Clinical And Mechanistic Studies On The Inhibition Of TNBC Growth Through PI3K/AKT Pathway By Rujifang

Objective1.To explore the possible mechanism of Rujifang against triple negative breast cancer based on network pharmacology analysis.2.To evaluate the clinical efficacy and safety of Rujifang intervention on triple negative breast cancer patients through retrospective clinical research.3.Based on network pharmacology and clinical research,research on animal,cell and molecular levels was carried out to explore the effect of Rujifang on the growth of triple negative breast cancer and related molecular mechanisms.Methods1.Network pharmacology analysisFirstly,the effective ingredients and corresponding target genes of Rujifang were screened through traditional Chinese medicine network pharmacology in databases such as TCMSP.TNBC related disease genes were obtained using OMIM,Gencards,and DisGeNET databases.Then,the two intersect to obtain the key genes for the treatment of TNBC with Rujifang.Finally,the Metascape database was used to analyze the KEGG pathway and GO enrichment of target genes,and the molecular and pathway mechanisms of Rujifang against triple negative breast cancer were preliminarily determined through the analysis of molecular complexity detection(MCODE)module.2.Retrospective clinical study of Rujifang intervention in triple negative breast cancerThe retrospective cohort study design was used to conduct clinical research,collect the case data of patients diagnosed as triple negative breast cancer in the outpatient and inpatient departments of Foshan Hospital of Traditional Chinese Medicine and the First People’s Hospital of Foshan since January 1,2013.We screened out three negative breast cancer patients who met the standard,and divided them into the control group(standard adjuvant treatment after surgery)and the observation group(combined standard adjuvant treatment and Rujifang after surgery).The patients in the control group received standard adjuvant treatment for at least 4 weeks,and the observation group took Rujifang orally for six months on the basis of standard adjuvant treatment.The baseline data of patients with breast cancer were analyzed on the onset age,tumor size,pathological type,Ki67 expression level,operation mode,whether there was tumor metastasis,lymph node metastasis,etc.In terms of observation indexes,the disease-free survival period and long-term survival of patients were followed up and recorded,and tumor markers(CEA,CA15-3),KPS score,blood routine,liver and kidney function and other indexes were collected and compared for all patients at their first postoperative visit and 6 months after the visit.3.Evaluation and Mechanism Exploration of the Effectiveness and Safety of Rujifang in AnimalsThree negative breast cancer cells of mice were subcutaneously injected into the right shoulder and back of mice to build a three negative breast cancer model(subcutaneous transplant tumor model),which was divided into model group,cyclophosphamide positive control group,and Rujifang high,medium,and low dose groups.According to the grouping situation,physiological saline was administered by gavage,cyclophosphamide was intraperitoneally injected(30mg/kg),high(58g/kg),medium(29g/kg),and low dose(14.5g/kg)Rujifang was administered by gavage.We observed and compared the body mass and tumor volume of each group of mice at different time points.After 28 days,the mice were euthanized,the tumors were removed and weighed,and the tumor inhibition rate of each group of mice was calculated.After the end of administration,the levels of alkaline phosphatase,alanine aminotransferase,aspartate aminotransferase,albumin,creatinine,and peripheral blood red blood cells,hemoglobin,white blood cells,neutrophils,lymphocytes,monocytes,and platelets in tumor bearing mice were measured to evaluate the impact of Rujifang on the safety of tumor bearing mice.Exploration of mechanism of action:Preparation of Rujifang medicated serum.26 SD rats were divided into two groups,with 13 rats in each group.The dosage of Rujifang in rats was calculated based on the body surface area of humans and animals(14.61g/kg),and blood was taken for 7 consecutive days to prepare Rujifang medicated serum.The mice and human triple negative breast cancer cells 4T1 and MD-MBA-231 were cultured respectively,and divided into blank serum group and Rujifang drug containing serum group.The effects of different concentrations(2.5%,5%,7.5%,10%,15%)of Rujifang drug containing serum and action time(24h,48h,72h)on the activity of 4T1 and MD-MBA-231 cells were detected by CCK8 method.The effect of different concentrations of Rujifang containing serum on the cell cycle of breast cancer was evaluated by low cytometry.We identified the relevant mechanisms of Rujifang’s anti TNBC effect through animal tumor tissue testing.Firstly,some tumor tissues were stained with Ki67 and TUNEL to observe the proliferation and apoptosis of cells in the tumor.Then the mRNA expression of cell cycle related genes(including CDK2,CDK4,P21,and P27)in tumor tissue was detected by RT PCR method.Finally,Western Blot method was used to detect the expression of PI3K/AKT pathway proteins in tumor tissue.Results1.According to network pharmacology,Rujifang contains 68 active ingredients,corresponding to 247 target genes.There are a total of 2800 TNBC related disease genes.The effective components of Rujifang correspond to the intersection of target genes and TNBC related disease genes,resulting in 170 overlapping genes,which are key genes that Rujifang may have therapeutic effects on TNBC.By analyzing 170 key genes through KEGG pathway,GO enrichment,protein interaction(PPI),and molecular complexity detection(MCODE)modules,we found that the mechanism of Rujifang in treating TNBC may be a synergistic effect of multiple components,targets,and pathways.Among them,the PI3K/AKT signaling pathway may be the main potential pathway for Rujifang to exert anti TNBC effects.2.In a retrospective clinical study,there was no significant difference in baseline data between the two groups of patients(including age of onset,tumor size,pathological type,surgical method,histopathological type,vascular infiltration status,pathological stage,maximum tumor diameter,axillary lymph node metastasis status,Ki-67 index,CEA,CA15-3)(P>0.05);In the efficacy evaluation,the recurrence metastasis rate of patients in the control group was 38.3%and the mortality rate was 29.4%,while the recurrence metastasis rate of patients in the observation group was 17.1%and the mortality rate was 12.2%;After taking two courses of milk accumulation formula,there was no significant difference in KPS scores between the observation group before and after treatment(P>0.05);The relevant serum tumor markers showed significant differences in CEA and CA15-3 between the control group and the observation group after treatment(P<0.05).In terms of safety evaluation,there were no significant changes in the blood routine and liver and kidney function of patients before and after taking Rujifang(P>0.05).3.In the experimental study,after the three negative breast cancer tumor bearing mice were treated with Rujifang at different doses for a certain period of time,the results showed that the tumor volume of tumor bearing mice decreased after the treatment of Rujifang at different doses,and showed a significant dose dependence(P<0.001).On the 28th day,the tumor weight of mice in different doses of Rujifang group significantly decreased,and the larger the dose of Rujifang,the smaller the tumor weight of mice bearing tumors(P<0.001).Compared with the model group,the tumor inhibition rate of mice in the Rujifang group showed a dose-dependent(P<0.001).In terms of safety,compared with the model group,the red blood cell count,hemoglobin,white blood cell count,and lymphocytes of the cyclophosphamide group mice were significantly reduced(P<0.05 or P<0.01),while platelets were increased(P<0.05).The lymphocyte count of the Rujifang group was significantly increased(P<0.05),while there was no significant difference(P>0.05).The alkaline phosphatase,alanine aminotransferase,and alanine aminotransferase levels in the cyclophosphamide group decreased(P<0.01),while no statistical significance was found in the Rujifang group(P>0.05).In the mechanism exploration,the cell proliferation inhibition rate of different concentration groups treated with Rujifang significantly increased(P<0.01 or P<0.001),and its inhibitory effect showed an enhanced effect when the drug concentration increased and the action time prolonged.Rujifang reduced the proportion of G2 phase cells and blocked the cell cycle in the S phase.Rujifang significantly inhibited the mRNA expression of CDK2 and CDK4 in tumor tissue,but increased the mRNA expression of CDKI(P21 and P27)(P<0.05).Rujifang has a significant inhibitory effect on the expression of cyclin in breast cancer MD-MBA-231 cells:the expression of CDK2 and CDK4 protein was significantly reduced(P<0.05 or P<0.01),and the expression of P21 and P27 protein was significantly increased(P<0.01).Rujifang significantly reduced the protein expression of PI3K/AKT pathway related genes in breast cancer MD-MBA-231 cells:the protein expression of P-Akt,AKT,P-PI3K was significantly reduced(P<0.01).Conclusion1.Network pharmacology analysis shows that tumor cell growth,such as proliferation and apoptosis,is the potential core target of Rujifang.Rujifang inhibits the growth of triple negative breast cancer through the interaction of multiple targets,multiple pathways and multiple pathways,in which PI3K/Akt signaling pathway plays a major role.2.The intervention of Rujifang is expected to prolong the overall survival and disease-free survival of patients,improve their quality of life,reduce the levels of serum tumor markers CEA and CA15-3,and have good safety when combined with chemotherapy drugs3.Rujifang can significantly inhibit tumor growth in TNBC model mice,which is safe and effective.Rujifang exerts anti-tumor effects by regulating the PI3K/AKT pathway to block cell cycle and induce apoptosis.