血管活性肠肽对肺泡巨噬细胞M1/M2型极化及相关细胞因子的影响

Effects of Vasoactive Intestinal Peptide on M1/M2 Polarization and Related Cytokines in Alveolar Macrophages

目的观察血管活性肠肽(vasoactive intestinal peptide,VIP)对肺泡巨噬细胞(alveolar macrophages,AM) M1/M2极化和相关细胞因子的影响。方法分别采用脂多糖(lipopolysaccharide,LPS)联合γ-干扰素(interferon-γ,IFN-γ)、白细胞介素-4(interleukin 4,IL-4)联合白细胞介素-13(interleukin 13,IL-13)诱导AM M1/M2型极化,后用10–8~10–6 mol·L^(-1) VIP干预极化的AM,免疫荧光双标法测定M1型AM标记物CD86和促炎因子白细胞介素-6(interleukin 6,IL-6)的共表达,M2型AM标记物CD206和抗炎因子白细胞介素-10(interleukin 10,IL-10)的共表达;ELISA检测细胞上清中IL-6、肿瘤坏死因子-α(tumor necrosisfactor-α,TNF-α)、IL-10、精氨酸酶-1(arginase-1,Arg-1)的表达,RT-PCR检测巨噬细胞表面标记物CD86、CD206和巨噬细胞相关因子IL-6、TNF-α、诱导型一氧化氮合酶(inducible nitric oxide synthase,i NOS)、IL-10、Arg-1、几丁质酶-3样蛋白3(chitinase 3-like 3,Ym1)的表达。结果经LPS联合IFN-γ、IL-4联合IL-13诱导后,AM分别向M1/M2型极化,与M1型相关的促炎因子IL-6、TNF-α、i NOS和与M2型相关的抗炎因子IL-10、Arg-1、Ym1的激活和释放明显高于未经诱导的正常组(P<0.05);不同浓度VIP(10–8~10–6 mol·L^(-1))的干预,均可下调M1型AM和相关炎症因子IL-6、TNF-α、i NOS的活性和表达(P<0.05);上调M2型AM和相关抗炎因子IL-10、Arg-1、Ym1的活性和表达(P<0.05)。结论 VIP可抑制AM M1型极化,减少炎症因子IL-6、TNF-α、i NOS的激活和释放;促进AMM2型极化,提高抗炎因子IL-10、Arg-1、Ym1的激活和释放,提示VIP在肺内炎症时可能通过调控AM M1/M2型极化,抑制炎症反应,对肺组织起保护性作用。

OBJECTIVE To observe the effects of vasoactive intestinal peptide(VIP) on M1/M2 polarization and related cytokines of alveolar macrophages(AM). METHODS Lipopolysaccharide(LPS) combined with interferon-γ(IFN-γ) and interleukin-4(IL-4) combined with interleukin-13(IL-13) were used to induce M1/M2 type polarization of AM, and then VIP(10-8-10-6 mol·L^(-1)) was used to intervene the polarization of AM. The co-expression of M1 type AM marker CD86 and pro-inflammatory factor interleukin 6(IL-6) and co-expression of M2-type AM marker CD206 and anti-inflammatory factor interleukin 10(IL-10) was determined by immunofluorescence double standard method. Expression of IL-6, tumor necrosis factor-α(TNF-α), IL-10 and arginase-1(Arg-1) in cell supernatant was detected by ELISA. The macrophage surface markers CD86, CD206 and macrophage related factors IL-6, TNF-α, inducible nitric oxide synthase(iNOS), IL-10, Arg-1 and chitinase 3-like 3(Ym1) were detected by RT-PCR. RESULTS After induction by LPS combined with IFN-γ and IL-4 combined with IL-13, AM was polarized to M1/M2 type respectively. The activation and release of pro-inflammatory factors IL-6, TNF-α and iNOS related to M1 and anti-inflammatory factors IL-10, Arg-1 and Ym1 related to M2 were significantly higher than those in the uninduced normal group(P<0.05). Interference with different concentrations of VIP(10-8-10-6 mol·L^(-1)) could down-regulate the activity and expression of M1 type AM and related inflammatory cytokines IL-6, TNF-α and iNOS(P<0.05). The activity and expression of M2 type AM and related anti-inflammatory factors IL-10, Arg-1 and Ym1 were up-regulated(P<0.05). CONCLUSION VIP can inhibit AM M1-type polarization and reduce the activation and release of inflammatory cytokines IL-6, TNF-α and i NOS. It can promote AM M2-type polarization and increase the activation and release of anti-inflammatory factors IL-10, Arg-1 and Ym1, suggesting that VIP may inhibit the inflammatory response and play a protective role in lung tissue by regulating the M1/M2-type polarization of AM in the process of pulmonary inflammation.