自噬抑制剂巴弗洛霉素A1对巨噬细胞极化的影响
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摘要
目的:探讨自噬下游抑制剂巴弗洛霉素A1(bafilomycin A1,Baf A1)对小鼠单核巨噬细胞RAW264.7极化的影响。方法:用Baf A1作用于小鼠单核巨噬细胞RAW264.7,酶联免疫吸附实验检测M1/M2极化相关细胞因子的含量;流式细胞术检测细胞表面M1/M2极化相关标志物;免疫荧光观察自噬小体形成情况;Western blot检测自噬相关蛋白水平变化。结果:Baf A1处理后,RAW264.7细胞分泌的促炎性细胞因子肿瘤坏死因子α(TNF-α)显著增高(P<0.01),而抑炎性细胞因子IL-10和IL-13的含量变化无显著差异。Baf A1处理的细胞表面标志物中CD197和HLA-DR(M1标志物)双阳性率与对照组相比显著升高(P<0.05),说明Baf A1可以诱导巨噬细胞向M1方向极化。免疫荧光结果显示,Baf A1处理后细胞自噬小体显著增多(P<0.05)。Western blot结果显示Baf A1处理后,自噬相关蛋白LC3-Ⅱ表达显著升高(P<0.05),而P62蛋白表达无显著差异。在自噬激活剂雷帕霉素处理组中,自噬体同样显著增多(P<0.05),但P62蛋白表达显著降低(P<0.05)。结论:巴弗洛霉素A1可以诱导小鼠单核巨噬细胞RAW264.7向M1方向极化,可能与其抑制自噬下游自噬溶酶体的形成有关。
AIM: To investigate the effect of bafilomycin A1(Baf A1) on polarization in mouse macrophages RAW264.7. METHODS: The macrophages RAW264.7 were treated with Baf A1, the concentration of M1/M2 polarization related cytokines were determined by ELISA. The markers of M1/M2 polarization in the macrophages were determined by flow cytometry. The formation of autophagicbody was observed by immunofluorescence. Western blot was used to detect the expression of autophagy related protein levels. RESULTS: The concentration of M1 related proinflammatory cytokine tumor necrosis factor-α(TNF-α) was increased significantly after Baf A1 intervention(P<0.01). However, the concentration of M2 related anti-inflammatory cytokines IL-10 and IL-13 showed no significant difference. The double positive rate of CD197 and HLA-DR(M1 marker) positive cells in Baf A1 treated group were significant higher than that in control group(P<0.05), indicated that Baf A1 induced polarization of macrophage to M1. The results of immunofluoresence showed that the autophagosomes formation was notable increased in Baf A1 group(P<0.05), meanwhile Western blot results showed the expression of autophay related protein LC3-Ⅱ but not P62 was marked up-regulated(P<0.05). For autophagy activator rapamycin(Rapa) treated group, autophagosome formation was also significant increased(P<0.05), but the expression of P62 was notable down-regulated(P<0.05). CONCLUSION: Baf A1 induces polarization of mouse macrophage RAW264.7 to M1, which may be related to the inhibitory effect on the formation of autolysosome.
AIM: To investigate the effect of bafilomycin A1(Baf A1) on polarization in mouse macrophages RAW264.7. METHODS: The macrophages RAW264.7 were treated with Baf A1, the concentration of M1/M2 polarization related cytokines were determined by ELISA. The markers of M1/M2 polarization in the macrophages were determined by flow cytometry. The formation of autophagicbody was observed by immunofluorescence. Western blot was used to detect the expression of autophagy related protein levels. RESULTS: The concentration of M1 related proinflammatory cytokine tumor necrosis factor-α(TNF-α) was increased significantly after Baf A1 intervention(P<0.01). However, the concentration of M2 related anti-inflammatory cytokines IL-10 and IL-13 showed no significant difference. The double positive rate of CD197 and HLA-DR(M1 marker) positive cells in Baf A1 treated group were significant higher than that in control group(P<0.05), indicated that Baf A1 induced polarization of macrophage to M1. The results of immunofluoresence showed that the autophagosomes formation was notable increased in Baf A1 group(P<0.05), meanwhile Western blot results showed the expression of autophay related protein LC3-Ⅱ but not P62 was marked up-regulated(P<0.05). For autophagy activator rapamycin(Rapa) treated group, autophagosome formation was also significant increased(P<0.05), but the expression of P62 was notable down-regulated(P<0.05). CONCLUSION: Baf A1 induces polarization of mouse macrophage RAW264.7 to M1, which may be related to the inhibitory effect on the formation of autolysosome.
引文
[1] Gourevitch D,Kossenkov AV,Zhang Y,et al.Inflammation and its correlates in regenerative wound healing:an alternate perspective[J].Adv Wound Care,2014,3(9):592-603.
[2] Nguyen KT,Seth AK,Hong SJ,et al.Deficient cytokine expression and neutrophil oxidative burst contribute to impaired cutaneous wound healing in diabetic,biofilm-containing chronic wounds[J].Wound Repair Regen,2013,21(6):833-841.
[3] Hsieh JY,Smith TD,Meli VS,et al.Differential regulation of macrophage inflammatory activation by fibrin and fibrinogen[J].Acta Biomater,2017,47:14-24.
[4] Fukushima H,Yamashina S,Arakawa A,et al.Formation of p62-positive inclusion body is associated with macrophage polarization in non-alcoholic fatty liver disease[J].Hepatol Res,2018,48(9):757-767.
[5] Deretic V,Saitoh T.Autophagy in infection,inflammation and immunity[J].Nat Rev Immunol,2013,13(10):722-737.
[6] 钟娟,青姚,吴曙粤,等.厄贝沙坦通过诱导自噬减轻db/db小鼠肝脏脂肪变[J].中国病理生理杂志,2018,34(3):521-527.
[7] Bai YD,Yang YR,Mu XP,et al.Hydrogen sulfide alleviates acute myocardial ischemia injury by modulating autophagy and inflammation response under oxidative stress[J].Oxid Med Cell Longev,2018,2018(6):1-17.
[8] 赵亚男,刘明,张玥,等.糖尿病溃疡中Wnt/β-catenin信号通路表达的变化[J].中国病理生理杂志,2015,31(11):2033-2038.
[9] Guo Y,Lin C,Xu P,et al.AGEs induced autophagy impairs cutaneous wound healing via stimulating macrophage polarization to M1 in diabetes[J].Sci Rep,2016,6:36416.
[10] Ryter SW,Cloonan SM.Autophagy:a critical regulator of cellular metabolism and homeostasis[J].Mol Cells,2013,36(1):7-16.
[11] Mizushima N,Yoshimori T.The role of Atg proteins in autophagosome formation[J].Annu Rev Cell Dev Biol,2011,27:107-132.
[12] Salminen A,Kaarniranta K.Inflammaging:disturbed interplay between autophagy and inflammasomes[J].Aging,2012,4(3):166-175.
[13] Liu K,Zhao E,Ilyas G,et al.Impaired macrophage autophagy increases the immune response in obese mice by promoting proinflammatory macrophage polarization[J].Autophagy,2015,11(2):271-284.
[2] Nguyen KT,Seth AK,Hong SJ,et al.Deficient cytokine expression and neutrophil oxidative burst contribute to impaired cutaneous wound healing in diabetic,biofilm-containing chronic wounds[J].Wound Repair Regen,2013,21(6):833-841.
[3] Hsieh JY,Smith TD,Meli VS,et al.Differential regulation of macrophage inflammatory activation by fibrin and fibrinogen[J].Acta Biomater,2017,47:14-24.
[4] Fukushima H,Yamashina S,Arakawa A,et al.Formation of p62-positive inclusion body is associated with macrophage polarization in non-alcoholic fatty liver disease[J].Hepatol Res,2018,48(9):757-767.
[5] Deretic V,Saitoh T.Autophagy in infection,inflammation and immunity[J].Nat Rev Immunol,2013,13(10):722-737.
[6] 钟娟,青姚,吴曙粤,等.厄贝沙坦通过诱导自噬减轻db/db小鼠肝脏脂肪变[J].中国病理生理杂志,2018,34(3):521-527.
[7] Bai YD,Yang YR,Mu XP,et al.Hydrogen sulfide alleviates acute myocardial ischemia injury by modulating autophagy and inflammation response under oxidative stress[J].Oxid Med Cell Longev,2018,2018(6):1-17.
[8] 赵亚男,刘明,张玥,等.糖尿病溃疡中Wnt/β-catenin信号通路表达的变化[J].中国病理生理杂志,2015,31(11):2033-2038.
[9] Guo Y,Lin C,Xu P,et al.AGEs induced autophagy impairs cutaneous wound healing via stimulating macrophage polarization to M1 in diabetes[J].Sci Rep,2016,6:36416.
[10] Ryter SW,Cloonan SM.Autophagy:a critical regulator of cellular metabolism and homeostasis[J].Mol Cells,2013,36(1):7-16.
[11] Mizushima N,Yoshimori T.The role of Atg proteins in autophagosome formation[J].Annu Rev Cell Dev Biol,2011,27:107-132.
[12] Salminen A,Kaarniranta K.Inflammaging:disturbed interplay between autophagy and inflammasomes[J].Aging,2012,4(3):166-175.
[13] Liu K,Zhao E,Ilyas G,et al.Impaired macrophage autophagy increases the immune response in obese mice by promoting proinflammatory macrophage polarization[J].Autophagy,2015,11(2):271-284.