| [Purpose] To observe whether RES can affect the metastatic ability of NPC by regulating the polarization state of tumor-associated macrophages and to explore its mechanism.[Methods] (1) Induction of M2 macrophages: PMA was used to induce M0 macrophages from human leukemia monocytes THP-1 , FCM was employed to detect M0 macrophages marked by CD11B;nasopharyngeal carcinoma CNE2 cells were co-cultured with M0 macrophages,FCM was used to detect M2-type macrophages marked by CD163 and CD206, and qPCR was used to detect IL-10 and TGF-β1. (2) Effects of RES on the polarization state of macrophages: M2 macrophages were treated with 40 μM resveratrol for 24 hours, FCM was used to detect the proportion of M2 macrophages expressing CD163 and CD206, as well as M1 macrophages expressing CD80 and CD86; qPCR was used to detect the changes in CD163, TGF-β1, and IL-10;WB was used to detect the expression levels of PI3K and p-AKT proteins.(3) Effects of RES on the metastasis ability of NPC cells by regulating macrophages: NPC cells were cultured for 24 hours in M2-CM and RES-M2-CM respectively,transwell assay was used to detect the migration and invasion abilities ,WB was used to detect the expression levels of MMP2, MMP9, E-cadherin, Vimentin proteins in the two groups of cells.[Results] (1) Successful induction of M2 macrophages: during the process of PMA-induced THP-1 cell transformation, changes in cell morphology were observed under an optical microscope, and FCM detection revealed an increase in cells expressing CD11B； after 24 hours of co-culture with CNE2-CM, FCM detection found an increase in the number of M2 macrophages expressing CD163 and CD206, and qPCR detection suggested elevated expression of CD163, TGF-β1, and IL-10 cytokines. (2) After RES treatment of M2 macrophages for 24 hours, the number of macrophages expressing CD163 and CD206 decreased（p＜0.001）, while the number of macrophages expressing CD80 and CD86 increased（p＜0.01）; the expression levels of CD163, TGF-β1, and IL-10 decreased（p＜0.01）, WB experiments revealed a decrease in the expression levels of PI3K and p-AKT proteins（p＜0.05）. (3) The Transwell experiment revealed that the invasive and migratory abilities of NPC cells were enhanced in witch co-cultured with M2-CM but were weakened in witch co-cultured with RES-M2-CM（p＜0.001）. The WB experiment showed that the expression of E-cadherin in NPC cells decreased in witch co-cultured with M2-CM but increased in witch co-cultured with RES-M2-CM; the expression levels of Vimentin, MMP2, and MMP9 increased in witch co-cultured with M2-CM but decreased in witch co-cultured with RES-M2-CM（p＜0.05）.All the differences were statistically significant.[Conclusion] RES can reverse M2-type macrophages into M1-type macrophages through the PI3K/AKT signaling pathway; RES can suppress the migration of nasopharyngeal carcinoma by inhibiting EMT via increasing the expression level of E-cadherin and decreasing the expression level of Vimentin,it can also inhibit the invasion ability of NPC by down-regulating the expression levels of MMP2 and MMP9. |
