The Molecμlar Mechanism Of The Prevention Of Breast Cancer By XIAOPI Formula Based On E2/ROS/Nrf2 Signaling Pathway

Objective1.In vivo:To investigate the inhibitory effect of XIAOPI on MMTV-PyMT transgenic mice spontaneous breast cancer and the mRNA expression of the related pathway,and to understand the role of XIAOPI in the prevention of breast cancer.2.In vitro:17 β-estradiol(E2)was used to induce normal breast epithelial cells MCF-10A and MCF-12A cells to simulate the cytological process of breast cancer.To observe the effect of XIAOPI on the oxidative index and biological phenotype of breast cells induced by E2,and to explore the molecular mechanism of XIAOPI on the prevention of breast cell carcinogenesis.Methods1.Therapeutic effect of XIAOPI on MMTV-PyMT mice and the regulation of E2/ROS/Nrf2 signaling pathwayTwelve female MMTV-PyMT mice were randomly divided into the control group and XIAOPI group after 3 weeks of adaptive feeding.The corresponding drugs or physiological saline were administered intragastrically every day for 12 weeks.Samples were taken at 9 weeks and 15 weeks respectively.1.1 Detection of blood biochemical indicators in mice:the level of estrogen,reactive oxygen species,8-OHdG,total antioxidant capacity,superoxide dismutase,catalase,glutathione peroxidase,and pathological morphology of breast tissue and lung tissue to evaluate the efficacy of XIAOPI.The expression of Nrf2,HO-1,NQO1,and laminin in breast tissue was detected by immunohistochemistry to evaluate the therapeutic effect of XIAOPI on preventing breast cancer.1.2 The expression of Nrf2,HO-1,NQO1 mRNA in breast tissue was detected by PCR.2.Protective effect of XIAOPI on E2 induced normal breast cells and its regulatory effect on E2/ROS/Nrf2 signaling pathway2.1 Using MTT assay,wound-healing assay,plate cloning assay,and flow cytometry assay to investigate whether XIAOPI could affect the proliferation,migration,cloning ability,and apoptosis level of normal mammary epithelial cells.2.2 DCFH-DA fluorescence probe method was used to detect the reactive oxygen species(ROS)levels,and the level of lactate dehydrogenase(LDH)leakage in the cell supernatant fluid was measured by trace enzyme method,the activity of catalase(CAT)was detected by visible light,the activity of superoxide dismutase(SOD)was detected by hydroxylamine method,ELISA assay was used to measure the 8-OHdG level.To investigate whether XIAOPI affects the cytotoxicity,ROS level,oxidase activity,and DNA oxidative damage of E2 induced normal breast epithelial cells.2.3 Western-blot and Real-Time-PCR were used to detect the protein and mRNA levels of related pathway,and to verify the target of XIAOPI in prevention of breast cancer.Results1.Therapeutic effect of XIAOPI on MMTV-PyMT mice and the regulation of E2/ROS/Nrf2 signaling pathway.1.1 Compared with the control group,XIAOPI could inhibit the number and growth(weight and volume)of MMTV-PyMT mice,and the results of HE staining showed that XIAOPI could inhibit the development of breast tumors and lung metastasis.Immunohistochemical results showed that the expression level of Laminin in the XIAOPI group was significantly lower than that in the control group,while the XIAOPI group could promote the expression of NQO1 and HO-1,Nrf2.The serum biochemical indexes further showed that the levels of ROS,E2,and 8-OHdG in mice were significantly reduced,and the level of oxidase(CAT、SOD、GSH-PX、T-AOC)was significantly increased after the intervention of XIAOPI for 15 weeks.1.2 PCR results showed that in the XIAOPI group,the Nrf2 signaling pathway was activated,and the expression levels of Nrf2,HO-1,and NQ01 mRNA were significantly increased,indicating that XIAOPI can activate the E2/ROS/Nrf2 signaling pathway,regulate the oxidative stress,and prevent the breast cancer.2.Protective effect of XIAOPI on E2 induced normal breast cells and its regulatory effect on E2/ROS/Nrf2 signaling pathway2.1 MTT assay:(1)Estrogen promoted the growth of MCF-10A and MCF-12A cells within the concentration range of 0.5-10 nM,peak at 5 nM.(2)XIAOPI could inhibit the growth-promoting effect of estrogen induced MCF-10A and MCF-12A cells.2.2 The results of the DCFH-DA fluorescent probe showed that E2 can induce the production of ROS in MCF-10A and MCF-12A cells.After XIAOPI co-treatment,it can reduce the ROS level;LDH,CAT,SOD level show that E2 can induce the increase of cell LDH leakage,increase cell damage,and the activity of oxidase(SOD、CAT)is obviously inhibited.After XIAOPI co-treatment,the LDH leakage of cells is decreased,and the activity of oxidase(SOD、CAT)is increased;the results of ELISA showed that E2 could induce the level of 8-OHdG to increase DNA damage.After XIAOPI co-treatment,the level of 8-OHdG decreased significantly,indicating that XIAOPI can regulate E2 induced oxidative stress and reduce oxidative damage.2.3 Cell apoptosis results:Compared with the control group,the proliferation of MCF-10A and MCF-12A cells showed a significant decrease in apoptosis under the stimulation of E2;compared with E2 group,XIAOPI could reverse the induction of E2,increase the apoptosis rate and restore the normal apoptosis level.2.4 Cell migration and cloning ability detected by cell migration experiment and plate cloning experiment:compared with the control group,E2 significantly enhanced the migration ability and cloning ability of MCF-10A and MCF-12A.Compared with the E2 group,the enhanced migration ability and cloning ability of E2 were significantly inhibited after the addition of XIAOPI.2.5 Western-blot and Real-Time-PCR:E2 could inhibit the expression of Nrf2 and HO-1 protein in MCF-10A and MCF-12A cells,compared with the control group,the difference was statistically significant(P<0.05);after XIAOPI co-treatment,the inhibition effect of E2 was blocked and the expression level of Nrf2 and HO-1 protein increased,compared with the E2 group,the difference was statistically significant(P<0.05).Under the stimulation of E2,the expression of Nrf2,HO-1,and NQO1 mRNA in MCF-10A and MCF-12A cells were significantly lower than those in the control group(P<0.05).After XIAOPI co-treatment,Nrf2,HO-1,and NQO1 inhibited by E2 could be activated,compared with E2 group,the difference of mRNA expression was statistically significant(P<0.05).Conclusion1.XIAOPI has a good therapeutic effect on preventing the occurrence of breast tumor in MMTV-PyMT mice,it has the effect of inhibiting the occurrence,growth and lung metastasis of breast cancer,which is manifested in the regulation of the internal environment of the body by reducing the level of E2,it is suggested that XIAOPI particles may prevent DNA oxidative damage by activating the E2/ROS/Nrf2 pathway and inhibiting oxidative stress,thus eliminating the accumulation of Ros and reducing the level of 8-OHdG,to prevent breast cancer.2.At the cellular level,XIAOPI can improve estrogen-induced oxidative stress and prevent DNA damage;inhibit the promotion of estrogen on breast epithelial cell proliferation,migration,and cloning ability;reverse the growth of breast epithelial cells stimulated by estrogen.Promote normal apoptosis of cells by activating the Nrf2 signaling pathway that is inhibited by estrogen,and inhibit the abnormal proliferation of cells.The mechanism may be that XIAOPI can inhibit the oxidative stress by activating the E2/ROS/Nrf2 pathway,to prevente the oxidative damage to DNA and thus prevent breast cell carcinogenesis.Comprehensivelly,this study have found that XIAOPI has a good regulatory effect on estrogen,has a good curative effect in reducing oxidative stress,eliminating harmful substances such as reactive oxygen species,preventing DNA oxidative damage.The molecular mechanism of its preventive effect related to the regulation of the E2/ROS/Nrf2 signaling pathway,which provides experimental basis for exploring the effective and safe methods to prevent breast cancer.