Intervention Of Shuganjianpi Formula On Depressive Breast Cancer In Mice Based On Pyroptosis

BackgroundBreast cancer is the most common malignant tumor in adult women.Emotional disorders are often accompanied by breast cancer.It is characterized by depression.anxiety,dysphoria,sleep disturbance,fatigue,and guilt.It seriously affects the physical and mental health of patients,interpersonal relationships,quality of life and tumor treatment outcomes.At present,there is no unified normative diagnosis and treatment guide for breast cancer depressive disorder.The clinical diagnosis and treatment of depression are mainly based on oral western medicine.However.due to the side effects of western medicine,some drugs may increase the risk of recurrence and metastasis of breast cancer.In addition,patients have higher resistance and poor compliance,and clinical efficacy is often unstable.Since ancient times,traditional Chinese medicine has attached importance to the role of emotional factors in diseases.It is good at distinguishing diseases from syndrome differentiation under the guidance of the overall concept.It has the characteristics of high safety and small side effects.The patient has high acceptance and clinical effects are obvious,Play an important role in the prevention and treatment of breast cancer.Inflammatory cytokines are a key role in the formation of depression.The inflammatory microenvironment is an important material basis for the development of tumors.Myeloid-derived inhibitory cells(MDSCs)are a large number of inhibitory cells in the microenvironment of malignant tumors.The body's anti-tumor cellular immune response has a strong inhibitory effect.Pyroptosis is one of the ways of programmed cell death.It plays a two-sided role in the regulation of tumors:as an effective defense mechanism for the host,when the cell contents of the tumor cells are ruptured and discharged into the stroma.the immune response of the motility is eliminated.Cytoinflammatory factors,but if the tumor pyroptosis is uncontrolled and unrestricted,the induction of inflammation and waterfall response in the tumor microenvironment will aggravate tumor progression.The damage-related molecular patterns(DAMPs)released during the process of coronation can amplify MDSC in large quantities,and the released IL-1? is an important activating factor of MDSC.Thus,pyroptosis-MDSC-cytokines may be an important mechanism of breast cancer depression.Regulating pyroptosis,inhibiting MDSC production,inactivating it or promoting its differentiation and maturation is a potential therapeutic target for tumor depression.Shugan Jieyu is a common treatment method for traditional Chinese medicine treatment of stagnation syndrome.Shuganjianpi Formula is the experience of treating breast cancer in the Oncology Department of Guang'anmen Hospital of China Academy of Chinese Medical Sciences.On the basis of the tumor,combined with the classic name Fang Xiaoyao scattered condensate,the main drugs include Chinese thorowax root,Debark peony root,Turmeric root tuber,Akebia trifoliata Koidz,Common selfheal fruit-spike,Milkvetch root,Sanqi,Licorice,etc.,the clinical application has a good effect The preliminary research of the research group showed that Shuganjianpi Formula or gemcitabine chemotherapy in the treatment of mice with depressive breast cancer can inhibit the proliferation of MDSC and NKT,slow the early apoptosis of CD8+T cells,up-regulate the ratio of CD4+/CD8+,down-regulate factors such as IL-4,IL-6,IL-13,TGF-?,and IFN-y,improve the quality of life and prolong survival of mice with depression.Therefore,we propose that Shuganjianpi Formula may improve breast cancer depression by regulating the cell death pathway,intervening in the massive release of inflammatory factors,and limiting the expansion and activation of MDSC.ObjectiveTo observe the intervention effect of Shuganjianpi Formula on mice with depression-bearing tumors by establishing a mouse model of depression-type breast cancer combined with disease syndrome,and to explore the key links of Shuganjianpi Formula in pyroptosis regulation of MDSC.Research methodsBasic experimental research is divided into three parts:Part 1:Establish and evaluate the mouse tumor-bearing model;Part 2:Observe the intervention effect of Shuganjianpi Formula on depression-bearing mice;Part 3:Based on pyroptosis.explore the mechanism of Shuganjianpi Formula in the Treatment of Depressive Breast Cancer.The description is as follows:Part 1:Establish and evaluate the mouse tumor-bearing model1.84 female BALB/c mice were inoculated with 2×105 4T1-luc cells under the right second breast pad.After 24 hours,they were randomly divided into 7 groups(Chemotherapy Group.Chinese Medicine Group,Chinese and Chemotherapy Combination Group,Depression Tumor-bearing Group.Simple Tumor-bearing Group,Simple Depression Group.Normal Control Group).12 mice in each group,the average was divided into 2 cages,and were raised in the Experimental Animal Center of Guang'anmen Hospital according to the following model administration:(1)Gemcitabine Chemotherapy Group(GEM):Inoculation of tumor cells and modeling of chronic mild unpredictable stress depression.The mice were treated with 120 mg/kg of gemcitabine,ie,on the 1st and 7th day after inoculation of the cells,respectively,within 1 hour after the solution was prepared into a concentration of 12 mg/mL with 0.9%sodium chloride injection.Intraperitoneal injection was performed twice.(2)Shuganjianpi Group(SGJP):Inoculation of tumor cells and modeling of chronic mild unpredictable stress depression.Shuganjianpi Formula(Personal dosage:Chinese thorowax root 10g.Debark peony root 15g,Turmeric root tuber12g.Akebia trifoliata Koidz 15g.Common selfheal fruit-spike 15g,Milkvetch root 30g.Powder 5g,Licorice 6g.one dose per day)According to the basis of the previous experiment,the dosage of mice per kilogram of body weight is equivalent to 15 times the clinical dose of adults(60 kg).The mice were given the Shuganjianpi Formula for the next 28 days from the next day of inoculation.(3)Chinese and Chemotherapy Combination Group(GEM+SGJP,abbreviated as:G+S):Inoculate tumor cells and perform chronic mild unpredictable stress depression modeling.The animals in this group were treated with chemotherapy twice as in(1),and the traditional Chinese medicine was intragastrically administered for 28 days as in(2).(4)Depression Tumor-bearing Group(DT):Inoculation of tumor cells and modeling of chronic mild unpredictable stress depression.During the experiment,there was no treatment within the scope of animal ethics.(5)Pure tumor-bearing group(PT):only tumor cells were inoculated,chronic mild and unpredictable stress depression modeling was not performed.During the experiment, according to the specifications of the experimental animal center,normal feeding, and free feeding.There are no treatments within the scope of animal ethics.(6)Pure Depression Group(PD):no tumor cells were inoculated,only chronic mild and unpredictable stress depression modeling was performed.No treatment is available.(7)Normal Control Group(NC):tumor cells were not inoculated,and chronic mild unpredictable stress depression modeling was not performed.According to the specifications of the experimental animal center,normal feeding and free feeding.2.The success of depression model was judged by sucrose water test,open field test,and determination of 5-HTiaR in mouse hippocampus.Part 2.Observing the intervention effect of Shuganjianpi Formula on mice with depression tumors.1.Monitor the body weight change of the mice,observe the tumor growth,measure the tumor diameter change,weigh the tumor weight after taking the material,and calculate the tumor inhibition rate.2.Flow cytometry was used to detect the content of MDSC and its typing in mouse spleen.3.Transmission electron microscopy was used to observe the morphological changes of tumor cells in mice with depressive breast cancer.Part 3.Based on cell death,explore the mechanism of Shuganjianpi Formula in the treatment of depressive breast cancer.1.RT-PCR detection of MDSC upstream microRNA:miR-146a,miR-155,miR-21.2.High-performance liquid suspension microarray mice serum cytokines IL-1?,IL-18,IL-6,IL-17A,TNF-a and so on.3.Western-blot detection of cell death pathway NLRP3,Caspase-1,ASC and other proteins.Results1.Established and improved 2x105 cells of breast cancer mice and vaccinated animals were molded.2.The open-field test showed that the animal activity level was Pure Depression Group>Normal Control Group>Pure Tumor Group>Chinese and Chemotherapy Group>Chinese Medicine Group>Depression Tumor Group>Chemotherapy Group.There was no significant difference between the Pure Depression Group and the Pure Tumor Group(P>0.05).There was a significant difference between the Depression Group and the Normal Group(P<0.001).There was a significant difference between the Chinese Medicine Group and the Pure Depression Group(P<0.05).There was a significant difference between the Chinese and Chemotherapy Group and the Pure Depression Group(P<0.001).There was a significant difference between the Chinese Medicine Group and the Chemotherapy Group.The combination of Chinese and Chemotherapy Group was significantly more significant than the Chemotherapy group(P<0.05).3.The expression of 5-HT1AR in the hippocampus is:Normal Group>Chinese and Chemotherapy Group>Chinese Medicine Group>Pure Depression Group>Pure Tumor Group>Chemotherapy Group>Depression Tumor Group,indicating chronic mild unpredictability Radical stimulation caused a decrease in 5-HT1AR content.Chemotherapy alone could not improve the effect.The effect of traditional Chinese medicine alone was better than that of chemotherapy alone.The combination of traditional Chinese medicine and western medicine significantly improved the 5-HT1AR content caused by CUMS.4.The tumor diameter of each tumor group showed a trend of increasing tumor length with the tumor-bearing time.In the first 8 days,the tumor diameter of each group did not change much,and there was no significant difference between the groups.There was no statistical significance after comparison(P>0.05).After 8 days,the tumor diameter showed the following trend:Depression Tumor Group>Pure Tumor Group>Chinese Medicine Group>Chemotherapy Group>Chinese and Chemotherapy Group.After 20 days,the tumor diameter of Chinese and Chemotherapy Group was smaller than that of the Chemotherapy Group.The difference was statistically significant(P<0.05).5.Bioluminescence small animal in vivo imaging observation of photon number changes in tumor-bearing animals,showing the same trend with tumor diameter changes,namely:Depression Tumor Group>Pure Tumor Group>Chinese Medicine Group>Chemotherapy Group>Chinese and Chemotherapy Group(P<0.05).6.(1)Total content of MDSC:Depression Tumor Group>Pure Tumor Group>Chinese Medicine Group>Chemotherapy Group>Chinese and Chemotherapy Group>Pure Depression Group>Normal Group.There was a significant difference in the ratio among Pure Tumor Group,Depression Tumor Group and Normal Group(P<0.001).There was a significant difference between the Chemotherapy Group and the Chinese Medicine Group and the Depression Group(P<0.001).(2)Ly6C Ly6G-%of MDSC:Chinese and Chemotherapy Group>Pure Depression Group>Normal Group>Chinese Medicine Group>Chemotherapy Group>Pure Tumor Group>Depression Tumor Group.There was no statistical significance between the Pure Depression Group and the Normal Group(P>0.05).There was a significant difference between the Pure Tumor Group and the Depression Tumor Group compared with the Normal Group(P<0.001).The Chinese and Chemotherapy Group had a comparison with the Depression Tumor Group.There was no significant difference between the Chemotherapy Group and the Chinese Medicine Group and the Depression Tumor Group(P>0.05).(3)Ly6G Ly6C-%of MDSC:Depression Tumor Group>Pure Tumor Group>Chinese Medicine Group>Chemotherapy Group>Chinese and Chemotherapy Group>Normal Group>Pure Depression Group.There was a significant difference between the Pure Tumor Group and the Depression Tumor Group(P<0.001).There was no statistical significance between the Pure Depression Group and the Depression Tumor Group(P>0.05).7.Transmission electron microscopy showed that each group had different degrees of nuclear shrinkage,organelle vacuolation,autophagy endocytosis,etc.The overall lesions were ranked as follows:Chinese and Chemotherapy Group(very mild lesions)<Chinese Medicine Group(mild Lesions)<Pure Tumor Group(moderate lesions)<Chemotherapy Group(severe lesions)<Depression Tumor Group(severe lesions).8.(1)Content of miR-146a in mouse tumor tissues:Chinese Medicine Group>Pure Tumor Group>Chinese and Chemotherapy Group>Chemotherapy Group>Depression Tumor Group.There was a significant difference between the Depression Tumor group and the Pure Tumor Group(P<0.01).There was a significant difference between the Chinese Medicine Group and the Depression Tumor Group(P<0.001).(2)The content of miR-155 in mouse tumor tissues:There was a significant difference between the Depression Tumor Group and the Pure Tumor Group(P<0.001).There were significant differences between the Chemotherapy Group the Chinese Medicine Group and the Depression Tumor Group(P<0.05).There was a significant difference between Chinese and Chemotherapy Group and the Depression Tumor Group(P<0.01).There was no significant difference among the three groups:The Chemotherapy Group,the Chinese Medicine Group,and the Chinese and Chemotherapy Group(P>0.05).(3)The content of miR-21 in tumor tissues of mice:There was a significant difference between the Depression Tumor Group and the Pure Depression Group(P<0.001).The Chemotherapy Group,the Chinese Medicine Group,the Chinese and Chemotherapy Group,and the Depression Tumor Group were compared.The difference was statistically significant(P<0.05).There was no significant difference between the three groups in the Chemotherapy Group,the Chinese Medicine Group and the Chinese and Chemotherapy Group(P>0.05).9.Serum cytokine levels in mice:(1)IL-1?:Compared with the Depression Tumor Group,the Chinese Medicine Group,the Chemotherapy Group,and the Chinese and Chemotherapy Group have extremely significant differences.(P<0.001);(2)IL-18:Chinese and Chemotherapy Group<Depression Tumor Group<Chinese Medicine Group<Pure Tumor Group<Chemotherapy Group<Normal Group<Pure Depression group,the content of Chinese Medicine Group increased statistically significant(P<0.05);(3)IL-6:The drug was reduced to some extent after the intervention,and the most significant decrease was observed in the TCM group(P<0.001).(4)IL-17A:Compared with the Depression Tumor Group,the levels of IL-17A in the Chemotherapy group and the Chinese and Chemotherapy Group were significantly increased(P<0.001).(5)TNF-a:Compared with the Depression Tumor Group,the TNF-a content in the Chemotherapy Group and the Chinese and Chemotherapy Group Both were significantly elevated(P<0.001).10.Expression of tumor protein in mice:(1)NLRP3:There was a significant difference between the Chemotherapy Group and the Chinese and Chemotherapy Group and the Depression Tumor group(P<0.001).There was no difference between the Chinese Medicine group and the Depression Tumor Group.(2)Caspase-1:The combination of the Chinese and Chemotherapy Group was significantly different from the Depression Tumor Group(P<0.05),and there was no difference between the other groups(P>0.05).(3)ASC:There was significant statistical significance in the Chinese Medicine Group and Depression Tumor Group(P<0.05).There was a significant difference between the Chinese and Chemotherapy Group and the Depression Tumor Group(P<0.01).Conclusion1.Shuganjianpi Formula can significantly improve the depressive state of depressive breast cancer mice.Combined chemotherapy can control the growth of tumors,remodel the function of MDSC,and regulate the micro-environment of tumor inflammation.2.Shuganjianpi Formula can regulate microRNA and reduce the content of MDSC in the spleen of tumor-bearing mice by regulating the expression of pyroptosis and inflammatory factors,thus reversing the local immunosuppressive state of tumors.3.The pyroptosis pathway of NLRP3-based inflammatory bodies may be an important mechanism of Shuganjianpi Formula on depressive breast cancer.