLR3 protein(P<0.05).The serum containing Vascular Softening Pills lowered the downstream signal of TLR9 MyD88,p-NF-κB and IRF7 mRNA expression levels(P<0.05).Vascular Softening Pills containing serum can regulated the expressions of LC3 II,Beclin,Akt,mTOR mRNA downstream of TLR3(P<0.05).Conclusion:Vascular Softening Pills regulate TLR9,downstream NF-κB and IRF7 signaling pathways,and then regulate macrophage M2/M1 balance and TLR3 to affect PI3K/Akt/mTOR signaling pathway,thereby regulating macrophage autophagy and inhibiting atherosclerosis the occurrence and development of hardening.
引文
[1] Spirig R,Tsui J,Shaw S.The Emerging Role of TLR and Innate Immunity in Cardiovascular Disease[J].Cardiol Res Pract,2012,2012:181394.
[2] Liu T,Gao YJ,Ji RR.Emerging role of Toll-like receptors in the control of pain and itch[J].Neurosci Bull,2012,28(2):131-144.
[3] Shen P,Jiang TW,Lu HQ,et al.Combination of Poly I:C and arsenic trioxide triggers apoptosis synergistically via activation of TLR3 and mitochondrial pathways in hepatocellular carcinoma cells[J].Cell Biol Int,2011,35(8):803-810.
[4] 王和峰,翟纯刚,庞文会,等.PI3K/Akt /mTOR信号通路在巨噬细胞自体吞噬及动脉粥样硬化斑块不稳定中的作用机制[J].中国病理生理杂志,2013,29(3):390-397.
[5] 张志鑫,李彦杰,秦合伟,等.基于PI3K/Akt /mTOR信号通路调控巨噬细胞自噬探讨黄芪甲苷抗动脉粥样硬化的作用机制[J].中草药,2017,48(17):3575-3581.
[6] Moore K J,Sheedy F J,Fisher E A.Macrophages in atherosclemsis:A dynamic balance[J].Nat Rev,2013,13(10):709-721.
[7] Nagareddy P R,Murphy A J,Stirzaker R A,et al.Hyperglycemia promotes myelopoiesis and impairs the resolution of atherosclerosis[J].Cell Metab,2013,17(5):695-708.
[8] 晏浩,徐建军,李文林,等.MicroRNA-30a调控Beclin-1对缺氧复氧乳鼠心肌细胞的保护效应[J].中国病理生理杂志,2012,28(4):583-588.
[9] 翟纯刚,季晓平,王和峰. 抑制PI3K/Akt /mTOR信号通路对兔原代巨噬细胞自体吞噬的影响及机制[J]. 中国动脉硬化杂志,2014,22(1):7-12.
[10] Williams HJ,Fisher EA,Greaves DR.Macrophage differentiation and function in atherosclerosis:opportunities for therapeutic intervention[J].J Innate Immun,2012,4(5-6):498-508.
[11] ST?GER JL,GOOSSENS P,DE WINTHER MP.Macrophage heterogeneity:relevance and functional implications in atherosclerosis[J].Curr Vasc Pharmacol,2010,8(2):233-248.
[12] Chen Y,Liu W,Sun T,Huang Y et al.1,25-Dihydroxyvitamin D promotes negative feedback regulation of TLR signaling via targeting microRNA-155-SOCS1 in macrophages[J].J Immunol,2013,190(7):3687-3695.
[13] Zhang E,Wu Y.Dual effects of miR-155 on macrophages at different stages of atherosclerosis:LDL is the key?[J].Med Hypotheses,2014,83(1):74-78.
[14] Duramad O,Fearon KL,Chanq B,et al.Inhibitors of TLR-9 act on multiple cell subsets in mouse and man in vitro and prevent death in vivo from systemic inflammation[J].J Immunol,2005,174(9):5193-5200.
[15] 刘建东,秦合伟.血管软化丸治疗急性脑梗死合并颈动脉粥样硬化斑块60例[J].中国民族民间医药杂志,2017,26(18);88-91.
[2] Liu T,Gao YJ,Ji RR.Emerging role of Toll-like receptors in the control of pain and itch[J].Neurosci Bull,2012,28(2):131-144.
[3] Shen P,Jiang TW,Lu HQ,et al.Combination of Poly I:C and arsenic trioxide triggers apoptosis synergistically via activation of TLR3 and mitochondrial pathways in hepatocellular carcinoma cells[J].Cell Biol Int,2011,35(8):803-810.
[4] 王和峰,翟纯刚,庞文会,等.PI3K/Akt /mTOR信号通路在巨噬细胞自体吞噬及动脉粥样硬化斑块不稳定中的作用机制[J].中国病理生理杂志,2013,29(3):390-397.
[5] 张志鑫,李彦杰,秦合伟,等.基于PI3K/Akt /mTOR信号通路调控巨噬细胞自噬探讨黄芪甲苷抗动脉粥样硬化的作用机制[J].中草药,2017,48(17):3575-3581.
[6] Moore K J,Sheedy F J,Fisher E A.Macrophages in atherosclemsis:A dynamic balance[J].Nat Rev,2013,13(10):709-721.
[7] Nagareddy P R,Murphy A J,Stirzaker R A,et al.Hyperglycemia promotes myelopoiesis and impairs the resolution of atherosclerosis[J].Cell Metab,2013,17(5):695-708.
[8] 晏浩,徐建军,李文林,等.MicroRNA-30a调控Beclin-1对缺氧复氧乳鼠心肌细胞的保护效应[J].中国病理生理杂志,2012,28(4):583-588.
[9] 翟纯刚,季晓平,王和峰. 抑制PI3K/Akt /mTOR信号通路对兔原代巨噬细胞自体吞噬的影响及机制[J]. 中国动脉硬化杂志,2014,22(1):7-12.
[10] Williams HJ,Fisher EA,Greaves DR.Macrophage differentiation and function in atherosclerosis:opportunities for therapeutic intervention[J].J Innate Immun,2012,4(5-6):498-508.
[11] ST?GER JL,GOOSSENS P,DE WINTHER MP.Macrophage heterogeneity:relevance and functional implications in atherosclerosis[J].Curr Vasc Pharmacol,2010,8(2):233-248.
[12] Chen Y,Liu W,Sun T,Huang Y et al.1,25-Dihydroxyvitamin D promotes negative feedback regulation of TLR signaling via targeting microRNA-155-SOCS1 in macrophages[J].J Immunol,2013,190(7):3687-3695.
[13] Zhang E,Wu Y.Dual effects of miR-155 on macrophages at different stages of atherosclerosis:LDL is the key?[J].Med Hypotheses,2014,83(1):74-78.
[14] Duramad O,Fearon KL,Chanq B,et al.Inhibitors of TLR-9 act on multiple cell subsets in mouse and man in vitro and prevent death in vivo from systemic inflammation[J].J Immunol,2005,174(9):5193-5200.
[15] 刘建东,秦合伟.血管软化丸治疗急性脑梗死合并颈动脉粥样硬化斑块60例[J].中国民族民间医药杂志,2017,26(18);88-91.
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