| The mammary gland is the place where milk is synthesized and secreted,bovine mammary epithelial cells(bMECs)are the main cells for the synthesize and secrete milk.The proliferation and apoptosis of mammary epithelial cells in pregnancy lactation cycle are in different levels of dynamic balance.In this cycle,the rate of proliferation and apoptosis of mammary epithelial cells has great changes,this normal physiological change directly affects or determines the length of lactation period and the milk yield of bovine.Meanwhile,the milk yield of bovine is also affected by the number and activity of bMECs.Therefore,it is of great significance to study the mechanism of mammary gland epithelial cell proliferation and mammary gland development.Kisspeptin(Kp)is a kind of neuropeptide hormone encoded by KISS1 gene,it’s precursor can be cleaved into a bioactive peptide containing 54 amino acids,namely Kp-54.Further cleavage of Kp-54 resulted in the formation of small peptides such as Kp-10,the small peptides of Kp family can specifically bind to G protein-coupled receptor 54(GPR54).It was found that KISS1/GPR54 system was mainly involved in the regulation of cell proliferation,initiation of puberty,gonadal hormone secretion,reproductive activities and other important life activities.Thus,is it possible that Kp-10 may mediate GPR54 and downstream signal molecules to regulate the proliferation of mammary epithelial cells,and then regulate the development of mammary gland.At present,there is no relevant report at home and abroad.Therefore,in this experiment,firstly,the effect of Kp-10 on the proliferation of bMECs was studied,and then the specific mechanism of Kp-10 on the proliferation of bMECs was studied by adding Kp-10 inhibitors.In order to get closer to the environment in vivo,the results of cell experiments were further verified by the method of bovine mammary gland culture in vitro.After studying the effect of Kp-10 on the proliferation of bMECs,the effect of Kp-10 on the development of puberty mammary gland was studied in C57 female mice in vivo.Firstly,CCK-8 technique was used to detect the effect of Kp-10 of different concentrations on the proliferation of bMECs,the results showed that Kp-10 of 10 nM and 100 nM could significantly promote the proliferation of bMECs.In the subsequent experiments,Kp-10 of 100 nM was selected as the experimental concentration.Flow cytometry was used to detect cell cycle,RT-PCR to detect P21 gene expression,Western blot to detect Cyclin D1 and Cyclin D3 protein expression.The results showed that 100 nM Kp-10 could promote the transformation of bMECs from G1 to G2 and S phase,inhibit P21 gene expression and promote Cyclin D1 and Cyclin D3 protein expression.GPR54 is the receptor of Kp-10,Western blot was used to detect GPR54 protein expression,the results showed that Kp-10 promoted GPR54 protein expression.In order to further study the mechanism of Kp-10 promoting the proliferation of bMECs,the inhibitor of Kp-10,Peptide-234,was selected to study whether Kp-10 promoted the proliferation of bMECs by activating GPR54,the results showed that Peptide-234 could inhibit the proliferation of bMECs promoted by Kp-10.Further study showed that Kp-10 could activate the signal pathways of protein kinase B(AKT),mammalian target of rapamycin(mTOR),extracellular regulated protein kinase 1/2(ERK1/2)in bMECs.AKT,mTOR and ERK1/2 inhibitors can inhibit the proliferation of bMECs promoted by Kp-10,it is suggested that Kp-10 can promote the proliferation of bMECs by activating AKT,mTOR and ERK1/2 signaling pathways.Meanwhile,Kp-10 can activate Rb,promote protein expression of E2F1,reduce the complex formed by Rb and E2F1,and promote E2F1 target gene expression.In order to get closer to the environment in vivo,the cell experimental results were further verified by culturing the mammary gland of bovine,the results showed that Kp-10 could activate AKT,mTOR and ERK1/2 signal pathways in the mammary gland of bovine.In addition,Kp-10 could promote the protein expression of Cyclin D1,Cyclin D3 and proliferating cell nuclear antigen(PCNA)in the mammary gland.Inhibition of GPR54 inhibited the above effects of Kp-10.Then,AKT,mTOR and ERK1/2 inhibitors can inhibit the protein expression of Cyclin D1,Cyclin D3 and PCNA in mammary gland promoted by Kp-10.Finally,we injected 100 μL 6 μM of Kp-10 intraperitoneally into C57 female mice 30 d after birth,10 times in a row every 2 d.The experimental mice breaking the neck to death after 21 d,the mammary gland was taken for whole tissue staining,the results showed that Kp-10 could promote the formation of mammary duct branches.Western blot detected the expression of Cyclin D1,Cyclin D3 and PCNA protein,the results showed that Kp-10 could promote Cyclin D1,Cyclin D3 and PCNA protein expression in mice mammary gland.The results showed that Kp-10 could promote the mammary epithelial cells proliferation and the mammary gland development. |
