Study Of Effect Of Saccharomyces Cerevisiae Mannan On Expression Of ?-defensin-1 In Ruminal Epithelial Cells Of Sheep And Its Signaling Pathway

The rumen epithelium of sheep serves as an immune interface with the environment and secretes antimicrobial peptides with bactericidal function against various pathogens.Defensins,which are normally produced in the body,have a broad spectrum of antimicrobial activity.Sheep ?-defensin-1(SBD-1)is one of them and is widely expressed throughout the gastrointestinal tract.Therefore,SBD-1 may play an important role in the innate immunity of the gastrointestinal tract of sheep.Our previous research showed that Saccharomyces cerevisiae and its whole cell wall can induce the expression of SBD-1 in ovine rumen epithelial cells(ORECs).Mannan as a main component of the cell wall of Saccharomyces cerevisiae has the functions of stimulating innate and adaptive immune responses.Therefore,this experiment first studied the culture,cryopreservation and resuscitation methods of ORECs,and then the effect of Saccharomyces cerevisiae mannan on the expression of ORECs SBD-1 was studied.Finally,the signaling pathway activated by mannan-induced SBD-1 expression was explored.The specific research contents and results are as follows:(1)Digestion of rumen tissue by trypsin continuous digestion for 7 different times(40,30,20,10,10,8 and 5 min),and the rumen tissues and cells after each digestion were observed to determine the degree of digestion.It was found that the cells were collected after the fourth digestion and primary culture was performed.At the same time,the cultured cells were purified by differential digestion and differential adherence,and then the cultured cells were confirmed to be ORECs by immunohistochemistry,immunofluorescence,cell growth curve and RT-PCR.Then we found that the cryoprotection effect was better when the ratio of the frozen solution was VFBS:VDMEM:VDMSO=9:0:1 by detecting the viability of the cells after resuscitation and the adherence rate of 24 h.Finally,the biological activity of the resected ORECs was tested.The results showed that the cells after resuscitation also have the biological characteristics of primary ORECs,which can be used in subsequent experimental studies.(2)After the ORECs were stimulated with different concentrations(0,10,50,100,200 and 400 g/mL)of mannan for 8 h,the optimal concentration of mannan-induced SBD-1 expression was screened.Then the ORECs were stimulated with the optimal concentration of mannan for different time(0,2,4,8,12 and 24 h),the best time for mannan to induce SBD-1 expression was screened.Both qPCR and ELISA results showed that mannan could induce the expression of SBD-1 in ORECs,and the expression of SBD-1 was maximized when the concentration of 50 g/mL mannan stimulated ORECs for 4 h.(3)Western blot,RT-PCR,immunohistochemistry,immunofluorescence,qPCR and ELISA were used to study the signal pathway activated by mannan-induced SBD-1 expression.The results showed that the membrane receptor Dendritic-cell-associated C-type lectin 2(Dectin-2)and the signal adaptor protein Spleen tyrosine kinase(Syk)were expressed in ORECs,and the cells were treated with Dectin-2 and TLR-2 siRNA or specific blocking antibody attenuated mannan-induced SBD-1 expression,and the inhibitory effect of Dectin-2 on SBD-1 is more obvious.Subsequent treatment of cells with Syk siRNA or specific inhibitors was also found can reduce mannan-induced SBD-1 expression.Finally,treatment of cells with specific inhibitors of p38,JNK,ERK1/2 and NF-?B can significantly reduce the expression of SBD-1 induced by mannan,and the effect of p38 pathway inhibitor was the most obvious.This study successfully cultivated ORECs through the determination of digestion time.When the preparation ratio of the frozen solution is VFBS:VDMEM:VDMSO=9:0:1,the cryoprotection effect on the ORECs is better.And the cells after resuscitation also have the biological characteristics of primary ORESs.In addition,mannan can induce the expression of SBD-1 in ORECs,and the membrane receptors Dectin-2 and TLR-2,the signal adaptor protein Syk,and the downstream signaling pathways p38,JNK,ERK1/2 and NF-?B are involved in the expression of SBD-1 induced by mannan,but may be mainly mediated by the Dectin-2-Syk-p38 signaling pathway.