| Salpingitis is the one of most common and seasonal reproductive system diseases in sheep, and is caused by exogenous microorganisms infection, specifically bacterial infection. It has been known that lipopolysaccharide (LPS), the main constituent of bacteria, could induce the immune response.Beta defensin is a kind of antibacterial peptide and widely expressed in epithelial tissues of mammals and birds, which has important infection resisting function. Physiologically, beta defensins show broad-spectrum antibacterial, antiviral and anti-cell toxicity properties. The research of beta defensin has important theoretical significance for the interpretation of the mammalian immune regulation mechanism.In recent years, more and more studies show that beta defensin could be induced by certain microorganisms. However, it is still unknown whether lipopolysaccharide (LPS) released by the bacterium could regulate the expression of sheep beta defensin-1 (SBD-1) and the possible mechanisms involved.1. To improve the ovine oviduct epithelial cell culture system, we used 0.05% trypsin to digest isolated oviduct, and cultured the primary cells at different time based on their physiological property. The cultured cells were identified by the methods of morphology and immunofluorescence. The growth curve of cultured third generation of ovine oviduct epithelial cells was proved to be normal and efficient.2. QRT-PCR was performed to examine the expression of SBD-1 and SBD-2 in ovine oviduct epithelial cells. The results showed that only SBD-1 was highly expressed, while SBD-2 was not detectable in the ovine oviduct epithelial cells. We used immunohistochemistry method to examine the cellular localization of SBD-1 in ovine oviduct. The results showed that SBD-1 was specifically expressed in the epithelial cells but not in the stromal cells. We next treated the cultured ovine oviduct epithelial cells with LPS (10 ng/mL、50 ng/mL、100 ng/mL、200 ng/mL and 1 mg/mL) and harvested at different time (0h、1h、3h、6h、12 h and 24 h). The qRT-PCR analysis showed that LPS could significantly upregulate the expression of SBD-1 in a concentration-and time-dependent manner. Moreover, it was found that the expression of SBD-1 reached the maximum level when treated with 100 ng/mL LPS at 12 h.3. The oviduct epithelial cells were treated with LPS to investigate the function of MAPK signaling in the LPS induced SBD-1 mRNA expression. The western blot results showed that the expression of P-P38, P-ERK1/2 and P-JNK was increased at 20 min,10 min and 10 min respectively. Moreover, immunohistochemistry results showed that P38, JNK and ERK1/2 was mainly expressed in the epithelial cells, and slightly expressed in the stromal cells. We also found that the expression of SBD-1 induced by LPS was dramatically reduced in the ovine oviduct epithelial cells when the cells were pretreated with P38 MAPK inhibitors. Attenuation of JNK signaling (SP600125) did not affect LPS induced SBD-1 expression in the ovine oviduct epithelial cells, while attenuation of ERK signaling (PD98059) was slightly reduced as compared with the inhibition of P38 MAPK signaling4.PCR method was performed to examine the expression of TLR4. The results showe d that mRNA levels of TLR4 could be detected in ovine oviduct epithelial cells. We next used TLR4 specific antibody to neutralize the function of TLR4. QRT-PCR and Western Blot results showed that neutralization of TLR4 could markedly decrease the expression of p-P38 and SBD-1 induced by LPS.Our findings indicate that the regulatory effects of LPS on SBD-1 in the ovine oviduct epithelial cells are mainly through TLR4 receptor and P38 MAPK signaling. |
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