| Objective:Oral squamous cell carcinoma（OSCC）accounts for over 90% of head and neck squamous cell carcinoma and the incidence rate is expected to increase by 30% by around 2030.Because OSCC is prone to cervical lymph node metastasis and the mechanism of metastasis is not clear,its patient mortality remains high,and the fiveyear survival rate is only 40%-50%.It is necessary to further clarify the mechanism of metastasis and find more effective treatment strategies.As the most abundant noncancer cell type in the tumor microenvironment（TME）,tumor-associated macrophages（TAMs）are broadly classified into two phenotypes,M1 with anti-tumor properties and M2 with pro-tumor properties.M2 type TAMs as the main polarized phenotype are indispensable during the initiation and progression of OSCC.Resveratrol（RES）is a natural estrogen from plants with antitumor activity.Research suggests that resveratrol is expected to be a drug for treating OSCC.Therefore,the objective of this study is further explore the role of the spleen tyrosine kinase（Syk）pathway in this regulation by investigating the effect of resveratrol on the invasion and metastasis of OSCC and the regulation of the polarization state of tumor associated macrophages,so as to elucidate the mechanism of resveratrol in the treatment of oral squamous cell carcinoma,and to provide experimental basis and potential strategies for the treatment and prognosis of oral squamous cell carcinoma.Methods:1.Human OSCC cell CAL27 conditioned medium（CAL27 conditional medium,CAL27-CM）was collected to induce mouse macrophage RAW264.7.TAMs related cytokines m RNA and protein expression levels were detected by q RT-PCR and ELISA.The expression level of TAMs marker protein was detected by immunofluorescence.The morphological changes of TAMs were observed under optical microscope.CAL27-CM induced RAW264 7 to obtain M2 TAMs was verified.2.CCK-8 method was used to detect the effects of different concentrations of RES intervention on the survival of TAMs and macrophages.Intervention of M2 TAMs with safe concentration of RES and simultaneous addition of RES intervention in RAW264 induced by CAL27-CM.The migration capability and invasiveness of OSCC CAL27 cells were detected by wound healing assay and Transwell test.The effects of RES on the migration and invasion of CAL27 cells through TAMs were analyzed.3.RES was used to intervene M2 TAMs and RAW264.7.TAMs related cytokines m RNA and protein expression levels were detected by q RT-PCR and ELISA.The expression level of TAMs marker protein was detected by immunofluorescence.The morphological changes of TAMs were observed under optical microscope.The regulation of RES on the polarization state of TAMs was analyzed.4.RES was used to intervene M2 TAMs and RAW264.7,the protein expression levels of Syk and p-syk were detected by Western blot.The Syk inhibitor entospletinib（GS-9937）was used to intervene in the induction of RAW264.7 by CAL27-CM.TAMs related cytokines m RNA and protein expression levels were detected by q RT-PCR and ELISA.The expression level of TAMs marker protein was detected by immunofluorescence.The regulation of RES on the polarization state of TAMs through Syk pathway was analyzed.5.GS-9937 was used to intervene in the induction of RAW264.7 by CAL27-CM.The migration capability and invasiveness of OSCC CAL27 cells were detected by wound healing assay and Transwell test.The effect of inhibiting Syk pathway in TAMs on the migration and invasion of cal27 cells was analyzed.Results:1.Compared with untreated RAW264.7,M2 marker cytokines IL-10,Arg-1 and TGF-β m RNA expression in TAMs cultured with CAL27 conditioned medium（CAL27-CM）up-regulated significantly,the protein expression of IL-10 increased significantly,the fluorescence intensity of CD206 was significantly enhanced.Under optical microscope,RAW264.7 was round and aggregated,while TAMs were larger and extended pseudopodia,and more dispersed.It showed that M2 phenotype TAMs could be obtained from RAW264.7 after CALl27-CM intervention.2.The results of CCK-8 experiment showed that in R-RES group and RES group,the survival rate of TAMs cells with concentration less than 20 μM was greater than80%,while the survival rate of 40 μM cells decreased significantly.Transwell migration and invasion experimental results showed that,compared with the control group（0μM）,the number of CAL27 cells passing through the chamber in the R-RES group（20μM）and RES group（20μM）was significantly reduced.TAMs conditioned medium（TAMsCM）treated with resveratrol was collected and treated CAL27.Wound healing assay results showed that,the scratch area of CAL27 was significantly reduced in the R-RES group（20μM）and RES group（20μM）compared with control group（0μM）.Stated that TAMs after resveratrol treatment obviously inhibited the migration capability and invasiveness of CAL27.3.QRT-PCR results showed that,M2 type marker cytokine（TGF-β,IL-10）m RNA expression levels were reduced,whereas M1 type signature cytokine（TNF-α,IL-12,i NOS）m RNA was increased in the R-RES group（20μM）compared with control group（0μM）.The m RNA expression levels of M1 signature cytokines TNF-α,IL-12 and i NOS were significantly increased,and M2 signature cytokines TGF-β,IL-10 and Arg-1 were significantly decreased in TAMs suggested by q RT-PCR of the RES group（20μM）compared with control group（0μM）.ELISA results showed that the TNF-αprotein secretion level was significantly increased,and the IL-10 protein secretion level was significantly decreased in the R-RES group（20μM）and RES group（20μM）compared with control group（0μM）.Immunofluorescence results showed that the fluorescence intensity of M2 type marker CD206 was weakened,and the expression of M1 type marker CD86 was enhanced in the RES group（20μM）and R-RES group（20μM）compared with control group（0μM）.Optical microscopy revealed that after resveratrol treatment,TAMs showed a reduced number of pseudopodia and a close to round morphology,which was significantly different from control group（0μM）.Stated that on the one hand,resveratrol could re-educate M2 TAMs into M1 phenotype,on the other hand,it could inhibit M2 polarization and promote M1 polarization of macrophages.4.Western blot showed that there was no change in Syk protein expression in the R-RES group（20μM）and RES group（20μM）compared with control group（0μM）,and p-Syk protein expression was significantly decreased,indicating that resveratrol could inhibit Syk protein phosphorylation in TAMs.TAMs were intervened with entospletinib（GS-9937）,a Syk inhibitor,and the results of q RT-PCR showed that M1 signature cytokine（TNF-α,IL-12,i NOS）m RNA levels increased,M2 type marker cytokines（IL-10,Arg-1）m RNA levels decreased,TGF-β expression was slightly elevated but not statistically significant in the GS-9937 group（1μM）compared with control group（0μM）.ELISA results showed that the TNF-α protein secretion level was significantly increased,and the IL-10 protein secretion level was significantly decreased in the GS-9937 group（1μM）compared with control group（0μM）.The results of immunofluorescence showed that the expression of CD86,a marker of M1 type,was enhanced and that of CD206,a marker of M2 type,was attenuated in the GS-9937 group（1μM）compared with control group（0μM）.The results illustrated that resveratrol could regulate the polarization of TAMs by inhibiting the phosphorylation of Syk protein.5.Transwell migration and invasion experimental results show that,compared with control group（0μM）,the number of cells in the GS-9937 group（1μM）that CAL27 cells crossed the chamber was significantly decreased.In the wounding healing assay,the supernatant of TAMs after resveratrol treatment was collected and applied to CAL27 cells.The test datas suggested that the scratch area of CAL27 was significantly lower level in the GS-9937 group（1μM）compared with control group（0μM）.It indicated that inhibition of Syk pathway in TAMs obviously inhibited the migration capability and invasiveness of CAL27 cells.Conclusion:1.Treatment of CAL27-CM was able to induce RAW264.7 cells into TAMs with M2 phenotype;2.Resveratrol inhibited the migration and invasion of OSCC cells through TAMs;3.Resveratrol can regulate the polarization state of TAMs by re-educating M2 TAMs into M1 phenotype,promoting the polarization of macrophages to M1 phenotype and inhibiting its M2 polarization;4.Resveratrol regulated the polarization of TAMs by inhibiting the phosphorylation of Syk protein,and then inhibited the migration and invasion of CAL27 cells. |
