Study On The Mechanism Of Regulating Myeloid-derived Suppressor Cells In The Intervention Of Mice Model Of Breast Cancer With Persistent Depression By Shuganjianpi Formula

Background:Breast cancer has become one of the most common malignant tumors affecting women.With the rapid pace of life,changes in women’s social status,job competition and family pressure,the incidence of depression disorder is also on the rise.According to a survey by the world health organization,there are 350 million people with depression disorder in the world.Depression disorder is associated with a variety of diseases,and is one of the causes of breast cancer.It can promote the development of breast cancer,and also amplify inflammatory responses that touch on a variety of cytokines.Myeloid-derived suppressor cells（MDSCs）are a group of immature cells from the bone marrow with immunosuppressive function and have become the main regulator of immune response in various types of tumors,chronic inflammation,autoimmune diseases,infection,trauma and other pathological conditions.A large number of inflammatory factors will cause persistent and abnormal myeloid hyperplasia,activate and recruit MDSCs,make these immature myeloid cells deviate from the normal differentiation pathway,and stimulate the inhibitory activity.MDSCs through the expression of immune checkpoint surface molecule,nitric oxide,the release of active oxygen substances,regulating cytokines（IL-6,IL-10,IL–1β,etc.）,inducing tumor microenvironment of inflammation,promoting the transformation of the immune system into tolerance and escape,the change of tumor inflammatory microenvironment determines the tumor cell proliferation,invasion and metastasis.Therefore,down-regulation of MDSCs to promote their differentiation and maturation and loss of immune activity is a potential target for the treatment of tumors and related depression.There are more types of breast cancer,so the treatment methods are diverse.Ⅰperiod is given priority to with having breast surgery plus radiotherapy.Ⅱperiod first surgical diagnosis and treatment,postoperative in weigh chemotherapy according to pathology.Preoperative chemotherapy may be considered for patients with large masses and breast conserving tendency.Ⅲperiod is after neoadjuvant chemotherapy and surgery,postoperative again according to the clinical and pathological situation do put,chemotherapy.Ⅳperiod is given priority to with medical treatment.Since it is difficult for Chinese women to accept physical defects,and the side effects of chemotherapy and endocrine drugs are large,the compliance is poor and the clinical efficacy is unstable.Therefore,traditional Chinese medicine is chosen as the adjuvant treatment.Chinese medicine attaches great importance to the holistic view and combines disease differentiation with syndrome differentiation,which has an advantage in the prevention and treatment of breast cancer.According to Chinese medicine,emotion is the root cause of breast cancer,and liver qi stagnation is the core pathogenesis throughout.Shugan jieyu is the commonly used treatment of yu syndrome,Shuganjianpi Formula is the experience of the treatment of breast cancer in the oncology department of guang an men hospital,to the"taiping huimin and agent department"in the classic prescription free and easy,the main drugs are:bupleurum,hang bai paehao,yu jin,August zha,summer grass,raw astragalus astragalus,notoginseng,licorice.Previous studies have shown that the depression state of post-tumor depressive breast cancer mice can be significantly improved by Shuganjianpi Formula,and the combination of chemotherapy can control tumor growth,reshape MDSC function,and regulate the tumor inflammatory microenvironment.Moreover,by regulating the expression of pyroptosis and inflammatory factors,microRNA was further regulated and the content of MDSC in spleen of tumor-bearing mice was reduced to reverse the local immunosuppressive state of tumor.Depressive disorder often associated with breast cancer occurrence,development process,therefore,we propose a sustained depression may promote the occurrence and development of breast cancer by inflammatory microenvironment,and liver spleen may through the regulation of inflammatory factors and pathways,inducing pyroptosis,reverse the raise of MDSC and immunosuppressive function,restore antitumor immune T cells,so as to delay the progress of breast cancer and improve depressive state.Objectives:By establishing a mouse model of disease and syndrome of whole-course liver depression combined with breast cancer,the influence of liver depression on the progress of breast cancer was observed,and the mechanism of MDSC in spleen of depression-bearing tumor mice regulated by Shuganjianpi Formula based on cell death of coke was explored.Methods:The basic experiment was divided into three parts:1.Establish and evaluate the mouse model of breast cancer with persistent depression;2.To observe the intervention effect of Shuganjianpi Formula on 4T1-luc breast cancer cells and depressed mice;3.Based on the factors and proteins related to the pyroptosis pathway,the mechanism of Shuganjianpi Formula in the treatment of breast cancer mice with persistent depression was explored.Results:1.Establish and improve the mouse model of sustained depression disorder,and inoculate1.5×10⁵ cells of the mouse model of 4T1-luc breast cancer into the model.2.The results of sucrose water experiment showed that the drinking amount of sugar water in the normal group was basically stable,and the drinking amount of sugar water in the normal group showed a rising trend after 28 days of modeling,the drinking amount of sugar water in the other groups decreased with the time of modeling,and the drinking amount of the traditional Chinese medicine group and the traditional Chinese and western medicine group showed a stable and slightly rising trend at 21 days.In general,Normal Control Group>PT>Chinese and Chemotherapy Group>Pure Depression Group>Chinese Medicine Group≈Depression Tumor Group>Chemotherapy Group.In general,the total food intake:Pure Depression Group>Pure Tumor Group>Normal Control Group>Depression Tumor Group>Chemotherapy Group>Chinese and Chemotherapy Group>Chinese Medicine Group.The activity time of the open-field experiment showed that Normal Control Group>Pure Depression Group>Chinese and Chemotherapy Group≈Chemotherapy Group>Chinese Medicine Group>Pure Tumor Group>Depression Tumor Group.The results of sucrose water and open field experiments showed that mice in the depression model group were less addicted to sugar water and had less ability to explore new environment than mice in the normal control group,and the depression model was successfully established.And after the intervention of Chinese and western medicine,the symptoms of depression were alleviated.3.The hippocampus is an important limbic system,which is responsible for emotion and spatial memory.The reduction of 5-HT is an important factor of depression disorder,and the5-HT_1A receptor,as one of the subtypes,is involved in the regulation of depression and anxiety.In this study,the expression of 5-HT_1AR mean fluorescence density on the cell membrane surface of hippocampal neurons in the brain was as follows:Depression Tumor Group>Pure Tumor Group>Pure Depression Group≈Chemotherapy Group>Chinese and Chemotherapy Group>Chinese Medicine Group>Normal Control Group（P<0.01）.The results showed that the model of depression was successfully established,and the expression of depression could be reduced by Shuganjianpi Formula.4.Effects of rat serum containing Shuganjianpi Formula on the proliferation of mouse 4T1-luc breast cancer cells:compared with the blank serum group of>the low-dose group>the medium-dose group,there was a significant difference between the low-dose group and the blank serum group（P<0.01）.5.Tumor size and tumor inhibition rate:after the modeling,the tumor tissues of mice were removed subcutaneously and weighed.The average tumor weight was（1.99±0.72）g in Pure Tumor Group,（2.12±0.58）g in Depression Tumor Group,（0.58±0.45）g in Chemotherapy Group,（1.49±0.58）g in Chinese Medicine Group,（0.47±0.25）g in Chinese and Chemotherapy Group.There were statistically significant differences between the traditional Chinese medicine group,the chemotherapy group,the integrated Chinese and western medicine group and the depression-bearing tumor group（P<0.05）.From the perspective of tumor inhibition effect,Chinese and Chemotherapy Group>Chemotherapy Group>Chinese Medicine Group.6.In vivo imaging of small animals at each time period showed that with the extension of inoculation time,the number of photons in tumor-bearing mice increased by an order of magnitude,and tended to be stable on the fifth day.The results of transverse comparison of the number of photons among each group were as follows:Pure Tumor Group>Depression Tumor Group>Chinese Medicine Group>Chemotherapy Group>Chinese and Chemotherapy Group.There was a significant difference between the integrated Chinese and western medicine group and the chemotherapy group,and the change of photon quantity tended to be stable after 21 days.7.MDSC content detected by flow cytometry:in the tumor-bearing group and the depression-bearing group,MDSC expression of myeloid inhibitory cells was higher in the spleen,up to（37.25±7.53）%,but decreased after the intervention of traditional Chinese and western medicine.In general,Pure Tumor Group>Chinese Medicine Group>Depression Tumor Group>Chemotherapy Group>Chinese and Chemotherapy Group>Pure Depression Group>Normal Control Group;And with the intervention of integrated traditional Chinese and western medicine,the autoimmune cells rebounded.The percentages of CD8⁺T cells and NKT cells showed that there was a statistical difference between the combined Chinese and western medicine group and the chemotherapy group and the depression-bearing tumor group（P<0.01）.This indicated that the combination of liver-nourishing and spleen-strengthening prescription and gemcitabine had the effect of inhibiting MDSC cells and improving autoimmunity.8.Western blot was used to detect the proteins NLRP3,caspase-1,ASC,GSDMD,and there was a statistical difference between the combined traditional Chinese and Western medicine group and the chemotherapy group and the depression tumor-bearing group（P<0.01）.Differences in expression of inflammatory pathway related proteins TLR-4/MyD88/NF-κB in each group:Depression Tumor Group>Chemotherapy Group>Chinese and Chemotherapy Group（P<0.05）.9.The expression of IL-1βin serum of mice was detected by ELISA as follows:Chinese and Chemotherapy Group>Pure Depression Group>Normal Control Group>Chemotherapy Group>Chinese Medicine Group>Depression Tumor Group>Pure Tumor Group.The expression of IL-18 was as follows:Pure Depression Group>Normal Control Group>Chemotherapy Group>Chinese and Chemotherapy Group≈Chinese Medicine Group>Depression Tumor Group>Pure Depression Group.Il-1 and IL-18 were the main factors in the pyroptosis pathway,and the expression increased after the intervention of integrated Chinese and western medicine,indicating that pyroptosis of tumor cells was accelerated.Expression of inflammatory factors IL-6 and IL-10:compared with the depression-bearing group,the chemotherapy group,the traditional Chinese medicine group and the integrated Chinese and western medicine group all had significant differences（P<0.01）.The combination of Chinese and western medicine could reduce the pro-inflammatory factor IL-6 and increase the expression of anti-inflammatory factor IL-10.Conclusions:1.Sustained depression disorder can promote the growth of breast cancer,which can be significantly alleviated by Shuganjianpi Formula,and combined with chemotherapy to improve the inflammatory microenvironment of tumors and control the growth rate of tumors.2.Shuganjianpi Formula may down-regulate the proportion of MDSC in the spleen of tumor-bearing mice through the TLR-4/MyD88/NF-κB inflammatory pathway,affect the immunosuppressive microenvironment,and restore the anti-tumor immunity of CD8⁺T cells.3.NLRP3 entry into the cell promotes the separation of Caspase-1 from the ASC complex,and the release of IL-1βmay be the mechanism of the action of liver-nourishing and spleen-strengthening formula on the occurrence of pyroptosis in depression disorder breast cancer.