Detection Of NMB And Its Receptor In Mice And Its Effect On Leydig Cells

Neuromedin B(NMB),a neuropeptide purified from pigs' spinal cord in 1983,is a member of the mammalian Bombesin(BN)-like peptide family.In mammals,NMB plays a variety of physiological functions by binding with its Neuromedin B receptor(NMBR).NMB and its receptor are widely expressed in the central nervous system and peripheral tissues and organs of mammals,participating in a variety of biological functions,such as stimulating smooth muscle contraction;regulating normal or tumor cells growth as a growth factor;regulating food intake and energy response;mediating stress,anxiety and other behavioral responses;regulating hormone secretion;regulating reproduction and so on.Since the discovery of NMB,domestic and foreign researchers systematically detected the expression of NMB and its receptor in human,rat,pig,rabbit,chicken and other animals.Although NMB and its receptor have been studied in mouse,they mainly have concentrated in individual organizations.The expression of NMB and its receptor in various tissues and organs of mouse was not systematically detected.Therefore,the present study systematically detected the expression of NMB and its receptor in different organs and tissues of mice by molecular biological methods,and established the mouse testicular interstitial cell model in vitro to detect the effects of NMB on the proliferation and apoptosis of Leydig cells,as well as the synthesis and secretion of testosterone.The purpose of this study was to clarify the expression of NMB and its receptor in different organs and tissues of mice and the role of NMB in mouse Leydig cells for providing experimental data in application of NMB in animal reproductive regulation and providing an idea in application of other neuropeptides in animals.1.Detection of the expression of NMB and its receptor in different organs and tissues of mouse and the distribution of NMB and its receptor in testisIn order to detect the expression of NMB and its receptor in different organs and tissues of mouse,in this part,the expression of NMB and its receptor gene in various tissues and organs of mouse was systematically detected by quantitative real-time PCR(qPCR),and the expression of NMBR protein in mouse was systematically detected by Western blot.The results showed that NMB and NMBR were widely expressed in mouse,and NMB mRNA was highly expressed in pituitary,testis and ovary.The expression levels of NMBR mRNA and NMBR protein were highest in testis.Then,by preparing paraffin sections of mouse testicular tissue,the distribution of NMB and its receptor in mouse testicular tissue was detected by immunohistochemistry(IHC)method and the cell localization of NMBR in mouse testicular tissue was determined by immunofluorescence(IF).The results showed that NMB immunopositive reaction was mainly distributed in spermatogonia and Leydig cells in spermatogenic tubules,and NMBR immunopositive reaction was mainly distributed in Leydig cells,and NMBR was mainly localized in Leydig cells.These results suggest that NMB/NMBR may play an important role in Leydig cells.2.The effects of NMB on the synthesis and secretion of testosterone,as well as the proliferation and apoptosis of Leydig cellsTo study the effects of NMB in mouse Leydig cells,Leydig cells cultured model in vitro was established and cells were treated with different concentrations of NMB(0,0.1,1,10,100 and 1000 nM)for 24 h.Firstly,CCK-8 was used to detect the effects of different concentrations of NMB on the cell activity of Leydig cells,and flow cytometry was used to detect the effects of NMB on cell apoptosis.Western blot was used to detect the effects of different concentrations of NMB on the expression of proliferation related proteins(PCNA and Cyclin B1)and apoptosis related proteins(Bax and Cleaved caspase-3).The results showed that compared with the control group(0 nM NMB),NMB had no significant effect on cell activity and expression of proliferation-related proteins PCNA and Cyclin B1 in mouse Leydig cells.However,10 and 100 nM NMB could significantly promote apoptosis,100 nM NMB had the most significant promotion effect on Bax protein expression,and 1 nM NMB had the most significant promotion effect on Cleaved caspase-3 protein expression.The results showed that NMB had little effect on the proliferation of mouse Leydig cells,but a certain dose of NMB could promote the apoptosis of mouse Leydig cells.Subsequently,the effects of different concentrations of NMB on testosterone secretion in Leydig cells were detected by ELISA,and the protein expressions of key enzymes for testosterone synthesis(StAR,CYP11A1 and 3?-HSD)were detected by Western blot.The results showed that compared with the control group 1 and 10 nM NMB treatment significantly increased the concentration of testosterone,and 1 nM NMB can significantly increase the expression level of StAR protein,and 100 nM NMB has the most significant promotion effect on CYP11A1 protein,and 10 nM NMB can significantly increase the expression level of 3?-HSD protein.The results showed that a certain dose of NMB could regulate the synthesis and secretion of testosterone in mouse Leydig cells.Finally,the effect of NMB on intracytoplasmic Ca2+concentration was detected by flow cytometry,and on the expression of G-protein-coupled receptor pathwayrelated proteins(NMBR,PKA and PKC)by Western blot.The results showed that compared with the control group,the intracytoplasmic Ca2+concentration was significantly increased after 1 nM NMB treatment,and extremely significantly increased after 10 nM NMB treatment,while other NMB treatments had little effect on the change of Ca2+ concentration.In addition to 1000 nM,other concentrations of NMB could significantly increased the expression of NMBR protein.The pPKC/PKC ratio was significantly increased by NMB treatment at 10 nM,but the pPKC/PKC and pPKA/PKA ratios were not significantly affected by NMB treatment at other concentrations.Therefore,the binding of NMB to its receptor may affect the function of mouse Leydig cells through PKC pathway.In conclusion,NMB and its receptor are widely expressed in mouse,and highly expressed in testis,and mainly distributed in Leydig cells.NMB can promote apoptosis of Leydig cells and regulate testosterone synthesis and secretion.It was found that NMB may play a role by binding to its receptor to activate Ca2+-PKC pathway.