Study On Biological Effects Of Quercetin On Boviene Mammary Epithelial Cells

This experiment compared antioxidation and gene expression levels with different does of estradiol, quercetin and both of these two, respectively, on bovine mammary epithelial cells. The further study forced on the protective function of quercetin in cells under H2O2-induced oxidative stress.This thesis includes three sections.1. Effects of estrogen on antioxidation and HSP70expression in bovine mammary epithelial cellsExperimental groups were divided into solvent control group (CK) and Ⅰ-Ⅴ experimental groups, which were treated with estradiol (E2) at0.5,1,5,10and100nmol/L respectively. After cultured for4h,8h,12h the viability of mammary epithelial cells was assessed by MTT assay and the activities of lactate dehydrogenase (LDH) in culture medium was assayed by a colorimetric method. After cultured forl2h the activities of superoxide dismutase (SOD) and malonaldehyde(MDA) in the cells also detected by a colorimetric method. HSP70mRNA expression were determined by real-time fluorescent quantitative PCR. The purpose was to obtain the does of estradiol adapting to bovine mammary epithelial cells in vitro. The result showed that:1. Compared with the control group, Ⅰ-Ⅲ E2-treated groups showed increased cell viabilities (P<0.05), Ⅳ-ⅤE2-treated groups showed exhibited cell viabilities(P<0.05) and whose LDH activity was significantly higher (P<0.01).2. Compared with the control group, HSP70mRNA level and SOD activity in Ⅰ-Ⅲ E2-treated groups were higher (P<0.05). HSP70mRNA level and SOD activity in V E2-treated groups were lower (P<0.05). Conclusion:Low concentration of E2could promote the proliferation of mammary epithelial cells, induced expression of HSP70mRNA, which might play antioxidant role by increasing intracellular SOD activity. High concentration of E2inhibited proliferation and antioxidation of mammary epithelial cells, caused cell damage with long-term stimulation.5nmol/L E2can be used as the optimal concentration to keep normal gowth of bovine mammary epithelial cells cultured in vitro. 2. Effects of Quercetin on antioxidation and HSP70,BcⅠ-2/Bax expression in bovine mammary epithelial cellsExperimental groups were divided into solvent control group (CK) and I-V experimental groups with Que at12.5,25,50,100and200μmol/L respectively. Then cells were cultured in4h,8h and12h for the test of proliferation. The12h cells and culture medium were used to determin the content of malonaldehyde(MDA) and the acivities of Lactate dehydrogenase (LDH) and superoxide dismutase (SOD), as well as HSP70and Bcl-2/Bax mRNA expression. The Result showed:the cell proliferation was promoted in dose and time dependent way in I-IIIQue-treated groups, besides up-regulation of SOD activity and Bcl-2/Bax RNA expression and down-regulation of MDA and LDH. However, the opposite results were shown in V Que-treated group (P<0.05). Conclusion:The treatment of Que at12.5-50μmol/L could promote the proliferation and improve the antioxidant and antiapoptosis function for the protection. The result in100-200umoi/L Que-treat group was opposite, which play anti-estrogenic function.3. Study on the estrogenicity and antioxidative stress function of quercetin on bovine mammary epithelial cellsExperimental groups were divided into solvent control group (CK) and Ⅰ～Ⅴ experimental groups with5nmol/L E2and Que at12.5,25,50,100and200μmol/L respectively. Then cells were cultured in4h,8h and12h for the test of proliferation. The12h cells and culture medium were used to determine the content of MDA and the acivities of Lactate dehydrogenase (LDH) and superoxide dismutase (SOD), as well as HSP70mRNA expression.H2O2damage group:the cells were incultrued with200μmol/L H2O2for4h followed by subsequent incubation with DMEM for12h. Que pre-protecion group:prior to incubation with200μmol/L H2O2for4h, then the cells were cultured with50μmol/L Que for12h. Que post-protection group:after incubation with200μmol/L H2O2for4h, then the cells were cultured with50μmol/L Que for12h. Then cells for the test of content of MDA and the acivities of Lactate dehydrogenase (LDH) and superoxide dismutase (SOD). The purpose was to further study protective roles of Que under oxidative stress and effects of Que on cells proliferation, anti-oxidation and HSP70mRNA expression in mammary epithelial cells. Then the antiestrogen does of the estogennicity of Quercetin was selected in two different situations. Comparison with the results of experiment two:Que protects mammary epithelial cells against oxidative stress-induced injury through improving SOD activity and decreasing lipid reaction and the preventive effect was stronger than repairing effect. When only Que was used or used together with E2, the antiestrogen concentration range was50～100μmol/L and25～50umol/L respectively, and the effect on promoting proliferation and increasing antioxidation was more significant when both E2and Que were used.