艾灸干预促进创伤大鼠伤口愈合的机制研究
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摘要
目的:通过观察艾灸对参与创伤愈合炎性反应阶段的细胞因子表达的影响,探讨艾灸促进伤口愈合的机制。方法:SD雄性大鼠随机分为正常组(6只)、模型组(39只)和艾灸组(39只)。采用直径8mm的取皮器从大鼠背部正中线肩胛下角2cm处钻取全层皮肤制备皮肤创伤模型。造模后即刻至造模后第5天每天给予创伤局部25min的艾灸干预。采用免疫荧光染色观察模型组和艾灸组大鼠创伤局部组织血管重建情况。造模后第1、2、3、5天采用液态芯片法检测创伤模型大鼠血清中白细胞介素(IL)-1α、IL-1β、IL-6、IL-4、IL-10、IL-13、粒细胞集落刺激因子(G-CSF)、粒细胞-巨噬细胞集落刺激因子(GM-CSF)、巨噬细胞炎性蛋白(MIP)-1α、血管内皮生长因子(VEGF)的含量。结果:与模型组比较,造模后第1天艾灸组大鼠血清中IL-1α和IL-6的含量显著升高(P<0.05),第1、2天艾灸组血清中IL-1β的含量显著升高(P<0.05),第3天艾灸组血清中IL-1α和IL-6的含量显著降低(P<0.05)。艾灸组大鼠前3dMIP-1α的含量均升高,且在造模后第2天明显升高(P<0.05)。艾灸组大鼠造模后第1天IL-4、IL-10含量显著升高(P<0.05),造模后第3天显著降低(P<0.05)。艾灸组大鼠造模后第1、2天血清中VEGF的含量显著升高(P<0.01,P<0.05)。艾灸组大鼠造模后第5天创伤组织中VEGF的含量显著升高(P<0.01)。艾灸组大鼠造模后第5天创伤组织中新生血管数量明显增加。结论:艾灸通过调控促炎细胞因子促进创伤炎性反应的同时,也调控了抗炎细胞因子在这一过程中的作用,促进并提前结束创伤愈合的炎性阶段,使创伤愈合提前进入增殖修复期,从而促进伤口愈合。
Objective Our previous study demonstrated that moxibustion could promote skin wound healing in rats with full-thickness cutaneous wounds.The present study was designed to observe the effect of moxibustion on the levels of pro-inflammatory and anti-inflammatory cytokines,and proliferation of vascular endothelial cells,so as to explore its mechanisms underlying facilitating wound-healing.Methods A total of 84 adult SD male rats were randomly assigned to normal(n=6),model(n=39)and moxibustion(n=39)groups.The skin wound model was established by removal of a piece of full-thickness skin from the median line of the rats' back(about 2 cm below the shoulder blade).Moxibustion was applied to the surrounding area of the focus for25 min,once daily for 6 days.The blood samples were collected at day 1,2,3 and 5 after modeling for assaying serum contents of IL-1α,IL-1β,IL-6,IL-4,IL-10,IL-13,granulocyte-colony stimulating factor(G-CSF),granulocyte macrophagecolony stimulating factor(GM-CSF,an inflammatory mediator),macrophage inhibitory protein-1α(MIP-1α)and vascular endothelial growth factor(VEGF)with liquid chip methods,and the topical wound tissues were collected after trans-cardiac perfusion with 4% paraformaldehyde solution for detecting the expression of CD31 proteins by using immunofluorescence staining.Results After modeling and in comparison with the model group,the contents of serum IL-1αand IL-6 at the 1 st day were significantly increased(P<0.05),but notably decreased at the 3 rd day after modeling in the moxibustion group(P<0.05).The contents of serum IL-1βon day 1 and 2 were significantly higher in the moxibustion group than those of the model group(P<0.05).After moxibustion,the contents of serum IL-4 and IL-10 were significantly up-regulated on day 1 after modeling(P<0.05),and obviously down-regulated from the 3 rd day on(P<0.05).The contents of serum MIP-1αon the 2 nd day and serum VEGF on day 1 and 2,and wound-skin VEGF on day 5 were obviously up-regulated in the moxibustion group in comparison with the model group(P<0.05,P<0.01).Conclusion Moxibustion promotes the inflammatory response by regulating the levels of both pro-inflammatory and anti-inflammatory cytokines in the early phase of skin wounds in rats,which may be in favor of the transformation from the inflammatory phase to the proliferation phase in advance,promoting wound healing at last.
Objective Our previous study demonstrated that moxibustion could promote skin wound healing in rats with full-thickness cutaneous wounds.The present study was designed to observe the effect of moxibustion on the levels of pro-inflammatory and anti-inflammatory cytokines,and proliferation of vascular endothelial cells,so as to explore its mechanisms underlying facilitating wound-healing.Methods A total of 84 adult SD male rats were randomly assigned to normal(n=6),model(n=39)and moxibustion(n=39)groups.The skin wound model was established by removal of a piece of full-thickness skin from the median line of the rats' back(about 2 cm below the shoulder blade).Moxibustion was applied to the surrounding area of the focus for25 min,once daily for 6 days.The blood samples were collected at day 1,2,3 and 5 after modeling for assaying serum contents of IL-1α,IL-1β,IL-6,IL-4,IL-10,IL-13,granulocyte-colony stimulating factor(G-CSF),granulocyte macrophagecolony stimulating factor(GM-CSF,an inflammatory mediator),macrophage inhibitory protein-1α(MIP-1α)and vascular endothelial growth factor(VEGF)with liquid chip methods,and the topical wound tissues were collected after trans-cardiac perfusion with 4% paraformaldehyde solution for detecting the expression of CD31 proteins by using immunofluorescence staining.Results After modeling and in comparison with the model group,the contents of serum IL-1αand IL-6 at the 1 st day were significantly increased(P<0.05),but notably decreased at the 3 rd day after modeling in the moxibustion group(P<0.05).The contents of serum IL-1βon day 1 and 2 were significantly higher in the moxibustion group than those of the model group(P<0.05).After moxibustion,the contents of serum IL-4 and IL-10 were significantly up-regulated on day 1 after modeling(P<0.05),and obviously down-regulated from the 3 rd day on(P<0.05).The contents of serum MIP-1αon the 2 nd day and serum VEGF on day 1 and 2,and wound-skin VEGF on day 5 were obviously up-regulated in the moxibustion group in comparison with the model group(P<0.05,P<0.01).Conclusion Moxibustion promotes the inflammatory response by regulating the levels of both pro-inflammatory and anti-inflammatory cytokines in the early phase of skin wounds in rats,which may be in favor of the transformation from the inflammatory phase to the proliferation phase in advance,promoting wound healing at last.
引文
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[2] BONIAKOWSKI A E,KIMBALL A S,JACOBS B N,et al.Macrophage-mediated inflammation in normal and diabetic wound healing[J].J Immunol,2017,199(1):17-24.
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[5] RIDER P,CARMI Y,GUTTMAN O,et al.IL-1αand IL-1βrecruit different myeloid cells and promote different stages of sterile inflammation[J].J Immunol,2011,187(9):4835-4843.
[6] MCFARLAND-MANCINI M M,FUNK H M,PALUCH A M,et al.Differences in wound healing in mice with deficiency of IL-6versus IL-6receptor[J].J Immunol,2010,184(12):7219-7228.
[7] MOSSER D M,EDWARDS J P.Exploring the full spectrum of macrophage activation[J].Nat Rev Immunol,2008,8(12):958-969.
[8] REIBER G E,VILEIKYTE L,BOYKO E J,et al.Causal pathways for incident lower-extremity ulcers in patients with diabetes from two settings[J].Diabetes Care,1999,22(1):157-162.
[9] MIAO M Y,NIU Y W,XIE T,et al.Diabetes-impaired wound healing and altered macrophage activation:apossible pathophysiologic correlation[J]. Wound Repair Regen,2012,20(2):203-213.
[10] BREM H,TOMIC-CANIC M.Cellular and molecular basis of wound healing in diabetes[J].J Clin Invest,2007,117(5):1219-1222.
[11] OKIZAKI S,ITO Y,HOSONO K,et al.Vascular endothelial growth factor receptor type 1signaling prevents delayed wound healing in diabetes by attenuating the production of IL-1βby recruited macrophages[J].Am J Pathol,2016,186(6):1481-1498.
[12]孙琦,孙忠人,张秦宏,等,艾灸对大鼠创伤皮肤组织血管内皮细胞和血管内皮生长因子表达的影响[J].中国针灸,2014,34(7):679-684.
[13]孙立虹,梁玉磊,孙彦辉等.温和灸对大鼠慢性难愈性创面组织巨噬细胞及胶原表达的影响[J].针刺研究,2012,37(4):259-265.
[14]阚宇,张晓宁,于清泉,等.艾灸促进皮肤外伤愈合及对局部组织的修复作用[J].针刺研究,2019,44(4):290-294.
[15] DAVATELIS G,TEKAMP-OLSON P,WOLPE S D,et al.Cloning and characterization of a cDNA for murine macrophage inflammatory protein(MIP),a novel monokine with inflammatory and chemokinetic properties[J].J Exp Med,1988,167(6):1939-1944.
[16] KILGORE K S,SCHMID E,SHANLEY T P,et al.Sublytic concentrations of the membrane attack complex of complement induce endothelial interleukin-8and monocyte chemoattractant protein-1through nuclear factor-kappa B activation[J].Am J Pathol,1997,150(6):2019-2031.
[17] DIPIETRO L A,BURDICK M,LOW Q E,et al.MIP-1alpha as a critical macrophage chemoattractant in murine wound repair[J].J Clin Invest,1998,101(8):1693-1698.
[18] TANAKA T,NARAZAKI M,KISHIMOTO T.IL-6in inflammation,immunity,and disease[J].Cold Spring Harb Perspect Biol,2014,6(10):a016295.
[19] CHAMBERLAIN C S,LEIFERMAN E M,FRISCH K E,et al.The influence of interleukin-4on ligament healing[J].Wound Repair Regen,2011,19(3):426-435.
[20] MOSSER D M,ZHANG X.Interleukin-10:new perspectives on an old cytokine[J].Immunol Rev,2008,226:205-218.
[21] WISE L M,STUART G S,REAL N C,et al.Orf virus IL-10accelerates wound healing while limiting inflammation and scarring[J].Wound Repair Regen,2014,22(3):356-367.
[22] KIERAN I,KNOCK A,BUSH J,et al.Interleukin-10reduces scar formation in both animal and human cutaneous wounds:results of two preclinical and phase II randomized control studies[J]. Wound Repair Regen,2013,21(3):428-436.
[23] MAKITA N,HIZUKURI Y,YAMASHIRO K,et al.IL-10enhances the phenotype of M2macrophages induced by IL-4and confers the ability to increase eosinophil migration[J].Int Immunol,2015,27(3):131-141.
[2] BONIAKOWSKI A E,KIMBALL A S,JACOBS B N,et al.Macrophage-mediated inflammation in normal and diabetic wound healing[J].J Immunol,2017,199(1):17-24.
[3] WYNN T A,BARRON L.Macrophages:master regulators of inflammation and fibrosis[J].Semin Liver Dis,2010,30(3):245-257.
[4] DIPIETRO L A.Wound healing:the role of the macrophage and other immune cells[J].Shock,1995,4(4):233-240.
[5] RIDER P,CARMI Y,GUTTMAN O,et al.IL-1αand IL-1βrecruit different myeloid cells and promote different stages of sterile inflammation[J].J Immunol,2011,187(9):4835-4843.
[6] MCFARLAND-MANCINI M M,FUNK H M,PALUCH A M,et al.Differences in wound healing in mice with deficiency of IL-6versus IL-6receptor[J].J Immunol,2010,184(12):7219-7228.
[7] MOSSER D M,EDWARDS J P.Exploring the full spectrum of macrophage activation[J].Nat Rev Immunol,2008,8(12):958-969.
[8] REIBER G E,VILEIKYTE L,BOYKO E J,et al.Causal pathways for incident lower-extremity ulcers in patients with diabetes from two settings[J].Diabetes Care,1999,22(1):157-162.
[9] MIAO M Y,NIU Y W,XIE T,et al.Diabetes-impaired wound healing and altered macrophage activation:apossible pathophysiologic correlation[J]. Wound Repair Regen,2012,20(2):203-213.
[10] BREM H,TOMIC-CANIC M.Cellular and molecular basis of wound healing in diabetes[J].J Clin Invest,2007,117(5):1219-1222.
[11] OKIZAKI S,ITO Y,HOSONO K,et al.Vascular endothelial growth factor receptor type 1signaling prevents delayed wound healing in diabetes by attenuating the production of IL-1βby recruited macrophages[J].Am J Pathol,2016,186(6):1481-1498.
[12]孙琦,孙忠人,张秦宏,等,艾灸对大鼠创伤皮肤组织血管内皮细胞和血管内皮生长因子表达的影响[J].中国针灸,2014,34(7):679-684.
[13]孙立虹,梁玉磊,孙彦辉等.温和灸对大鼠慢性难愈性创面组织巨噬细胞及胶原表达的影响[J].针刺研究,2012,37(4):259-265.
[14]阚宇,张晓宁,于清泉,等.艾灸促进皮肤外伤愈合及对局部组织的修复作用[J].针刺研究,2019,44(4):290-294.
[15] DAVATELIS G,TEKAMP-OLSON P,WOLPE S D,et al.Cloning and characterization of a cDNA for murine macrophage inflammatory protein(MIP),a novel monokine with inflammatory and chemokinetic properties[J].J Exp Med,1988,167(6):1939-1944.
[16] KILGORE K S,SCHMID E,SHANLEY T P,et al.Sublytic concentrations of the membrane attack complex of complement induce endothelial interleukin-8and monocyte chemoattractant protein-1through nuclear factor-kappa B activation[J].Am J Pathol,1997,150(6):2019-2031.
[17] DIPIETRO L A,BURDICK M,LOW Q E,et al.MIP-1alpha as a critical macrophage chemoattractant in murine wound repair[J].J Clin Invest,1998,101(8):1693-1698.
[18] TANAKA T,NARAZAKI M,KISHIMOTO T.IL-6in inflammation,immunity,and disease[J].Cold Spring Harb Perspect Biol,2014,6(10):a016295.
[19] CHAMBERLAIN C S,LEIFERMAN E M,FRISCH K E,et al.The influence of interleukin-4on ligament healing[J].Wound Repair Regen,2011,19(3):426-435.
[20] MOSSER D M,ZHANG X.Interleukin-10:new perspectives on an old cytokine[J].Immunol Rev,2008,226:205-218.
[21] WISE L M,STUART G S,REAL N C,et al.Orf virus IL-10accelerates wound healing while limiting inflammation and scarring[J].Wound Repair Regen,2014,22(3):356-367.
[22] KIERAN I,KNOCK A,BUSH J,et al.Interleukin-10reduces scar formation in both animal and human cutaneous wounds:results of two preclinical and phase II randomized control studies[J]. Wound Repair Regen,2013,21(3):428-436.
[23] MAKITA N,HIZUKURI Y,YAMASHIRO K,et al.IL-10enhances the phenotype of M2macrophages induced by IL-4and confers the ability to increase eosinophil migration[J].Int Immunol,2015,27(3):131-141.