| Neuromedin B(NMB)is a member of the mammalian bombesin-like peptide family.It participates in the regulation of the synthesis and secretion of various hormones in the body by binding to its receptor(NMBR).Studies have shown that NMB and its receptors are expressed in the hypothalamus-hypophysis-target gland(thyroid,adrenal gland,testis and ovary),and were highly expressed in the hypophysis,and mainly exist in the hypophysis trophic hormone cells(thyrotropin cells,adrenocorticotropic hormone cells and gonadotropin cells),which can regulate the secretion of hypophysis trophic hormone.Previous study found that NMB and its receptors are also highly expressed in the hypophysis of mice,but the detailed study of NMB and its receptors in the hypophysis of mice is rarely reported.Therefore,the hypophysis of mice was selected as the research object.The expression and distribution of NMB and its receptors in mice hypophysis were systematically detected by molecular biology and morphology.The localization of NMBR in mice hypophysis trophic hormone cells was detected by immunofluorescence(IF).To establish an in vitro culture model of primary mice pituitary cells(MPC).The effects of different concentrations of NMB on the synthesis and secretion of MPC-related trophic hormones(ACTH,TSH,FSH and LH)were detected by qPCR,ELISA and Western Blot.The effects of different concentrations of NMB on the proliferation and apoptosis of MPC cells were further detected by CCK-8,flow cytometry and Western Blot.(1)Expression and distribution of NMB and its receptor in mice hypophysisIn order to detect the expression of NMB and NMBR in mice hypophysis,the expression of NMB and NMBR genes and proteins in mice hypophysis was systematically detected by qPCR and Western Blot.The results showed that the expression of NMB mRNA and protein in each part of mice hypophysis(adenohypophysis and neurohypophysis)was basically the same.NMBR mRNA and protein were mainly expressed in hypophysis.Subsequently,the distribution of NMB and NMBR in the mice hypophysis was further detected by IHC.The results showed that NMB was widely distributed in the hypophysis,both in the adenohypophysis and the neurohypophysis,while NMBR was mainly distributed in the adenohypophysis and concentrated in the distal chromatophil cells.(2)Distribution of NMBR in mice hypophysis trophic hormone cellsIn order to detect the distribution of NMBR in mice hypophysis trophic hormone cells,To detect the co-localization of NMBR and ACTH/TSHβ/FSHβ/LHβ protein in mice hypophysis by IF.The results showed that NMBR was co-expressed with ACTH/TSHβ/FSHβ/LHβ protein in cells,indicating that NMBR existed in thyrotropin cells,adrenocorticotropic hormone cells and gonadotropin cells.It provides a theoretical basis for the study of NMB/NMBR on the synthesis and secretion of hypophysis hormones.(3)Effect of NMB on hormone synthesis and secretion of MPCIn this study,we successfully established an in vitro culture model of primary mice adenohypophysis cells.To prove that NMBR protein was expressed in MPC cells by Western Blot,and it was found that NMB could promote the expression of NMBR in MPC.In order to detect the effect of NMB on the hormones of MPC,MPC was stimulated with different concentrations(0,0.1,1,10,100 and 1000 nM)of NMB for 24 h.The concentrations of TSH,ACTH,FSH and LH in the supernatant of cell culture medium were detected by ELISA.The results showed that 1,10 and 100 nM NMB inhibited TSH secretion(P<0.01 or P<0.05),0.1,1 and 10 nM NMB promoted ACTH secretion(P<0.01 or P<0.05),and NMB had little effect on LH.Except for 0.1 nM NMB,other concentrations of NMB could inhibit FSH secretion.The results of qPCR and Western Blot detection showed that the effects of a certain concentration of NMB on the mRNA and protein expression of TSHβ,POMC/ACTH,FSHβ and LHβ were similar to those of trophic hormone secretion.(4)Effects of NMB on proliferation and apoptosis of MPC cellsIn order to further detect the effect of NMB on the growth of MPC,the effects of different concentrations of NMB on the proliferation and apoptosis of MPC cells were detected by CCK-8,flow cytometry and Western Blot.The results showed that NMB had no significant effect on the number of MPC cells and the expression of proliferation-related proteins PCNA and Cyclin B1,but each concentration of NMB could inhibit apoptosis and inhibit the expression of apoptosis-related proteins Bax and Caspase-3.In summary,NMB and its receptors are widely expressed and distributed in the hypophysis of mice.NMBR exists in hypophysis hormone-stimulating cells.NMB can regulate the synthesis and secretion of hormones in hypophysis cells,affect cell proliferation and apoptosis,and provide experimental data for the development of NMB and NMBR related products and provide experimental data for applications in animal stress,reproduction,and growth. |
