Study On The Mechanism Of Kidney-tonifying Traditional Chinese Medicine In Regulating The Niche Of Breast Cancer Bone Metastases

Bone metastasis is a common complication in patients with advanced breast cancer,which is often found in the spine,pelvis and metaphysis,and often occurs frequently,accompanied by pain,pathological fracture,hypercalcemia and other bone related events,which seriously affects the quality of life of patients.The treatment methods of bone metastasis include bone protectors,chemotherapy,radiotherapy,traditional Chinese medicine,etc.Bisphosphonates and dinofimab are good therapeutic agents in the treatment of bone metastasis,and are helpful to the auxiliary treatment of early breast cancer.In the mechanism of bone metastasis,the latest research suggests that before the clinical symptoms of bone metastasis,tumor cells can break the bone homeostasis state composed of osteoblasts and osteoclasts in the bone microenvironment,thus forming the bone metastasis niche,which is related to the mutual regulation of breast cancer cells and osteoblasts,immunosuppression in the niche,hypoxia state and so on.Traditional Chinese medicine(TCM)is effective in the prevention and treatment of bone metastasis of breast cancer,which can prevent the occurrence of bone metastasis early,delay the progress of bone metastasis,and improve the quality of life of patients.The team summarized the clinical experience of Li Peiwen,a famous Chinese medicine in China,found that the traditional Chinese medicine belonging to lung meridian can prevent and control lung metastasis,liver metastasis can be prevented and treated by liver meridian medicine,and the kidney meridian medicine can prevent and control bone metastasis.Through the experiment of rats,it is further proved that the curative effect of kidney tonifying herbs in the treatment of bone metastasis is far better than other meridian tropism drugs.On the basis of clarifying the different action mechanism of different drugs on bone tumor,we have doubts about the therapeutic effect of different properties(cold and heat)of Chinese Herbal Medicinal on bone tumor.Therefore,we should further explore the action mechanism of Tonifying Kidney Yin,tonifying kidney yang and tonifying both yin and Yang in regulating bone micro-environment.Objective:1.Combined with clinical experience,expound the TCM pathogenesis theory and treatment of bone metastasis of malignant tumor,and preliminarily explore the therapeutic mechanism of kidney tonifying herbs through network pharmacology.2.Through the animal model of bone metastasis and cell co-culture model,demonstrate the changes of bone metastasis niche in the process of bone metastasis,and the relationship between breast cancer cells and osteoblasts.3.On the basis of exploring the mechanism of early intervention of kidney tonifying Chinese Herbal Medicinal on bone metastasis,the difference of curative effect of kidney tonifying yin method,kidney tonifying yang method,yin and Yang double tonifying method in the prevention and treatment of malignant tumor bone metastasis was clarified.Methods:1.Systematically sumarize the ancient literature on bone tumor related diseases,as well as the modern doctors’ understanding and medication experience of bone metastasis,and summarize its etiology,pathogenesis and treatment.The theory of TCM pathological changes of bone metastasis was put forward,and the treatment and medication were explored.The network pharmacology was used to analyze the representative kidney tonifying drug"Epimedium-Cistanche deserticola",to explore the mechanism of kidney tonifying traditional Chinese medicine,and to provide a certain direction for the development of basic experiments.2.MDA-MB-231-luc and 4T1-luc cells were injected into left ventricle,tibia and caudal artery to establish models.The success rate and the process of metastasis were evaluated by small animal imaging technology.3.Establish bone metastasis model,explore the curative effect of kidney tonifying drugs in preventing and treating bone metastasis by using in vivo imaging technology and Micro-CT,and analyze the regulating effect of kidney tonifying drugs on bone metastasis niche from serum markers of bone turnover,osteoclast count and lymphocyte subsets expression.4.Immunohistochemistry,Western blotting and RT-qPCR were used to explore the regulatory effects of kidney tonifying herbs on Mesenchym alepithelial transition(MET)and Dkk-1/BMP2 signaling pathway in breast cancer cells.Results:1.The pathogenesis of bone tumor is "the invasion of qi deficiency and pathogenic factors,and bone tumorigenesis due to the injury of apoplexy".The location of the disease is in bone,which has the characteristics of deficiency in origin and excess in sign.The pathological process can be summarized as four stages:kidney deficiency and essence deficiency,cancer evil entering bone,the combination of healthy and evil,and inducing evil Qi to cause disease.In terms of treatment strategy,under the guidance of the theory of"Preventive treatment of diseases",the treatment of bone metastasis can be divided into three stages:initial stage,middle stage and final stage.2.Through network pharmacology,it was found that the kidney tonifying herbs(Epimedium-Cistanche deserticola)affected the survival,differentiation and maturation of breast cancer cells and osteoblasts by intervening in gap junctions and adhesion,and regulated the expression of Dkk-1,BMP2,CDH1,CDH2,Runx2 and other genes and proteins.3.Comparison of different models:left ventricular injection can simulate the early pathological process of breast cancer bone metastasis,and its success rate is about 40%,and also causes death of mice.In vivo imaging shows that fluorescence imaging of the metastasis site can be observed about 20 days after the model is made.The success rate of tibial injection was 100%,and the injury was small in mice.When different cell lines were used for the model making by tail artery injection,mda-mb-231-luc cells were mostly concentrated in the lung of nude mice and formed lung metastasis.When 4T1-Luc cells were used,the cancer cells gathered in the tail back of mice and formed local bone metastasis.4.Kidney tonifying herbs can regulate the niche of bone metastasis.The tibial injection model of bone metastasis of breast cancer was established.Through small animal imaging and Micro-CT detection,it was found that kidney tonifying herbs can inhibit the progress of bone metastasis of breast cancer,improve bone volume fraction,trabecular thickness,Trabecular number and trabecular separation.In addition,it can promote the expression of CD4+and CD8+,reduce the expression of serum bone turnover markers BALP,PINP and blood calcium,and inhibit the activity of osteoclasts.Among them,Yin Yang shuangbu recipe has the most obvious effect(P<0.05).5.Osteoblasts promoted the mesenchymal epithelial transformation of cancer cells in bone metastasis niche.Compared with the blank control group,the protein expressions of E-cad,N-cad and Cx43 in the model group were up-regulated(P<0.05).Yin Yang shuangbu formula could down regulate the expression of E-cad and Cx43(P<0.05),but the regulatory effect on N-cad was not obvious(P>0.05).The expression of Dkk-1 in breast cancer cells was high.The expression of Dkk-1 was decreased after the treatment with Yin Yang shuangbu formula,while the expression of BMP2,β-catenin and Runx2 was up regulated(P<0.05).6.The co-culture model of MDA-MB-231 cells and MC3T3-E1 cells was established to verify the mechanism of Yinyang shuangbu formula improving bone metastasis niche.Compared with the monoculture group,the proliferation of MDA-MB-231 cells was accelerated,while the growth of MC3T3-E1 cells was inhibited.Yin Yang shuangbu formula can inhibit cancer cells and promote osteoblast proliferation.WB and real-time PCR analysis showed that the protein and gene expression of E-cad,N-cad,Cx43 and Dkk-1 were significantly up-regulated(P<0.05),while the expression of β-Catenin,BMP2 and Runx2 were down regulated.Yin Yang shuangbu formula can reverse the MET of breast cancer cells and promote the maturation and differentiation of MC3T3-E1 cells.Conclusion:1.Combing the ancient literature related to bone tumor and summarizing the clinical experience of modern doctors are helpful to deepen the understanding of the pathogenesis of bone metastasis.It accords with the clinical practice and has guiding significance for the differentiation of symptoms and signs.Combined with clinical practice,TCM understanding of pathological changes of bone metastasis can be summarized into four stages.The treatment is divided into early stage,middle stage and late stage,which is conducive to the prevention and treatment of clinical bone metastasis.2.Through network pharmacology,the mechanism of "Epimedium-Cistanche deserticola" in the prevention and treatment of bone metastasis of breast cancer is preliminarily clarified,which provides a certain basis and research direction for the follow-up experimental research.3.MDA-MB-231-luc and 4T1-luc cells were injected into left ventricle,tibia and caudal artery to establish bone metastasis model.The tibia injection method had the highest success rate.4.Different kidney tonifying methods(kidney yang tonifying,kidney Yin tonifying and Yin Yang tonifying)have different curative effects on the prevention and treatment of bone metastasis of breast cancer,and Yin Yang tonifying formula has the best curative efect.5.Yinyang shuangbu formula can regulate the niche of bone metastasis,reduce the expression of E-cad,n-cad,Cx43 and Dkk-1,and up regulate the expression of BMP2,β-Catenin and Runx2.6.Establish the co-culture model of breast cancer cells and osteoblasts.Yin Yang shuangbu formula can reverse the MET of cancer cells and promote the maturation and differentiation of osteoblasts through Dkk-1/BMP2 pathway.