Effect of Pregnane X Receptor on CYP3A29 Expression in Porcine Alveolar Macrophages during Mycoplasma hyopneumoniae Infection.

Mycoplasma hyopneumoniae (M. hyopneumoniae, Mhp) is the causative agent of mycoplasma pneumonia of swine (MPS). M. hyopneumoniae infection causes inflammation in pigs and leads to considerable economic losses in the pig industry. Pregnane X receptor (PXR) is a pluripotent gene regulatory protein that plays an important role in regulating cytochrome P-450 (CYP) in pigs in the context of inflammatory responses, drug metabolism, homeostasis, etc. We previously reported that cytochrome P450 3A29 (CYP3A29) expression was significantly upregulated in pigs infected with M. hyopneumoniae compared with healthy control pigs. This experiment mainly focused on identifying the role of PXR in the regulation of CYP3A29 and inflammatory factors after M. hyopneumoniae infection by establishing pig alveolar macrophage (PAM) cells in which PXR was overexpressed or silenced. Our results showed that the overexpression of PXR could significantly improve the protein and the mRNA expression levels of CYP3A29 with and without M. hyopneumoniae infection in PAM cells. After the expression of PXR was inhibited, protein and mRNA expression levels of CYP3A29 were significantly reduced with and without M. hyopneumoniae infection in PAM cells. Moreover, PXR can regulate the mRNA expression levels of IL-6 and IL-8 during M. hyopneumoniae infection of PAM cells. In conclusion, these results suggest that PXR positively regulates CYP3A29 expression during the inflammatory response caused by M. hyopneumoniae infection.

Transcriptome sequencing analysis of porcine MDM response to FSL-1 stimulation.

Mycoplasma infection can cause many diseases in pigs, resulting in great economic losses in pork production. Innate immune responses are thought to play critical roles in the pathogenesis of mycoplasma disease. However, the molecular events involved in immune responses remain to be determined. Hence, the object of this study was to use RNA-Seq to investigate the gene expression profiles of the innate immune response mediated by FSL-1 in pig monocyte-derived macrophages (MDMs). The results revealed that 1442 genes were differentially expressed in the FSL-1 group compared with the control groups, of which 777 genes were upregulated and 665 genes were downregulated. KEGG pathway analysis showed that the upregulated genes were mainly involved in innate immune-related pathways including the TNF signaling pathway, cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway, Jak-STAT signaling pathway, chemokine signaling pathway, NOD-like receptor signaling pathway and NF-kappa B signaling pathway. The downregulated genes were only involved in the cGMP-PKG signaling pathway and glycerophospholipid metabolism. Our results showed that FSL-1 stimulation activated the TLR2 signaling pathway and resulted in diverse inflammatory responses. FSL-1 induced the transcription of numerous protein-coding genes involved in a complex network of innate immune-related pathways. We speculate that TNF, IL1B, IL6, NFKB1, NFKBIA, CXCL2, CXCL8, CXCL10, CCL2, CCL4 and CCL5 were the most likely hub genes that play important roles in the above pathways. This study identified the differentially expressed genes and their related signaling pathways, contributing to the comprehensive understanding of the mechanisms underlying host-pathogen interactions during mycoplasma infection and providing a reference model for further studies.

Investigation of Eph-ephrin A1 in the regulation of embryo implantation in sows.

Eph A1 and ephrin A1 (Eph-ephrin A1) is a key receptor-ligand pair of Eph-ephrin system, which plays important roles in the migration and adhesion of cells, tissue morphogenesis and vasculogenesis in mammals. In order to investigate the regulation of Eph-ephrin A1 during porcine embryo implantation, the expressions of mRNA and protein of Eph-ephrin A1 were detected in different reproductive tissues from twelve sows during embryo implantation period on pregnancy day 13, 18 and 24, respectively. Functions of Eph-ephrin A1 on the migration and adhesion of porcine endometrial epithelial cells were analysed by RNA interference (RNAi), transwell migration assays and MTT assays. Results showed that mRNA levels of Eph-ephrin A1 were highly expressed in endometrial attachment site when compared to other reproductive tissues (p < 0.05) and were peaked on pregnancy day 18 during embryo implantation (p < 0.05). Protein levels of Eph-ephrin A1 were highly expressed in endometrial attachment site and were peaked on pregnancy day 18 (p < 0.05). Eph-ephrin A1 proteins were located in endometrial luminal epithelium, stroma of attachment site and inter-attachment site during embryo implantation, and the protein levels were higher during implantation compared to pre-implantation or post-implantation. Furthermore, silencing ephrin A1 gene significantly reduced the migration and adhesion capacity of porcine endometrial epithelial cells. These findings suggest that the Eph-ephrin A1 protein likely targets endometrial attachment site to enhance the migration and adhesion of porcine endometrial epithelial cells around pregnancy day 18 during pregnancy in sows.

CYP1A1 mediates the suppression of major inflammatory cytokines in pulmonary alveolar macrophage (PAM) cell lines caused by Mycoplasma hyponeumoniae.

Mycoplasmal pneumonia is a lung infection disease caused by Mycoplasma hyopneumoniae in swine. We previously reported that Cytochrome P450 1A1 (CYP1A1) expression was significantly downregulated in pigs infected with M. hyopneumoniae compared to the healthy controls. In this study, pulmonary alveolar macrophage (PAM) cell lines with CYP1A1 overexpression or siRNA-mediated CYP1A1 silencing were used to explore the biological function and regulatory mechanism of CYP1A1 gene expression changed on the inflammatory response of pigs infected with M. hyopneumoniae. The results showed that the cells overexpressing CYP1A1 infected with M. hyopneumoniae led to a rapid increase in PPAR-γ expression, which resulted in decreasing the levels of several inflammatory cytokines including IL-1ß, IL-6, IL-8 and TNF-α. On the contrary, this effect was just opposite in CYP1A1-RNAi cells infected with M. hyopneumoniae. We suggest that CYP1A1 suppress the inflammatory response caused by M. hyopneumoniae infection, via PPAR-γ signaling pathway in pigs.

Long form leptin receptor and SNP effect on reproductive traits during embryo attachment in Suzhong sows.

To ascertain whether the long form leptin receptor (LEPR) affects the regulation of embryo attachment and whether there are LEPR Single Nucleotide Polymorphisms (SNPs) associated with reproductive traits in pigs, Real-time qPCR was used to detect relative abundance of LEPR mRNA pattern in different tissues of Suzhong sows during the embryo attachment period (pregnancy day 13, 18 and 24) to the uterus, and PCR-RFLP as well as PCR-sequencing were used to investigate the coding sequence for SNPs of LEPR in a population of 512 Suzhong sows. Real-time qPCR results indicated that LEPR mRNA was present in all 22 tissues of pigs with differences in relative abundance of the LEPR mRNA (P<0.05). Among these tissues, the greatest relative abundance occurred at the endometrial attachment site (P<0.01), followed by the hypothalamus and most reproductive tissues (P<0.05), and there was a lesser relative abundance of the LEPR mRNA in the pituitary. During different embryo attachment periods, LEPR mRNA was greatest on Day 18 (attachment; P<0.05), followed by Day 24 (post-attachment), and relative abundance was least on Day 13 (pre-attachment). The prevalence of the LEPR mRNA in pregnant sows was greater than in non-pregnant sows (P<0.05). At the c.2856C>T locus of LEPR, Chi-square test results demonstrated that allele and genotype frequencies were in Hardy-Weinberg disequilibrium at this locus, PCR-RFLP results revealed that Genotype TT was greater than Genotype CC (P<0.05) for reproductive traits of TNB (Total Number Born) and NBA (Number Born Alive), which suggested that T allele at c.2856C>T locus has advantageous effects on litter size and litter weight in Suzhong pigs. In conclusion, the expression of the LEPR gene might be involved in the regulation of embryo attachment mechanisms in pigs, and the LEPR SNP c.2856C>T could be a molecular marker for improving litter size and litter weight in pig breeding.

Tissue expression of EphB2 and RNA-seq analysis during embryo im-plantation in Meishan pigs.

Embryo implantation is a key step affecting swine litter size, which is an important economic and reproduction trait in pigs. In order to investigate the effect of erythropoietin-producing hepatocellular receptor B2 (EphB2) on endometrium migration and attachment during swine embryo implantation, the mRNA and protein expression levels of EphB2 in endometrium implantation sites, endometrium non-implantation sites and ovary were detected in Meishan sows during pre-implantation, mid-implantation and post-implantation period using real-time quantitative PCR and Western blot. Differential expression genes were also analyzed in endometrium implantation sites and ovary during different implantation periods by RNA sequencing (RNA-seq) technology. The qRT-PCR and Western blot results showed that EphB2 mRNA and protein expression curve was the same in endomtrium implantation sites and endometrium non-implantation sites during pre-implantation, mid-implantation and post-implantation period, with a first increase followed by a decrease, and its expression level during mid-implantation was significantly higher than pre-implantation and post-implantation (P<0.01). In contrast, EphB2 mRNA and protein expression curve in ovary during pre-implantation, mid-implantation and post-implantation period showed a first decrease followed by an increase, and the expression levels were significantly different among different implantation periods (P<0.05). RNA-seq results indicated that EphB2 mRNA expression during mid-implantation was higher than that of pre-implantation extremely significantly in endometrium implantation sites (P<0.01), and was significantly higher than that of post-implantation in ovary (P<0.05). By and large, EphB2 might play an important role in swine embryo implantation, and it's a potential candidate gene for litter size in pigs.

Roles of SIRT1 in granulosa cell apoptosis during the process of follicular atresia in porcine ovary.

Ovarian follicular atresia is characterized by granulosa cell apoptosis, however, the exact mechanism is still unclear. Sirtuin 1 (SIRT1) is a NAD(+)-dependent deacetylase, which is associated with apoptosis in several of cell types, but its exact role in ovarian granulosa cell apoptosis is not clearly defined. In present study, we identified the involvement of SIRT1 in the process of follicle degeneration, which is known as "follicular atresia", both from in vivo models and cell culture data. The results of immunohistochemistry showed that SIRT1 was widely detected in non-apoptotic granulosa cells of follicles, but significantly decreased during the process of granulosa cell apoptosis. Quantitative real-time PCR and Western blot analysis showed that the expression levels of SIRT1 mRNA and protein were increased (P<0.05) during follicular atresia. In order to provide more evidences elucidating the roles of SIRT1 during the process of follicular atresia, granulosa cells were cultured in vitro with resveratrol which acts as a potent activator of SIRT1. Results showed that resveratrol caused a dose-dependent increase in both SIRT1 mRNA and protein levels. Meanwhile, apoptotic rate of granulosa cell was increased (P<0.01) in a dose-dependent manner. Additionally, resveratrol significantly increased the expression levels of Caspase-3 (P<0.01) and Bax mRNA (P<0.01), while Bcl-2 mRNA level was significantly decreased (P<0.01). Thus, our results suggest that SIRT1 may play important roles in the regulation of granulosa cell apoptosis during follicular atresia in porcine ovary.

Breed-linked polymorphisms of porcine toll-like receptor 2 (TLR2) and TLR4 and the primary investigation on their relationship with prevention against Mycoplasma pneumoniae and bacterial LPS challenge.

Toll-like receptors (TLRs) play a crucial role in innate immunity, serving as pattern-recognition receptors and the first barrier in host defense against microbial infections. Genetic variations of TLR2 and TLR4 are closely associated with a variety of infectious diseases, particularly lung diseases. In this study, we detected six and four single nucleotide polymorphisms (SNPs) in the coding sequences of porcine TLR2 and TLR4 genes, respectively. Only SNP 1027C>A of TLR4 was shown to be markedly biased in Western and Oriental pig populations. Hence, the susceptibility of pigs with different genotype at position 1027C>A to Mycoplasma hyopneumoniae (Mhp) infection was investigated, and changes to the expression of TLR2, TLR4, TNF-α and IL-1ß were monitored. The results showed that there was no significant difference in susceptibility to Mhp infection between AA and CC individuals despite expression levels for all detected genes of the challenge groups being significantly higher than the corresponding control groups. Furthermore, porcine alveolar macrophages of different genotype were collected and stimulated by lipopolysaccharide. We found that the expression of TLR2, TLR4, TNF-α and IL-1ß genes were enhanced to different levels by lipopolysaccharide stimulation. TLR2 and TLR4 gene expressions and their rates of increase of 1027CC pigs were significantly higher than for 1027AC pigs (P < 0.01), while TNF-α and IL-1ß expressions were significantly lower than for 1027AC pigs (P < 0.01). We predict that allele C at position 1027 of the TLR4 gene contributes to the pig's immune response to gram-negative bacterial infections.

Effect of FTO Expression and Polymorphism on Fat Deposition in Suzhong Pigs.

Fat mass and obesity associated gene (FTO) plays an important role in appetite control and energy consumption in human and mice. In order to examine FTO expression influence on fat deposition in Suzhong pigs, FTO mRNA expression was detected in 16 tissues by RT-PCR, FTO protein expression was detected in 5 tissues by western blot, and association of FTO polymorphism with meat quality traits was analyzed in Suzhong populations with 714 records. RT-PCR results revealed that FTO mRNA was expressed in all sixteen tissues with significant differences (p<0.05), expression in backfat was significantly higher than that of any other tissue (p<0.05), and expression in longissimus dorsi muscle had the second highest significance level (p<0.05). Western blot results demonstrated that FTO protein was highly expressed in backfat and longissimus dorsi muscle. Furthermore, FTO mRNA and protein expression in tissues of high-fat pigs was significantly higher than that of low-fat pigs (p<0.05), suggesting FTO expression had advantageous effects on fat deposition. FTO polymorphism results evidenced that at A227G locus, G allele seemed to have advantageous effects on fat deposition, indicating it could be a significant candidate gene for improving pork quality in Suzhong pigs.

Cloning, sequencing and identification of single nucleotide polymorphisms of partial sequence on the porcine CACNA1S gene.

CACNA1S gene encodes the alpha1 subunit of the calcium channel. The mutation of CACNA1S gene can cause hypokalemic periodic paralysis (HypoKPP) and maliglant hyperthermia synarome (MHS) in human beings. Current research on CACNA1S was mainly in human being and model animal, but rarely in livestock and poultry. In this study, Yorkshire pigs (23), Pietrain pigs (30), Jinhua pigs (115) and the second generation (126) of crossbred of Jinhua and Pietrain were used. Primers were designed according to the sequence of human CACNA1S gene and PCR was carried out using pig genome DNA. PCR products were sequenced and compared with that of human, and then single nucleotide polymorphisms (SNPs) were investigated by PCR-SSCP, while PCR-RFLP tests were performed to validate the mutations. Results indicated: (1) the 5211 bp DNA fragments of porcine CACNA1S gene were acquired (GenBank accession number: DQ767693 ) and the identity of the exon region was 82.6% between human and pig; (2) fifty-seven mutations were found within the cloned sequences, among which 24 were in exon region; (3) the results of PCR-RFLP were in accordance with that of PCR-SSCP. According to the EST of porcine CACNA1S gene published in GenBank (Bx914582, Bx666997), 8 of the 11 SNPs identified in the present study were consistent with the base difference between two EST fragments.