肺岩宁颗粒对肿瘤相关巨噬细胞及Lewis肺癌细胞侵袭能力的影响

The Influence of Fei Yan Ning Granule on Invasion of Lewis lung Cancer Cells by Regulating Tumor-Related M acrophages

目的:观察中药制剂肺岩宁颗粒对肿瘤微环境中的关键成分肿瘤相关巨噬细胞的作用及其对小鼠Lewis肺癌细胞侵袭能力的调节作用。方法:采用肺岩宁颗粒给SD大鼠灌胃制备药物血清;利用Lewis肺癌细胞制作条件培养基(Conditioned medium,CM)并诱导小鼠巨噬细胞RAW264.7细胞转化为肿瘤相关巨噬细胞;侵袭实验检测肺岩宁颗粒对Lewis肺癌细胞侵袭能力的影响;ELISA法检测细胞上清中白细胞介素-10(interleukin-10,IL-10)、白细胞介素-12(interleukin-12 p40,IL-12)、转化生长因子-β(transforming growth factor-β,TGF-β)水平;Western blot检测肿瘤细胞中基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)、基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)、核转录因子kappa B p65(unclear factor ka PPa B p65,NF-κB p65)蛋白表达。结果:侵袭实验:DDP组、肺岩宁颗粒组的细胞侵袭能力受到抑制,这两组穿过膜的Lewis细胞个数比模型组少(P<0.05),在此基础上发现肺岩宁颗粒组穿过膜的Lewis细胞个数比DDP组少(P<0.05)。ELISA法:DDP组、肺岩宁颗粒组两组细胞上清液的IL-10的含量水平比模型组低(P<0.05),而肺岩宁颗粒组细胞和DDP组比较,肺岩宁颗粒组细胞上清液IL-10的含量水平比DDP组高(P<0.05);肺岩宁颗粒组细胞上清IL-12水平明显高于模型组(P<0.05),而且肺岩宁颗粒组细胞上清IL-12水平高于DDP组(P<0.05);DDP组、肺岩宁颗粒组细胞上清TGF-β1水平均明显低于模型组(P<0.05);与模型组比较,DDP组、肺岩宁颗粒组细胞中MMP-2、MMP-9、NF-κB p65的表达均降低,且肺岩宁颗粒组表达更低。结论:肺岩宁颗粒能抑制Lewis肺癌细胞侵袭能力,其机制可能是通过对NF-κB信号通路的干预进一步调节肿瘤相关巨噬细胞,促进M2向M1转化、抑制M2,抑制血清中IL-10、TGF-β1,提升IL-12水平,抑制MMP-2、MMP-9的产生。

Objective：To observe the influence of Fei Yan Ning Granule（ FYNG） on tumor-related macrophages,the key components of tumor microenvironment and the regulating effect on the invasion ability of Lewis lung cancer cells in mice. Methods：The drug serum was prepared by gavage of FYNG on SD rats. Lewis lung cancer cells were used to make Conditioned medium（ CM）and induce the RAW264. 7 cells of mouse macrophages to be transformed into tumor related macrophages. The effect of FYNG on the invasion ability of Lewis lung cancer cells was detected by the method of invasion. The levels of interleukin-10（ IL-10）,interleukin-12 P40（ IL-12 P40） and transforming growth factor beta（ beta） in the cell supernatant were detected by ELISA. The protein expression of matrix metalloproteinase-9（ MMP-9） and nuclear transcription factor kappa B p65（ p65） were detected. Results：The invasion experiment showed that the invasive abilities of the DDP group and the FYNG group were inhibited. The number of Lewis cells passing through the membrane was less than that of the model group（ P〈0. 05）. On this basis,the number of Lewis cells passing through the membrane in the FYNG was less than that of the DDP group（ P〈0. 05）. ELISA method：The levels of IL-10 in cell supernatant of two groups in group DDP and FYNG group were lower than that of the model group（ P〈0. 05）,but the level of IL-10 in the cell supernatant of FYNG group was higher than that of the DDP group（ P〈0. 05）,and the level of IL-12 in the cell supernatant of the FYNG group was obviously higher than that of the model group（ P〈0. 05）. The level of IL-12 in cell supernatant of FYNG group was higher than that of group DDP（ P〈0. 05）,and the levels of TGF-beta 1 in DDP group and FYNG group were significantly lower than that of model group（ P〈0. 05）. Western blot detected MMP-2,MMP-9 and NF-κB p65 in the cells：compared with the model group,the expressions of MMP-2,MMP-9 and NF-κB p65 in the DDP group and FYNG were all decreased,and the expression of FYNG was lower. Conclusion：FYNG can inhibit the invasion ability of Lewis lung cancer cells in vitro,which may be mediated by NF-κB signaling pathway to regulate tumor-related macrophages and their secreted cytokines.