The Intervention And Biological Mechanism Of Vitamin D3 On Experimental Hyperplasia Of Mammary Glands In Rats

Objective Breast hyperplasia of mammary glands（HMG）is a common disease of women in reproductive age and is also one risk factor for breast cancer.However,the study on the treatment and biological mechanism of HMG remains unclear.In order to gain a new insight in HMG,in this study,we used vitamin D3 to interfere with HMG model rats,and to explore the intervention effect and biological mechanism of VD3 on HMG.Material and Method（1）10 SD female rats were grouped in blank control group（Ctrl,n=5）and hyperplasia model group（Hyper,n=5）randomly,while the Hyper group was injected estradiol benzoate（E2）0.5 mg/kg for 25 days,following by injecting progesterone（P）4 mg/kg for 5 days.The body weights,heights and diameters of nipples,and the thickness of the mammary glands of rats were measured.The serum estradiol2（E2）,progesterone（P）and the prolactin（PRL）were detected by Enzyme-Linked Immunosorbent Assay（ELISA）.（2）Collect the ipsilateral second pair of mammary glands tissues,one part was fixed by 4%paraformaldehyde（PMA）for pathology analysis,the other part was detected the expression of estrogen receptorα（ERα）and vitamin D3receptor（VDR）by Western blot.（3）42 SD female rats were divided into 6 groups randomly,named the blank control group（Ctrl,n=5）,hyperplasia model group（Hyper,n=5）,negative control group（Negative,n=5）and VD3 groups（VD3,n=27）（n=9 in low,medium,and high dose groups）.HMG was established in all groups except the Ctrl group,and from the 10^th day of modeling,the Negative group was injected with 0.9%Na Cl and VD3 was given to the VD3 groups at the same time.After 30-day administration,the body weights,heights,diameters of nipples,and the thickness of the mammary glands of rats were measured.（4）The serum of the rats in each group was collected,the content of FSH,LH,E2,P,T,PRL and VD3 were detected by ELISA.（5）Collect the ipsilateral second pair of mammary glands tissues of rats,the slicemorphological characteristics were analyzed by HE stain,and the expression of ERα、PR、EGFR、PCNA、VDR were detected by IHC stain.（6）Similarly,the total protein was harvested from the second pair of mammary glands of rats,then the expression of ERα,PR,EGFR,PCNA,VDR were measured by Western blot.Results（1）Compared with those in the Ctrl group,the height and diameter of nipples in the Hyper group which intraperitoneal injection with E2 25 days and P for 5 days were increased（P<0.05）,the height of nipples increased from 1.40±0.10mm to 2.47±0.42 mm,and the diameter increased from 1.06±0.17mm to 1.90±0.17mm;and the thickness of mammary gland increased from 1.85±0.20mm to 2.24±0.40mm;（2）The ELISA results showed that serum E2,T and VD3 were significantly increased in the Hyper group,however,the P,FSH,PRL or LH remained no significant change between the two groups.（3）The results of HE stain showed that the volume of mammary glands and the number of lobules were increased in the Hyper group,as well as,the lobular ducts expanded and the ductal epithelium thickened,moreover,the epithelial cells were arranged disorderly compared with the Ctrl group,showing typical characteristics of HMG.（4）The IHC stain showed that the expressions of ERα,VDR,PR,EGFR and PCNA were significant upregulated in the Hyper group,and the expressions of ERα,VDR,PR,EGFR and PCNA in the mammary glands tissues measured by Western blot showed the same results as IHC stain.（5）Compared with the Hyper group,the three VD3 groups displayed much decrease of the thickness of the mammary glands tissues and of the height and diameter of nipples,while the MVD showed even better.（6）The serum E2,T,LH and VD3 were markedly decreased in all VD3 groups（P<0.05）,E2 and LH displayed lower in MVD group than other two groups,while the FSH,PRL and P seemed no significance between the Hyper group and VD3 groups.（7）The IHC results showed that the expressions of ERα,PR,EGFR and PCNA were decreased in three VD3 groups,however,the VDR was increased in the three VD3 groups.（8）Western Blot demonstrated that both ERαand PR were reduced in mammary glands tissues in the LVD,MVD and HVD groups,while significantly enhanced the VDR level.Moreover,the expressions of EGFR and PCNA were obviously downregulated in the three VD3 groups.Conclusion（1）Intraperitoneal injection of 0.5 mg/kg/d E2 25 days,combined with4mg/kg/d P 5 days in female rats can effectively increase the mammary gland thickness,nipple diameter,and nipple height,as well as increase the serum E2,T,and VD3,showing typical mammary gland tissue hyperplasia characteristics.（2）The expressions of ERα,VDR,PR,EGFR,and PCNA significantly increased in HMG rats.（3）VD3 could markedly decreased the mammary gland thickness,nipple diameter,and nipple height of rats,with the decrease of serum E2,T,and LH.（4）The expressions of ERα,PR,EGFR and PCNA were decreased,accompanied by the increase of VDR under the VD3intervention.It is suggested that VD3 could be used in HMG treatment.