温阳散结汤含药血清通过NF-κB通路调控肿瘤相关巨噬细胞极化对Lewis肺癌的影响

Effect of Wenyang Sanjie Decoction medicated serum of Lewis lung cancer by regulating the polarization of tumor-associated macrophages through the NF-κB pathway

目的:观察温阳散结汤对Lewis肺癌小鼠肿瘤生长及瘤组织中巨噬细胞M2型极化的影响,并探索其调控NF-κB信号通路抑制肿瘤相关巨噬细胞M2型极化,对Lewis肺癌细胞侵袭、迁移能力的影响。方法:建立C57BL/6小鼠Lewis肺癌移植瘤模型,温阳散结汤干预14 d后观察小鼠瘤重和肺转移灶,计算肺转移抑瘤率,免疫组化染色检测小鼠瘤组织中巨噬细胞M2型表面标志物CD206的表达;温阳散结汤灌胃SD大鼠制备含药血清,采用IL-13干预RAW264.7小鼠巨噬细胞建立巨噬细胞M2型极化模型,CCK-8法检测温阳散结汤含药血清对巨噬细胞增殖的影响,流式细胞术和Western-blot检测CD206的表达,RT-PCR检测巨噬细胞M2型标志基因的mRNA表达,ELISA法检测细胞上清中IL-1β、IL-10、TGF-β和TNF-α的水平变化,Western-blot检测氧化物酶体增殖物激活受体γ(PPARγ)、核因子-κB p65(NF-κB p65)和磷酸化核因子-κB p65(pNF-κB p65)的蛋白表达;收集温阳散结汤含药血清处理的M2型巨噬细胞条件培养基,将其作用于Lewis肺癌细胞,通过Transwell侵袭迁移实验检测Lewis肺癌细胞的侵袭和运动迁移能力。结果:温阳散结汤干预后,明显减少了小鼠瘤重和肺转移灶数,并抑制了瘤组织CD206的阳性表达(P<0.05);温阳散结汤含药血清干预后,IL-13诱导的M2型巨噬细胞中F4/80^(+)CD206^(+)的细胞比例和CD206蛋白表达明显减少,MRC1、Arg1、Ym1的mRNA表达水平显著降低,细胞中TNF-α、IL-1β的含量明显升高,IL-10、TGF-β的含量明显降低,同时明显下调了细胞PPARγ、NF-κB p65、pNF-κB p65的蛋白表达及pNF-κB p65/NF-κB p65比值(均P<0.05);温阳散结汤含药血清处理的M2型巨噬细胞条件培养基干预后,Lewis肺癌细胞迁移和侵袭细胞数明显减少(P<0.05)。结论:温阳散结汤具有抑制肺癌肿瘤生长和转移的作用,其作用机制可能与调节NF-κB通路,抑制IL-13诱导的RAW264.7细胞M2型极化,从而抑制Lewis肺癌细胞侵袭和迁移能力相关。

Objective:To observe the effect of Wenyang Sanjie Decoction on tumor growth and M2 polarization of macrophages in Lewis lung cancer mice,and to explore its regulation of NF-κB signaling pathway inhibits M2 polarization of tumor associated macrophages and its effect on the invasion and migration of Lewis lung cancer cells.Methods:The transplanted tumor model of Lewis lung cancer in C57BL/6 mice was established.After intervention with Wenyang Sanjie Decoction for 14 days,the tumor weight and lung metastasis were observed,the tumor inhibition rate of lung metastasis was calculated,and the expression of macrophage M2 surface marker CD206 in mouse tumor tissue was detected by immunohistochemical staining.The drug containing serum was prepared by intragastric administration of Wenyang Sanjie Decoction in different doses to SD rats.IL-13 was used to intervene the macrophages of RAW264.7 mice to establish the macrophage M2 polarization model.The effect of the drug containing serum of Wenyang Sanjie Decoction on the proliferation of macrophages was detected by CCK-8 method,and the expression of M2 surface marker CD206 of macrophages was detected by flow cytometry.The mRNA expression of M2 marker gene in macrophages was detected by RT-PCR,the content of IL-1β,IL-10,TGF-βand TNF-αin cell supernatant was detected by ELISA.Western-blot was used to detect the level of oxidase proliferator activated receptorγ(PPARγ),Nuclear factor-κB p65(NF-κB p65)and phosphorylated nuclear factor-κB p65(pNF-κB p65).The macrophage conditioned medium treated with Wenyang Sanjie Decoction containing drug serum was collected and acted on Lewis lung cancer cells.The invasion,movement and migration ability of Lewis lung cancer cells were detected by Transwell test.Results:After the intervention of Wenyang Sanjie Decoction,the tumor weight and the number of lung metastases in mice were significantly reduced,and the positive expression of CD206 in tumor tissue was inhibited(P<0.05).After the intervention of Wenyang Sanjie Decoction containing serum,the proportion of F4/80^(+)CD206^(+)cells in M2 macrophages induced by IL-13 decreased significantly,the mRNA expression levels of MRC1,Arg1 and Ym1 decreased significantly.The content of and TNF-α,IL-1βin cells was significantly increased,and IL-10,TGF-βsignificantly decreased.Significantly down-regulated the protein expression of cell PPARγ,NF-κB p65,pNF-κB p65 and the ratio of pNF-κB p65/NF-κB p65(all P<0.05).After the intervention of M2 macrophage conditioned medium treated with Wenyang Sanjie Decoction containing drug serum,the migration and invasion of Lewis lung cancer cells decreased significantly(P<0.05).Conclusion:Wenyang Sanjie Decoction can inhibit the growth and metastasis of lung cancer.Its mechanism may be related to regulating NF-κB pathway,inhibiting IL-13-induced M2 polarization of RAW264.7 cells and inhibiting the invasion and migration of Lewis lung cancer cells.