Expression Characteristics Of Porcine Intestinal Antimicrobial Proteins And The Molecular Mechanism Of RegⅢγ

The intestinal tract of piglets produce a series of antimicrobial proteins（AMPs）with multiple functions,such as antibacteria,maintaining the homeostasis of intestinal microbiota,regulating immune function,and promoting the proliferation of intestinal epithelial cells.At present,many studies focus on the functions of specific AMPs and there is no systemic report on the identification and expression characteristics of intestinal antimicrobial proteins in pigs.RegⅢγis a novel multifunctional antimicrobial protein,which plays an important role in the innate immunity of the intestine.At present,the relevant research is mainly concentrated in humans,mice,rats,and has a certain understanding of the regulation and function of RegⅢγexpression and its molecular mechanism,but there are few reports of RegⅢγin pigs,especially on its function.Therefore,this study take piglets of different days of age as the research object,and preliminarily identified the intestinal AMP genes and expression characteristics of piglets.At the same time,cultured porcine intestinal epithelial cells（IPEC-J2）are used as research objects to explore the effects of recombinant porcine RegⅢγon cell proliferation,antioxidant and apoptosis.Methods and results are as follows:Real-time quantitative PCR is used to detect intestinal AMPs from birth to weaning and analyze the changes in protein expression.As a results,60 porcine intestinal antimicrobial proteins are identified,according to the relative expression level,it is divided into 16 high expressions,11 medium expressions,and 33 low expressions;Among them,RegⅢγis the highest expression in the small intestine of pigs and gradually increases with the time of development,but the expression level decreases after weaning,followed by a significant increase.In this experiment,prokaryotic recombinant expression and purification of porcine RegⅢγis performed,The effect of RegⅢγon IPEC-J2 cell viability in recombinant pigs is determined by MTT method,and the changes of cell proliferation-related genes are detected by q RT-PCR method.At the same time,H₂O₂ was selected to establish IPEC-J2 cell injury model.MTT method was used to screen out the optimal concentration and time of H₂O₂treatment.The cells were co-treated with the optimal concentration of H₂O₂ and RegⅢγto detect the changes in antioxidant capacity and expression levels of apoptosis-related factors.The test results show that:（1）Recombinant porcine RegⅢγcan promote the proliferation of IPEC-J2 cells at the optimal concentration of 40μg/m L,and can increase the m RNA expression levels of EGFR,ERK1 and ERK2,and inhibit the m RNA expression of AKT1.The m RNA expression levels of CDK2,CDK4,Cyclin E and Cyclin D are also increased.（2）H₂O₂ could significantly reduce the activity of IPEC-J2 cells and the optimal induction condition was 0.5 m M H₂O₂ concentration for 12h.When the cells were co-treated with recombinant pig RegⅢγ,the contents of ROS,MDA and LDH decreased,the activities of SOD and GPx enzyme increased,and the apoptosis rate decreased significantly.（3）The m RNA expression levels of apoptosis-related molecules caspase-3,Caspase-8,Caspase-9 and Bax are significantly decreased,the m RNA expression levels of anti-apoptotic molecules Bcl-2 are significantly increased,and the protein expression level of Caspase-8 is significantly decreased.In summary,we systematically identified 60 intestinal AMPs with different expression levels,and found that most of the AMPs with high expression levels were expressed and tended to be stable throughout the whole intestinal and developmental cycle.Among them,the high expression recombinant porcine RegⅢγcan promote the proliferation of IPEC-J2cells and has antioxidant damage and anti-apoptosis effects.