| Masititis is one of the most common diseases in dairy cow industry,which has a high incidence in the perinatal period.The main cause of mastitis is bacterial infection.Nucleotide binding oligomerization domain?NOD?2 is a significant member in the NOD like receptor?NLR?family,which involves in immune defense of the host.17?-Estradiol?E2?and progesterone?P4?are reproductive hormones with immunomodulatory function.During the perinatal period,the high incidence of infectious diseases is associated with dramatic change of their concentration.In view of this,we will investigate the effects of E2 and P4 on the expression and function of NOD2 in bovine mammary epithelial cells?BMEC?.Experimental methods are as follows:?1?We used collagennase I digestion to culture BMEC.Pure BMEC were obtained by differential typsinzation and differential velocity adherent technique.In order to identify BMEC,we detected the expression of cytokeratin?CK?-18 by immunofluorescence and the expression of ?-casein?CSN2?by western bloting.Cells were transferred to 15 th passage and then resuscitated after cryopreservation to observe the growth status.?2?We divided the BMEC into three groups: E2 group,P4 group and muramyl dipeptide?MDP,the specific ligand of NOD2?group.In these three groups,BMEC were treated with different concentrations of E2,P4 and MDP,respectively.Then,the growth status of the cells were odserved.The interleukin?IL?8 m RNA expression of BMEC in E2 group and P4 group and the NOD2 m RNA expression in MDP group were detected by quantitative real-time polymerase chain reaction?q RT-PCR?.?3?The experimental groups were shown as follows: control group?medium only?,E2/P4 group?BMEC incubated with E2/P4?,MDP group?BMEC incubated with MDP?,E2/P4+MDP group?BMEC incubated with E2/P4 following with MDP?and MDP+E2/P4 group?BMEC incubated with MDP following with E2/P4?.BMEC were assessed for m RNA expression of NOD2 and its downstream molecules?receptor-interacting protein kinase?RIP/RIPK?2,IL-1?,IL-6,IL-8 and tumor necrosis factor?TNF???.We also detected the expression of NOD2 protein.Experimental results are as follows:?1?The purified cells had a typical cobblestone-like morphology.CK-18 expressedpositively and the CSN2 expression was stable.The BMEC of 15 th passage were recovered,andhad well adherent-grew.The cells can be used for subsequent experiments.?2?There were no effect of E2 and P4 on BMEC growth.The BMEC IL-8 m RNA expression was decreased with the E2 concentration increased.However,when the concentration of E2 reached 10-8 mol/L,the decreased trend was not obvious.After BMEC was treated with P4,the expression of IL-8 m RNA decreased.The IL-8 m RNA expression increased with the increase of P4.The increased trend was not obvious when the concentration of P4 reached 10-7 mol/L.The level of NOD2 m RNA expression in BMEC was positively correlated with the concentrations of MDP.When MDP concentration reached 15 ?g/m L,a small amount of BMEC damaged and detached from bottom.When MDP concentration reached 20 ?g/m L,a large amount of BMEC detached and floated from bottom.Therefore,the 10-8 mol/L E2,10-7 mol/L P4 and 10 ?g/m L MDP were used as the concentrations of the subsequent experiments.?3?NOD2 and its downstream molecules m RNA expression reduced in the E2/P4+MDP group and MDP+E2/P4 group when compared with MDP group?P < 0.01 or P < 0.05,with the exception of IL-6 m RNA expression in MDP+P4 group,P > 0.05?,but they were still higher than that in E2/P4 group and untreated control group?P < 0.01?.Compared with E2/P4+MDP group,NOD2 and its downstream molecules m RNA expression increased in MDP+E2/P4 group?P < 0.01 or P < 0.05?.There was a reduction in NOD2 protein in the E2+MDP group and MDP+E2 group when compared with MDP group?P < 0.01?.In conclusion,E2 and P4 have inhibitory effects on the NOD2 and its downstream molecules m RNA expression which are induced by MDP,E2 also can inhibit the expression of NOD2 protein.This may be one of the causes of the high incidence of mastitis in the perinatal period. |
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