| Objective:Observe peripheral blood myeloid derived suppressor cells (MDSC) expression level in breast cancer patients with depressive disorder; reveal the correlation between depressive disorder and disease progression in breast cancer. Establish animal model of combination of disease and syndrome, and observe the intervention effect of Shuganjianpi formula.Analyze the role of MDSC and Natural killer T cells (NKT) in immune remodeling model.Method:Clinical observation:Observed31cases of breast cancer from oncology department in Guang An Men Hospital of China Academy of Chinese Medical Sciences by Hamilton depression scale (HAMD) and case observation. Flow Cytometry was used to detect the expression levels of MDSC in breast cancer patients.Animal experiments:112BALB/c mice were randomly divided into7groups (normal control group, the pure depression group, the tumor-bearing group alone, depression tumor group, TCM group, chemotherapy group, Chinese medicine combined with chemotherapy group), the corresponding group were inoculated with4T1inflammatory breast cancer cells and were under chronic unpredictable mild stimulus for28consecutive days to establish the animal model of breast cancer depressive disorder. At the same time, drug intervention groups were administrated by Shuganjianpi formula and (or) Gemcitabine. Sucrose water test, open field test, weight monitoring, immunohistochemistry of hippocampal5-HT were used to evaluate the model of depression; tumor diameter, tumor weight, survival time were observed to calculated tumor inhibition rate and draw tumor growth curve. Small animal in vivo imaging techniques was used to monitor tumor growth. Flow cytometry analysis was used to detected MDSC%, NKT%and T cell subsets expression levels. ELISA was used to detect IL-13in peripheral blood. Magnetic bead sorting method was used to separate CD8+T cells and early apoptosis of CD8+T cells was detected by flow cytometry analysis.Results:(1) Clinical observation of31patients with breast cancer, the estimated percentage of cases of patients with major depressive disorder was64.51%. MDSC%in peripheral blood of breast cancer patients was detected by flow cytometry, and we found that MDSC%in stage Ⅲ、Ⅳ breast cancer patients was significantly higher than stage Ⅰ、Ⅱ patients, and MDSC%was positively related to the clinical staging of breast cancer patients. Compared with MDSC%(0.68±0.24%) in patients of depressive disorder score less than8points, MDSC%(0.84±0.27%) in patients of high depressive disorder scores were much higher (P>0.05).(2) Evaluation of animal models:In depression model group, the drinking amount of sucrose water, horizontal and vertical movement in the number of cells in the open field test, body weight, hippocampal5-HT expression levels were decline in different degrees (P<0.05),which suggested a successful depressed animal model.(3) Tumor inhibition rates in TCM group, chemotherapy group, TCM+chemotherapy group were26.02%,77.83%,82.13%separately, and TCM+chemotherapy group had the highest tumor inhibition rate. Group comparison:survival of traditional Chinese medicine combined chemotherapy with chemotherapy alone group> pure traditional Chinese medicine group> pure tumor group depression tumor group, traditional Chinese medicine combined with chemotherapy group life extension rate (37.33%) is greater than the pure traditional Chinese medicine group (11.55%) and chemotherapy alone group (17.79%).(4) In vivo, fluorescence values for all tumor-bearing mice were gradually increased with the number of day; depression tumor-bearing group fluorescence values were greater than21days after the tumor group alone. Traditional Chinese medicine combined with chemotherapy group, the chemotherapy alone group fluorescence value is always lower than the depressive tumor group, pure efficacy of traditional Chinese medicine (TCM) is apparent in the first15days of measurement, pure traditional Chinese medicine group<depressive tumor-bearing group. In the first28days of observation, the traditional Chinese medicine combined with chemotherapy group<chemofherapy alone group<pure traditional Chinese medicine group<depression tumor-bearing group.(5) MDSC%in spleen of depression model increased in different degree, in particular, MDSC%(69.09±2.56%) in tumor-bearing mice with depressive disorders was significantly higher than the normal control group (2.00±0.09%), and lymphocyte surface molecules CD3/CD4/CD8and CD4+/CD8+T cell ratio decreased in varying degrees (P<0.05).(6) In late stage4T1inflammatory breast cancer microenvironment, NKT%, and IL-13level in tumor-bearing group was significantly higher than the normal control group (P<0.01), NKT cells and IL-13showed a significantly positive correlation (r=0.617, P<0.01).(7) CD8+T cells in early apoptosis:normal control group (3.38%)<pure depression group (5.13%)<Chinese medicine combined with chemotherapy group (7.46%)<chemotherapy alone group (13.25%)<pure traditional Chinese medicine group (14.37%)<depression tumor group (15.91%).Conclusions:(1) Depressive disorder showed a high incidence in breast cancer patients, and peripheral blood MDSC%was closely related to disease progression in breast cancer. MDSC%in high depressive disorder score patients tended to increase.(2) Shuganjianpi formula may reduce the activity of tumor cells to inhibit tumor growth and prolong the survival time of tumor-bearing mice, especially when combined with chemotherapy.(3) Shuganjianpi formula combine with chemotherapy can inhibit the proliferation the MDSC and NKT cells, reduce the level of IL-13, CD8+T cells in early apoptosis mitigation, and improve body immunity. (4) Depressive disorder and tumor growth may inhibit immune function in different degrees, and its mechanism may be in advanced4T1inflammatory breast cancer microenvironment, NKT cells secret IL-13, and played a role in negative immunomodulatory together with MDSC. Both NKT and MDSC induced early apoptosis of CD8+T cells, thereby inhibit T-cell immune response and promote tumor progression. |
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