| BackgroundColon cancer is a malignant disease under the comprehensive role of multi-factors and multi-pathways.Compared with the previous studies focused on the direct effects of cancer cells and tumor microenvironment,chronic stress and abnormal immune function also play an important role in the occurrence and development of colon cancer.Chronic stress can cause abnormal regulation of neuroendocrine-immune network through the HPA axis(the core pathway of stress response).Recent studies found that chronic stress can activate the HPA axis to release CORT continuously,thereby causing dendritic cells to specifically overexpress the critical transcriptional regulator TSC22D3.This can lead to phenotypic and functional disorders of dendritic cells,create an immunosuppressive microenvironment,and weaken immune recognition and killing capacity.It eventually results in cancer cells proliferating and colon cancer progresses.This recurrence process is related to the pathogenesis of“Simultaneous Onset of Form,Qi and Spirit”.The Tongxieyao formula is effective in the treatment of mental-related bowel diseases,and previous studies have confirmed that it can delay the progression of colon cancer.However,further investigation is needed to determine whether it is related to chronic stress-induced immunosuppressive microenvironment.Focus on the role of chronic stress and abnormal immune function in colon cancer occurrence and development,and researching the effect of Tongxieyao formula regulates“Spirit”,assists“Qi”and treats“Form”to delay the colon cancer progression,which will provide empirical support for the concept of“Simultaneous Regulation of Form,Qi and Spirit”.Objective1.Confirming the correlative effect of Tongxieyao formula on delaying the evolution of colon cancer induced by chronic stress.2.Elucidating the regulatory effect of Tongxieyao formula on chronic stress-dendritic cells-colon cancer immune microenvironment.3.Revealing the empirical evidence of regulate“Spirit”and assist“Qi”to treat“Form”of the Tongxieyao formula.Methods1.Intervention effect of Tongxieyao formula on chronic stress colon cancer mouse model:mouse were randomly divided into the Control group,Model group,MIFE group,TXYF-L group,TXYF-M group and TXYF-H group.Using chronic restraint stress and subcutaneous tumor implantation of colon cancer CT26-Luc cells,a chronic stress colon cancer model was developed,and corresponding drug interventions were conducted.After 35days,eyeball blood was drawn and tumor tissue was removed:(1)The general information of mouse,such as weight,mental state,and reaction time,were observed;(2)Conducting behavioral tests using tail suspension test and forced swimming test to measure the degree of depression in mouse;(3)The serum levels of 5-HT,CORT,and NA stress hormones were measured using the ELISA method for mouse in each group;(4)Measuring the tumor volume and weight of mouse in each group,and observing the pathological changes in tumor tissue using H&E staining;(5)Using immunofluorescence to detect Ki67 expression in tumor tissue to determine tumor proliferation.2.The effect of Tongxieyao formula on the immune microenvironment of transplanted colon cancer tumor mouse under chronic stress:(1)The ratio of CD4~+/CD8~+T cells,Th1/Th2 cells,and M1 and M2 macrophages in peripheral blood and tumor tissues of mouse were determined by flow cytometry;(2)Detection of serum Th1/Th2 cytokines by ELISA.3.Effect and molecular mechanisms of Tongxieyao formula on the dendritic cell maturation of colon cancer mouse model induced by chronic stress:(1)The phenotypic maturation(CD80,CD86 and MHCII)and migration ability(CCR5,CCR7 and CXCR4)of dendritic cells in peripheral blood and tumor tissues of mouse were detected by flow cytometry;(2)Detection of IL-12 level in serum of mouse in each group by ELISA method;(3)RT-PCR and Western blot were used to separately detect TSC22D3 protein and m RNA expression levels in tumor dendritic cells.4.The reversed cross-validation of Tongxieyao formula’s regulatory effect on chronic stress-DC-colon cancer TIME cross-linking mechanism:Dexamethasone was used as the reversed validation control,and the optimal dose of Tongxieyao formula was selected for intervention in combination with previous experiments.Completing the validation study of Tongxieyao formula on the colon cancer mouse model induced by chronic stress using experimental methods from Experiments 1,2,and 3.Results1.Intervention effect of Tongxieyao formula on chronic stress colon cancer mouse model:compared to the control group,the model group mouse lost significant weight,had lost hair luster and lost hair,flagged spirit,curled up and didn’t want to move,and their sensitivity to external stimuli decreased with the prolongation of modeling time.With the prolongation of chronic restraint stress,the resting time of the tail suspension test and forced swimming test in the Model group was significantly longer than that in the CRC group.At the same time,chronic stress could significantly up-regulate CORT,while down-regulate 5-HT and NA.Tumor volume,small animal living imaging,tumor weight and H&E pathological staining were also measured.The results showed that chronic stress could significantly enhance colon cancer growth,mitosis and active proliferation of cancer cells.Compared with the Model group,the body weight of mouse increased in varying degrees,their mental state gradually improved,hair loss decreased,activity increased and food intake increased,after the intervention of Tongxieyao formula.Each group of mouse had a decrease in inactivity time,and their hormone levels were increased.In tumor volume,tumor weight,and pathological tests,it was shown that various doses of Tongxieyao formula could inhibit the growth of the volume and weight of colon cancer in varying degrees,and there was less pathological mitosis in tumor tissue,the expression of Ki67 was significantly downregulated.2.The effect of Tongxieyao formula on the immune microenvironment of transplanted colon cancer tumor mouse under chronic stress:in peripheral blood and tumor tissue,the proportion of CD4~+T cells in the Model group was significantly lower than that in the CRC group,while the proportion of Th2 cells was significantly upregulated.Furthermore,the proportion of M1 type macrophages in peripheral blood of model group decreased significantly,while the proportion of M2 type macrophages increased.After treatment with Tongxieyao formula,the proportion of T cells can be regulated to varying degrees,among them,the TXYF-M group was found most significant.Tongxieyao formula also could significantly increase the proportion of CD4~+T cells and CD4~+/CD8~+T cells,while increasing the proportion of Th1 cells and Th1/Th2 cells.Compared with the Model group,Tongxieyao formula could upregulate the proportion of M1 type macrophages,while the proportion of M2 type macrophages was decreased significantly.After chronic stress,the level of serum Th1 cytokines decreased and the level of Th2 cytokines increased in the Model group;after treatment with Tongxieyao formula,it could promote the secretion of Th1 type cytokines and inhibit the secretion of Th2 type cytokines.3.Effect and molecular mechanisms of Tongxieyao formula on the dendritic cell maturation of colon cancer mouse model induced by chronic stress:chronic stress stimulation could lead to a decrease in the proportion of CD80,CD86,and MHCII phenotypes of dendritic cells in the peripheral blood and tumor tissues of colon cancer mouse,while increasing the surface chemokine receptor CCR5,and decreasing CCR7 and CXCR4.This result indicates that chronic stress could induce phenotypic and functional maturation disturbance of dendritic cells.After the treatment with Tongxieyao formula,the proportion of CD80,CD86,and MHCII phenotypes of dendritic cells in peripheral blood and tumor tissue of mouse increased,and the surface chemokine receptor CCR5 decreased,while CCR7 and CXCR4 were upregulated.Meanwhile,Tongxieyao formula increased the serum IL-12 secretion compared to the Model group.Detection of TSC22D3 protein expression and m RNA level in dendritic cells of tumor tissue by Western blot and RT-PCR:chronic stress stimulation in the Model group upregulated the expression of TSC22D3protein and the content of m RNA of dendritic cells in tumor tissue.After the treatment with Tongxieyao formula,the expression of TSC22D3protein of dendritic cells in mouse tumor tissue was significantly reduced,while the content of TSC22D3 m RNA was downregulated,and the TXYF-M group had the best therapeutic effect.4.The reversed cross-validation of Tongxieyao formula’s regulatory effect on chronic stress-DC-colon cancer TIME cross-linking mechanism:in this experiment,dexamethasone was selected as a reverse validation control.When dexamethasone was administered based on a colon cancer model,which could lead to the change of chronic restraint stress-like changes in colon cancer mouse,such as curling up and not moving,stress hormone levels changed,and the tail suspension and forced swimming was prolonged.Based on the model group,mouse receiving dexamethasone showed a change in chronic restraint stress-like and stress hormones altered.At the same time,after administration of dexamethasone,the proportion of CD4~+/CD8~+T cells and Th1/Th2 cells in mouse decreased,and the dendritic cell maturation was impaired,leading to a final decrease in the body’s anti-tumor immune ability.When dexamethasone was administered simultaneously with Tongxieyao formula treatment,this method weakened the ability of Tongxieyao formula to activate T cells,reduced the improvement in the CD4~+/CD8~+T cell ratio and Th1/Th2 cell ratio in the body,weakened the maturation promoting effect of dendritic cells,and decreased the reduction level of TSC22D3 protein content and m RNA.Conclusions1.Mice on Tongxieyao formula could be improved in terms of stress states,hormone levels,and colon cancer growth inhibition.2.Tongxieyao formula increased the proportion of CD4~+/CD8~+T cells and M1/M2 macrophages,promoted the shift of Th1/Th2 immune balance to Th1,and improved the tumor immune microenvironment.3.A significant reduction in the expression of TSC22D3 protein and m RNA content of dendritic cells in mouse tumor tissue can be achieved by Tongxieyao formula,thereby regulating the phenotypic and functional maturation of dendritic cells.4.Tongxieyao formula improved the chronic stress,downregulated the expression and m RNA content of TSC22D3 protein in dendritic cells,and regulated the phenotype and functional maturation of dendritic cells.Meanwhile,it could enhance the body’s anti-tumor immune response through the activation of T cells,thereby exerting the effect of regulating“Spirit”,assisting“Qi”and treating“Form”to delay the colon cancer progression. |
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