Study On The Mechanism Of Haihua Tiaofei Prescription Regulating Neutrophils To Inhibit The Formation Of Lung Pre-metastatic Nich

Distant metastasis is an important cause of poor prognosis in malignant tumors,and the lung is one of the most frequently metastasized target organs.The organ specificity of distant metastasis of malignant tumors has aroused widespread thinking in the academic community.From the initial "seed soil" theory to the "blood dissemination" theory,and now the concept of "pre-metastasis niche" has been proposed,reflecting the deepening understanding of researchers on distant metastasis of tumors.One of the characteristics of pre metastatic niches is inflammation,and inflammatory state is a complex concept involving many cells,factors,proteins,etc.Therefore,exploring the mechanism of inflammation promoting the formation of pre-metastatic niches in the lung is of great significance.Traditional Chinese medicine(TCM)is an important treasure in Chinese medicine,and"treating diseases before they occur" is a huge advantage of TCM.Therefore,TCM has a good prospect in preventing and treating the formation of small habitats of lung pre metastasis.Inflammation is closely related to the concepts of "fire" and "heat" in traditional Chinese medicine.Combined with the physiological and pathological characteristics of the lung,we believe that the etiology and pathogenesis of the formation of lung pre-metastasis niches are fire poison attacking the lung,qi and body injuries,and phlegm and blood stasis intertwined.The treatment should be based on clearing away heat,supplementing qi,and resolving phlegm to remove blood stasis.Therefore,we have developed a compound Haihua Tiaofei Formula based on traditional Chinese medicine Haifushi and Xuanfuhua to prevent and treat lung metastasis from malignant tumors.Purpose:1.Taking breast cancer as an example,to explore the risk factors affecting the occurrence of lung metastasis of malignant tumor,to measure the clinical significance of inflammation related indicators in predicting the incidence of lung metastasis,to build a nomogram prediction model to screen high-risk groups with lung metastasis,so as to guide clinical treatment.2.To explore the intrinsic mechanism of LPS induced lung inflammation promoting the formation of lung pre metastasis niche.3.To explore the micro mechanism of Haihua Tiaofei Formula inhibiting the formation of lung pre-metastic niche.Methods:1.Retrospective analysis of the medical records of breast cancer patients in our department was conducted according to the nanoemission standard,and the absolute values of inflammation related indicators(neutrophils,lymphocytes,platelets)were collected three months before and after the diagnosis of the patients.The baseline information of each group was divided into two groups according to whether there was lung metastasis,followed by single and multi-factor binary logistic regression analysis,and the use of R language to build a nomogram prediction model for the final meaningful indicators,and draw calibration curves ROC curve,decision analysis curve,and clinical impact curve.2.Using the Integrated Pharmacology Platform(TCMIP)database and SwissTargetPrediction to obtain the effective components and corresponding targets of Haihua Tiaofei Formula,use DisGeNET and GeneCards disease databases to obtain malignant tumor lung metastasis disease targets,use R language to obtain intersection targets,and perform GO/KEGG analysis;The STRING database was used to analyze the intersection target protein interaction network,and the MCODE plug-in in Cytascape was used to screen core targets.After that,we analyzed the difference in the expression of core targets in tumor tissues and normal tissues adjacent to cancer,the impact on the prognosis of breast cancer patients,and the immune microenvironment,and finally used Autodock Vina software for molecular docking to predict the binding ability of drug components and core targets in Haihua Tiaofei Recipe.3.BALB/c mice were injected with 4T1-luc cells into the tail vein to build a lung metastasis model of breast cancer.LPS was inj ected into the nasal cavity to induce lung inflammatory reaction,which was divided into blank control group,inflammation model group,model group,aspirin group,and HHTF group.Observe the general state and weight of the mice.After modeling,live imaging was performed every 7 days.On the 22nd day,the number of metastatic nodules on the surface of the lungs was counted,and the right upper lung was taken for HE staining.4.The mice were subjected to eyeball extraction and blood extraction,and then centrifuged to measure ARG1 and IL-1β in the supernatant using ELISA Content;Take the left lung for flow cytometry to detect the proportion of N1 and N2 neutrophils;Take the right upper lung for immunohistochemical staining and semi quantitative analysis of MPO and CitH3 content;The activation of ERK/STAT3 signaling pathway was detected using qPCR and(phosphorylation)Western blotting.5.The co-culture model of human breast cancer cell MCF-7 and human neutrophil HL-60 was constructed by using Transwell chamber.Before co-culture,HL-60 cells were pretreated with LPS to induce inflammation.According to the growth inhibition rate of MCF-7 cells,the optimal intervention concentration and duration of Haihua Tiaofei Formula freeze-dried powder were determined.They were divided into MCF-7 single culture group,HL-60 single culture group,coculture model group,coculture inflammation group,and coculture HHTF group,and the migration and invasion ability of MCF-7 cells was tested.6.Centrifuge each group,collect the supernatant and cells,and use ELISA to detect ARG1 and IL-1β in the supernatant.The content of collected HL-60 cells and detect their N1 and N2 ratios by flow cytometry;The expression of CitH3 was detected by Western blotting.Results:1.Univariate and multivariate binary logistic regression analysis showed that neutrophils,lymphocytes,platelets and bone metastasis were closely related to lung metastasis of breast cancer(P<0.05),and neutrophils had the strongest predictive ability(OR:2.33,95%CI 1.33-4.08);The calibration curve indicates that the accuracy of the nomogram prediction model in this study is high;The AUC value of the ROC curve is 0.967,indicating that this model has good sensitivity and specificity;The decision analysis curve and clinical impact curve indicate that this model can better guide clinical treatment.2.Integrating pharmacological cues,AKT1,TP53,HIF1A,ESR1,HSP90AA1,IL-1β,TNF,IL-6,NFKB1,and IL-17 signaling pathways,hepatitis B related signaling pathways,and AGE-RAGE signaling pathways are important targets and signaling pathways for promoting lung metastasis in malignant tumors,which are closely related to inflammation;The prediction results of immune microenvironment showed that neutrophils were the key cells to promote the formation of lung pre-metastatic niche;Molecular docking suggests that Haihua Tiaofei Formula can effectively interfere with the expression of core targets to prevent and treat lung metastasis of malignant tumors.3.In vivo experiments,from the 7th day after modeling,compared with the model group,the fluorescence intensity of lung imaging in the inflammatory model group was significantly increased(P<0.05);From the 14th day,the fluorescence intensity of lung imaging in the HHTF group was significantly lower than that in the inflammatory model group and model group(P<0.05).4.In vivo experiments,compared with the model group,the expression of NETs markers CitH3,p-ERK1/2,and p-STAT3 protein in the lung tissue of mice in the inflammatory model group was significantly upregulated,while the expression of ARG1,IL-1β was not significantly upregulated.There was no statistical difference in the proportion of neutrophils and MPO between N1 and N2.Compared with the inflammatory model group,IL-1β in the HHTF group.The proportion of N2 neutrophils,MPO,CitH3,ERK1/2,and STAT3 mRNA,STAT3 protein expression,and phosphorylated protein expression of ERK1/2,and STAT3 were significantly reduced,with an effect similar to that of aspirin.5.In vitro experiments,the lowest survival rate of MCF-7 cells was 51.05 ± 12.11%when treated with freeze-dried powder of Haihua Tiaofei Formula at a concentration of 50 mg/mL for 48 hours.Compared with MCF-7 single cμLture group and co-cμLture model group,the migration or invasion ability of MCF-7 cells in co cμLture inflammation group was significantly enhanced(P<0.05).Compared with the co cultured inflammatory group,the migration and invasion ability of MCF-7 cells in the co-cultured HHTF group was significantly reduced(P<0.05).6.In vitro experiments,compared with the co-cultured model group,the expression of CitH3 protein in the co-cultured inflammatory group was significantly increased(P<0.05).There was no significant difference in the proportion of neutrophils between N1 and N2(P>0.05);Compared with the co cultured inflammatory group,the co-cultured HHTF group showed significant differences in ARG1,IL-1β.There were significant differences in the proportion of N1 neutrophils and the expression of CitH3 protein(P<0.05).Conclusions:1.Neutrophil,lymphocyte,platelet and bone metastasis are closely related to whether breast cancer has lung metastasis,of which neutrophil is the most influential factor.2.The nomogram model based on neutrophils,lymphocytes,platelets and bone metastasis has a good ability to predict whether breast cancer has lung metastasis.3.Integrated pharmacology and immune microenvironment analysis suggest that inflammation promotes the formation of lung pre metastasis niches,and neutrophils are a key factor in this process.4.In vivo and in vitro experiments have confirmed that LPS induced inflammation is mainly mediated by promoting the production of NETs by neutrophils and mediating the formation of lung pre-metastatic niches.5.Integrated pharmacology,molecular docking,and in vitro and in vivo experiments have shown that Haihua Tiaofei Formula can inhibit neutrophil function by interfering with multiple targets and pathways,thereby preventing the formation of lung pre metastasis niches.