Evaluation Of Shu-gan-jian-pi Method In The Treatment Of Breast Cancer And Mechanism Of Intervention Studies Of Breast Cancer In Mice With Pre-neoplastic Depressive Disorder

Background:At present,breast cancer has developed into the number one malignancy among new cases over the world.Breast cancer patients are often accompanied by depressive disorder before the onset.and during diagnosis,treatment,and long-term survival.Previous studies have found that chronic depression significantly increases the risk of cancer in patients,and that depression symptoms are negatively correlated with breast cancer survival."Shu-gan-Jian-pi" method is one of the important principles of breast cancer treatment,but the current level of evidence-based evidence is insufficient.Therefore,this study will systematically evaluate the therapeutic effect of this method on breast cancer.The Shu-gan-Jian-pi decoction is an empirical formula established by our department based on the method of smoothing the liver and strengthening the spleen.In the previous researches,We found that the Shu-gan-Jian-pi decoction can reduce the proportion of MDSC cells in tumor-bearing mice with persistent depression and post-tumor depression,and can inhibits the growth of tumors by activating NLRP3-mediated pyroptosis pathway.However,the effect of pre-tumor depression on the occurrence and development of breast cancer is still lacking in detailed studies.Therefore,this study stablishes a tumor-bearing mouse model of pre-tumor depression to further explore the effect of depression on the body's microenvironment,tumor occurrence and development,and the intervention of Shu-gan-Jian-pi decoction.Object:1.Carry out systematic reviews and Meta-analysis on the effectiveness and safety of the method of "Shu-gan-Jian-pi" in the treatment of breast cancer,and provide a higher level evidence of clinical application of the " Shu-gan-Jian-pi" method.2.Establish a tumor-bearing mouse model of pre-tumor depression to further clarify the effect of depression on the body's microenvironment and tumor growth.3.From the perspective of tumor immune microenvironment and NLRP3/caspase-1/GSDMD-mediated pyrolysis pathway,explore the intervention mechanism of Shu-gan-Jian-pi decoction on tumor-bearing mice with pre-tumor depression,and further explain the scientific connotation of the prevention and treatment of breast cancer by"Shu-gan-Jian-pi" method.It will provide a theoretical basis for prolonging the survival of advanced tumors by using the "Emotional Theory" of TCM.Method:1.Systematic review and meta-analysis of effectiveness and safety of "Shu-gan-Jian-pi" method in treating breast cancer:Search domestic and foreign databases,and collect the RCT study using the treatment of breast cancer by "Shu-gan-Jian-pi" method that meets inclusion and exclusion criteria from the establishment of the database until January 23,2021.The main observation indicators are ORR,DCR,KPS score,depression scale score,and incidence of adverse reactions.The quality of the literature is evaluated based on the criteria of the Cochrane Handbook.We adopt Review Manager 5.3 software for Meta-analysis.Chi-square to test heterogeneity:If I2?50%and P?0.1,a fixed-effects model is used for analysis;when I2>50%or P<0.1,a random-effects model is used for analysis;The results are presented in a forest plot.And sensitivity and publication bias analysis also be analysis.2.Study on the intervention effect and molecular mechanism of Shu-gan-Jian-pi decoction on breast cancer in mice with pre-tumor depression:(1)Construct and evaluate the pre-tumor depression and tumor-bearing breast cancer model:After successfully constructing a depression model through the chronic unpredictable stimulus method(CUMS),mice in the depression model group were randomly selected and inoculated with 4T1-Luciferase(4T1-Luc)breast cancer cell.Bodyweight,sucrose water partiality,behavioral evaluation,brain tissue 5-HT1AR expression,and success rate of tumor grafting were used as model evaluation indicators.(2)Study the effect of depression on the development of breast cancer:After the depression model was successfully constructed,? spleen HE staining,immune cell flow cytometry and serum inflammatory factor detection(ELISA method);?randomly select mice from normal control group and depression model group.Inoculated 4T1-Luc cells,and observed the body weight,sucrose water partiality,brain tissue 5-HT1AR expression,tumor volume in each group.Spleen HE staining,immune cell flow cytometry,and inflammatory factors based on ELISA will be carry out after the 21 days of tumor inoculation.(3)Study the intervention effect of Shu-gan-Jian-pi decoction on tumor-bearing mice with pre-tumor depression:after the 21 days of tumor inoculation in each group,observed mouse body weight,sucrose water bias,behavioral evaluation,brain tissue 5-HT1AR expression,tumor volume,tumor weight;and perform spleen HE staining,immune cell flow cytometry,and serum inflammatory factors detection(ELISA method).(4)Study the intervention mechanism of Shu-gan-Jian-pi decoction on tumor-bearing mice with pre-tumor depression:? In vitro experiment:cultured 4T1-Luc cells,and used CCK8 method to detect the proliferation rate of 4T1-Luc cells under the action of different drug-containing serums.Selected the concentration of drug-containing serum that significantly inhibits the proliferation of 4T1-Luc cells,setted up a normal control group and a drug-containing serum group,and used LDH cytotoxicity detection kit to detect the release of LDH,used ELISA method to detect the release of IL-10 and 11-18,and used Western blotting method to detect the expression of NLRP3,caspase-1 and GSDMD protein.? In vivo experiment:After 21 days of intervention in each group,Western blotting and immunohistochemistry were used to detect the expression of NLRP3,caspase-1,and GSDMD protein in tumor tissue,and immunofluorescence method was used to detect the expression of IL-1? in tumor tissue.Result:1.Systematic review and meta-analysis of effectiveness and safety of "Shu-gan-Jian-pi" method in treating breast cancerA total of 2294 patients were involved in the 29 randomized controlled studies of the method of "Shu-gan-Jian-pi" combined with chemotherapy and chemotherapy alone.Meta-analysis showed that the improvement rates of ORR,DCR,and KPS in the combined group were significantly better than those in the chemotherapy group.The RR values were 1.20,1.09,and 1.29,respectively,and the difference was statistically significant,P<0.05;The combination method could improve the depressive state,KPS score,CD4+T cell ratios of patients,which was better than those in the chemotherapy group.SMD were 0.68,0.54,0.98,respectively,and the difference was statistically significant with P<0.05.The incidence of gastrointestinal adverse reactions and liver and kidney injury in the combination group were lower than those in the chemotherapy group.The RR values were 0.79 and 0.44,respectively,and the difference is statistically significant with P<0.05.Finally,sensitivity analyses showed that excluding any research would not change the overall statistical effect,and would not affect the final statistical conclusion.The results of publication bias analysis showed that there was no publication bias in the improvement rates of ORR,DCR,and KPS.2.Study on the intervention effect and molecular mechanism of Shu-gan-Jian-pi decoction on breast cancer in mice with pre-tumor depression(1)Construction of pre-tumor depression breast cancer model:35 days after CUMS modeling,the total distance of movement,the distance of movement in the central area,and the stay time in the central area were significantly lower than those of the normal control group,and the stationary time is significantly longer than those of the normal control group.Comparing with the normal control group,the expression of 5HT1AR in the CA1 area of hippocampus was significantly reduced,P<0.05.On the 7th day after inoculation with 4T1-luc cells,the tumor nodules were palpable in each group,which proved that the pre-tumor depression type breast cancer mouse model was successfully constructed.(2)The effect of depression on the immune and inflammatory microenvironment:35 days after CUMS modeling,comparing with the normal control group,the proportion of NKT,MDSC,and TAM cells in the spleen increased significantly,and the expression of serum inflammatory factors IL-6,IL-1?,and IL-18 increased significantly,P<0.05.(3)Depression promotes tumor growth:21 days after tumor inoculation,the average tumor weight of the tumor-bearing group was 1.02±0.08 g,and the average tumor weight of the depressive tumor-bearing group was 1.26±0.13 g.The tumor promotion rate of depression in mice with breast cancer was 23.50%.(4)Depression aggravates the immune suppression and inflammatory microenvironment in tumor-bearing mice:Compared with the tumor-bearing group alone,the red and white marrow structure of the spleen tissue in the depression-bearing tumor group was blurred,and the splenic sinus was dilated.The proportions of TAM cells and MDSC cells were significantly increased in depression tumor-bearing group,and the difference was statistically significant compared with the simple tumor-bearing group,P<0.05.(5)Shu-gan-Jian-pi decoction can improve the depression state of mice:21 days after tumor intervention,sucrose water partiality of the Chinese medicine group and the Chinese medicine combined chemotherapy group was not statistically significant different comparing with the normal control group.Comparing with the depression and tumor-bearing group,the total distance of exercise in the Chinese medicine group and the Chinese medicine combined chemotherapy group was significantly longer,and the resting time was significantly shortened,P<0.05.Comparing with the chemotherapy group,the total distance of exercise in the Chinese medicine combined chemotherapy group is significantly longer,and the resting time was significantly shortened,P<0.05.Comparing with depression and tumor-bearing group,the expression of 5-HT1AR in brain tissue of the Chinese medicine group and the Chinese medicine combined chemotherapy group was significantly increased,and the difference was statistically significant,P<0.05.(6)Shu-gan-Jian-pi decoction can inhibit tumor growth,and improve the body's immune and inflammatory microenvironment:21 days after intervention,the tumor inhibition rates of the chemotherapy group,Chinese medicine group,and Chinese medicine combined chemotherapy group were 67.28%,16.08%and 81.29%.There were more macrophages in the spleen tissue of the Chinese medicine group,and the structure of the spleen in the Chinese medicine combined chemotherapy group was improved most obviously.C ompared with the depression and tumor-bearing group,the proportion of CD4+T cells,CD8+T cells,and NKT cells increase and the proportion of MDSC?TAM cells were significantly reduced in the spleen and tumor tissues of the Chinese medicine combined chemotherapy group.And compared with the depression and tumor-bearing group,the levels of INF-y and Granzyme B secreted by CD8+T cells in the spleen and tumor tissues in the Chinese medicine combined chemotherapy group were significantly increased(P>0.05).Compared with the depression tumor-bearing group and chemotherapy group,the expression of IL-1? in the traditional Chinese medicine group was significantly reduced(P<0.05).Compared with the chemotherapy group,the expression of IL-6 in the traditional Chinese medicine combined chemotherapy group was also significantly reduced(P<0.05).(7)Shu-gan-Jian-pi decoction can induce 4T1-luc breast cancer cell pyrolysis through the NLRP3/caspase-1/GSDMD pathway:?20%low-dose medicated serum inhibited the proliferation rate of 4T1-luc cells significantly(P<0.05).? Compared with the normal control group,after 20%low-dose medicated serum intervened in 4T1-luc cells for 48 hours,LDH release and IL-1? expression increased significantly(P<0.05);NLRP3 inflammasome protein,caspase-1 cleavage segment(p20),and GSDMD-N segment protein increase(P<0.05).? After 21 days of intervention,compared with the depression tumor-bearing group,the expression levels of NLRP3,Caspase-1 and GSDMD proteins in each intervention group were significantly increased.Western blotting results showed that the protein expression of Chinese medicine combined with chemotherapy group was significantly higher than that of depression tumor-bearing group and chemotherapy group,and the activated caspase-1 and GSDMD-N-terminal expression of Chinese medicine group was significantly higher than that of depression tumor-bearing group(P<0.05).The results of immunofluorescence detection showed that the expression of IL-1? increased after chemotherapy intervention,and the expression of IL-1? in the traditional Chinese medicine group was significantly reduced.Conclusion:1.Traditional Chinese medicine with the method of Shu-gan-Jian-pi as the main treatment principle may be an important treatment plan for breast cancer patients with depression.2.The mechanism of Shu-gan-Jian-pi decoction in inhibiting the occurrence and development of depression and tumor-bearing breast cancer may be that Shu-gan-Jian-pi decoction can induce breast cancer cell death through the NLRP3/caspase-1/GSDMD cell pyrolysis pathway.It may control the release of pro-inflammatory factors IL-6 and IL-1?,reduce the proportion of MDSC and TAM cells,and increase the proportion of NKT cells,CD8+ T cell function,and thereby reduce the impact of depression on the body's microenvironment and inhibit tumor growth.