Electroacupuncture At Zusanli Activates The Vagus Nerve To Regulate The Inflammatory Level And Anti-tumor Immune Function In Mice With Breast Cancer

BackgroundInflammation and immunity in tumor-bearing hosts are significantly related to the tumor progression.As an important risk factor of cancer,inflammation has a strong inhibitory effect on immunity through various mechanisms.One of the most important manner is to induce the accumulation and activation of myeloid-derived suppressive cells(MDSC),which sets a great obstacle for proliferation,migration and activation of CD8~+T cells and natural killer(NK)cells.Therefore,how to effectively control the inflammation,relieve the immunosuppression and restore the antitumor immunity has become an important strategy of cancer therapy.Acupuncture has good efficacy in enhance the antitumor immunity in cancer patients by benefiting many immune effector molecules and cells.In addition,acupuncture has been proven to be efficacious in alleviating inflammation.Accumulated studies have demonstrated favorable outcomes of electroacupuncture(EA)in a variety of diseases involving excessive inflammatory responses.According to multiple studies,the effect of EA at ST36 in repressing inflammation was mediated by eliciting efferent vagus nerve activity to inhibit excessive production of inflammatory cytokines.ObjectiveThe present study was designed to explore whether EA at ST36 was effective in alleviating tumor progression in breast tumor mice by regulating inflammation and immunity through the vagus nerve.Then,this study aims to provide evidences to widen the clinical application of acupuncture in cancer therapy.MethodsIn the first part,female BALB/c mice were randomly divided into control,model,and EA group.An orthotopic breast tumor model was established in mice of model and EA groups by subcutaneously injecting 4T1-luc2 mice breast tumor cells into the right second mammary fat pad.Mice in EA group received EA intervention at bilateral ST36(0.1 mA,2/15,30 min)once every other day.Tumor volume was measured and calculated using a Vernier caliper.Tumor location,area and total fluorescence intensity was measured using bioluminescence imaging.After the final intervention,the solid tumor and spleen were harvested to measure the weight.The second part assessed the level of inflammatory cytokines including tumor necrosis factor(TNF)-??interleukin(IL)-1? and IL-10 in the blood and local tumor using Mouse Pro-inflammatory V-Plex Tissue Culture Kit and western blotting.At the same time,HE staining was used to observe the inflammatory infiltration in local tumor tissues.In the third part,the proportion of CD8~+T cells and NK cells in the blood,spleen and local tumor was examined using flow cytometry.The expression of perforin and granzyme B in local tumor tissue was assessed using western blotting.In the fourth part,the proportion of MDSC in the blood,spleen and local tumor was analyzed using flow cytometry.The expression level of cyclooxygenase 2(COX-2)and arginase-1(Arg-1)in local tumor tissue was assessed by western blotting.As for the in vitro experiments,T cells isolated from the spleen of naive mice and MDSC purified from the spleen of tumor-bearing mice were co-cultured.CellTrace Far Red was applied to assessed the proliferation of T cells.Expression of CD25 molecules was measured to determine the activation level of T cells.The fifth part included three sections.The first one examined the effect of EA intervention on vagal efferent nerve of mice bearing breast tumor by examining the c-Fos expression of choline acetyltransferase-positive(ChAT+)neurons in the dorsal motor nucleus of the vagus(DMV)using the fluorescence immunohistochemistry staining.Meanwhile,acetylcholine(ACh)in the plasma and local tumor tissue was quantified using enzyme-linked immunosorbent assay(ELISA).The second section observed the immunity and inflammation of breast tumor mice after activation of ?7 subunit of the nicotinic acetylcholine receptors(?7nAchR).Mice injected with PBS or 4T1-luc2 cells were randomly allocated to the control,DMSO and PNU-282987 group.Mice in DMSO and PNU-282987 group were injected intraperitoneally once every other day with 100 ?L DMSO and PNU-282987(0.1 mg/kg),which is a selective agonist of ?7nAchR,respectively.Tumor location,area,total fluorescence intensity and weight was measured using Vernier caliper and bioluminescence imaging.A Mouse Pro-inflammatory V-Plex Tissue Culture Kit was used to quantified the level of inflammatory cytokines TNF-??IL-1? and IL-10 in the blood and local tumor.Flow cytometry was adopted to evaluate the proportion of CD8~+T cells?NK cells and MDSC in the blood and spleen.The last section adopted subdiaphragmatic vagotomy to verify participation of the vagus nerve in the efficacy of EA intervention on tumor-bearing mice.The subdiaphragmatic vagotomy and sham operation was performed in mice before implantation of 4T1-luc2 cells.Mice were randomly divided into Vagotomy-Model group(VNX),Vagotomy-EA group(VNX-EA),Sham-vagotomy-Model group(sVNX),Sham-vagotomy-EA group(sVNX-EA).EA intervention was applied in mice of VNX-EA and sVNX-EA group.Tumor location,area,total fluorescence intensity and weight was measured.Proportion of CD8~+T cells?NK cells and MDSC in theblood and spleen were evaluated using flow cytometry.ResultsThe first part showed that EA intervention ameliorated tumor growth.Compared with the model group,the tumor volume on the 18th and 22nd days was markedly decreased after EA intervention(P<0.05).Consistently,the fluorescent area and intensity of local tumors monitored by bioluminescence imaging were obviously lower in the EA group(P<0.05).Meanwhile,the solid tumor weight(P<0.01)was also significantly reduced after EA intervention.In addition,EA markedly alleviated splenomegaly(P<0.01).For the second part,systemic and local inflammation was alleviated after the EA intervention.Content of TNF-?,IL-1? and IL-10 in the blood significantly increased in tumor-bearing mice(P<0.001).Pro-inflammatory cytokines TNF-? and IL-1? in the serum(P<0.01)and local tumor tissues(P<0.05)decreased after EA intervention.But there was no obvious change of the anti-inflammatory cytokine IL-10 in both the serum and the local tumor(P>0.05).Meanwhile,tumor tissues from the EA group displayed a lower degree of inflammatory infiltration than that of model group.Results of the third part showed that EA at ST36 effectively promoted the antitumor immunity of tumor-bearing mice.The proportions of CD8~+T cells and NK cells in the blood and spleen were significantly reduced in breast tumor mice.Of note,EA significantly increased the ratio of CD8~+T cells(blood,P<0.01,spleen,P<0.05,local tumor,P>0.05)and NK cells(P<0.05)in the blood,spleen and local tumor.In addition,expression level of both perforin(P<0.05)and granzyme B(P<0.01)protein in local tumors were markedly increased after EA intervention.The fourth part showed that EA alleviated the immunosuppressive state of mice bearing breast mice.Tumor-bearing mice displayed extensive expansion of MDSC in the blood,spleen and local tumors(P<0.001).After EA intervention,the accumulation levels of MDSC in the blood(P<0.05),spleen(P<0.001)and local tumor(P<0.05)decreased significantly.Additionally,protein level of both COX-2 and Arg-1 in tumor tissue were notably reduced after EA intervention(P<0.05).Results of the in-vitro experiments showed that T cells co-cultured with MDSC from EA-treated mice displayed higher level of proliferation and activation than that co-cultured with MDSC from mice of model group.Results of the fifth part demonstrated the involvement of the vagus nerve in the regulation of EA on immunity and inflammation in mice bearing breast tumor.In the Section One,EA intervention not only led to substantial c-Fos expression in ChAT+neurons of DMV region,but also markedly increased the level of ACh in the plasma and local tumor tissue.The section section showed that the tumor volume was obviously decreased after intraperitoneal injection of PNU-282987(P<0.05).Likewise,the fluorescent area and intensity of local tumors monitored by bioluminescence imaging were obviously lower in the PNU-282987 group(P<0.05).Moreover,serum level of TNF-? and IL-1? in the mice of PNU-282987 group was much lower than the DMSO group(P<0.05),but there was no clear difference of IL-10 between the two groups(P>0.05).In the local tumor,level of IL-10 increased(P<0.05)after injection of PNU-282987,however,level of TNF-? and IL-1? has no obvious change(P>0.05).Furthermore,compared with DMSO group,the proportion of CD8~+T cells in the spleen of PNU-282987 group was clearly increased(P<0.05),but the NK cells had no significant change(P>0.05).What's more,the ratio of MDSC both in the blood and spleen was obviously decreased after the injection of PNU-282987(P<0.05).In the last section,mice in the sVNX-EA group displayed minor area,total fluorescent intensity(P<0.05)and weight(P<0.05)of tumor than that of mice in sVNX group.Moreover,the proportion of CD8~+T cells and NK cells in the blood and spleen increased while the MDSC deceased in the sVNX-EA group.However,there is no significant difference in the image area,total fluorescent intensity and weight of tumor,as well as the proportion of CD8~+T cells,NK cells and MDSC in the blood and spleen between the VNX and VNX-EA group.ConclusionThe current study revealed that EA intervention ameliorated tumor growth in breast tumor-bearing mice by inhibiting immunosuppressive MDSC and enhancing antitumor immunity,which was mediated by promoting CD8~+T cells and NK cells.The effect of EA might be attributed to activating the subdiaphragmatic vagus nerve to suppress systemic and local pro-inflammatory cytokines.