The Expression Of ?-defensin Gene 1 Stimulated By E₂ In Oviduct Epithelial Cells And Analysis Of Transcriptome Sequencing

Defensins are short peptides widely existing in the biological world that can kill microorganisms such as bacteria,fungi or viruses and have anti-tumor activity.They are important components of the innate immune system.Studies have shown that changes in estrogens in the blood of sheep can cause changes in the expression level of SBD-1 in the reproductive organs and are positively correlated.The study of the regulation of SBD-1expression in sheep oviduct epithelial cells by in vitro estrogens is of great significance for studying the relationship between estrogen and mucosal immunity.In this experiment,the effect of different concentrations of 17-?estradiol on the expression of oviduct epithelial SBD-1 was determined by RT-qPCR technique,and the appropriate additive amount and time were determined.The experimental results show that 10^-8mol/L is the best concentration of 17-?estradiol stimulation,3.5h is the highest expression time point,compared with the control group 0h significantly higher?P<0.05?,after 3.5h With the increase of time,the expression of SBD-1 gradually decreased,but it was higher than that of the control group.The analysis of transcriptome sequencing of the epithelial cells incubated with17-?estradiol was performed.The results showed that there were a total of 619 genes that were enriched in GO-Term and were significantly enriched in biological processes.There are oxidation-reduction process,toxin metabolic process,protein localization to kinetochore,cellular response to organic cyclic compound,inner ear development,etc.;genes that are significantly enriched in cellular components include extracellular vesicular exosome,extracellular space,and integral component of plasma membrane,cell surface,extracellular regions,etc.Genes that are significantly enriched for molecular function include protein binding,calcium ion binding,microtubule binding,oxidoreductase activity,and RAGE receptor binding.The KEGG enrichment analysis was performed on all the differential genes.The pik3r3,Akt and mapk3 genes related to SBD-1 expression were selected for validation.The expression levels of the three genes were gradually increased by RT-qPCR method and Western blotting method.The highest expression level of pik3r3 was found after the addition of 10^-8mol/L E₂ to the cells.At1.5h,the expression level at 3h also increased significantly.The highest expression level of Akt appeared at 2.5h,and the expression level at 3h also increased significantly.The highest expression level of mapk3 appeared at 2.5h,and the expression level at 3h had a significant increase.The RT-qPCR method was used to detect the expression of SBD-1 in pre-incubated PI3K-Akt,a key protein inhibitor of MAPK signaling pathway and then added E₂different time intervals,which verified the need for 17-?estradiol-promoted SBD-1expression.The involvement of PI3K-Akt and MAPK pathways.Conclusion:17-?estradiol can promote sheep oviduct epithelial cell proliferation and induce SBD-1 expression.When the concentration of 17-?estradiol was 10^-8mol/L,the expression of SBD-1 was the highest at 3.5h in vitro.17-?estradiol could induce the expression of pik3r3,Akt and mapk3 in sheep oviduct epithelial cells,and the expression level was.The highest points were 1.5h,2.5h and 2.5h,respectively;the non-genomic pathway of 17-?estradiol-promoted SBD-1 expression was via the PI3K-Akt and MAPK signaling pathways.