Antimicrobial innate immune molecules produced by the epithelial cells provide the host with a constitutive or immediately inducible defense mechanism against invading pathogens . Meanwhile, non-inflamed tubal and middle ear mucosa have been shown to contain relatively few immunocytes [15, 16]. Furthermore, because of the under-developed and premature adaptive immune system in young animals, the role of AIIMs in protecting against pathogens becomes important. This suggests that AIIMs protect the body during the neonatal and early postnatal periods when the adaptive immunity is not yet fully developed.
Among the AIIMs, defensins and cathelicidins are the dominant antimicrobial peptides found in neutrophils and epithelia as components of the early host defenses of mammals against infection, and these peptides have potent microbicidal activity against prokaryotic and eukaryotic pathogens as well as viruses . The expression of defensin in epithelial cells is mediated by certain hormones and is closely related to estrogenic hormone and progesterone . This suggests that E2 might be important in the modulation of ovine defensin expression in ovine oviduct epithelial cells. However, there is little information on how the E2 regulates the expression of defensins in ovine oviduct epithelial cells by E2.
We demonstrate that the gene of SBD-1 was expressed in ovine oviduct epithelial cells through the RT-PCR analysis and sequencing. In addition, using qPCR, there was a single peak in every dissociation curve (Figure 1A&B), and the amplification efficiencies of SBD-1 and β-actin were 0.996 and 0.990. Meanwhile, both slopes were −3.3. Based on this we also demonstrated that E2 could up-regulate the expression of SBD-1, especially when treated with 10-8 M E2 (Figure 1C&D). The mechanism by which E2 induces or activates the expression of SBD-1 is not precisely understood, but our data showed that the expression of SBD-1 still occurs in ovine oviduct epithelial cells left untreated. Therefore, E2 may be not necessary for the expression of SBD-1 but nay rather play a role as a promoting factor. Moreover, after stimulation with E2 at 10-8 M, the effect continued for at least 24 h (Figure 1C&D) and peaked at 3.5 h, while, the highest expression level had been sustained for 4 h from 2.5 to 6.5 h (Figure 1E). This data suggests that the expression was activated and reached it’s highest level simultaneously, but only for a short time, after which time it continued as a reduced-level, which was still much higher compared with the non-treated control. This shows that there might be a fast pathway, which could be activated quickly, in the expression of SBD-1 by E2.
Previous studies have suggested that the putative mechanisms of estrogen action have two mechanisms: a Non Classical Genomic Mechanism and a Classical Genomic Mechanism. Meanwhile, GPR30 and ERs might play important roles in the two mechanisms . Based on these results, we investigated the role and identity of GPR30 and ERs in the modulation of SBD-1 expression by E2 in ovine oviduct epithelial cells. We noted that the expression increased after addition of E2 and G1 from 2.5 to 5.5 h and with no change in the E2 only treatment from 6.5 to 7.5 h (Figure 2A&C). Meanwhile, after addition of E2 and ICI 182,780, the expression diminished quickly from 5.5 to 7.5 h (Figure 2B), however, the expression increased from 2.5 to 3.5 h (Figure 2D). This is consistent with the fact that ICI 182,780 is also an agonist at the membrane estrogen receptor GPR30 . These results suggest that E2-induced up-regulation of SBD-1 mRNA expression was dependent on GPR30 and ERs in ovine oviduct epithelial cells at different times. GPR30 plays an important role in Non Classical Genomic Mechanism, activating rapid pathways, while ERs acts with a Classical Genomic Mechanism controlling gene transcription.
In the Non Classical Genomic Mechanism, the cAMP/PKA pathways , PKC pathway  and NF-κB pathway [22, 23] play important roles in the putative mechanisms of estrogen action . Our data showed that there was almost no difference between the untreated and treated cells, which were added with the inhibitors of H-89, H-7, and PDTC either individually or in combination for 3.5 h (Figure 3). These results suggest that the three pathways were indispensable for the expression of SBD-1 by E2 in ovine oviduct epithelial cells. Furthermore, we also conclude that the three pathways should be activated simultaneously, or otherwise there would be no effect on the E2-induced up-regulation of SBD-1 expression.