Sirtuin 5 alleviates apoptosis and autophagy stimulated by ammonium chloride in bovine mammary epithelial cells.

Ammonia (NH3) is an irritating and harmful gas that affects cell apoptosis and autophagy. Sirtuin 5 (SIRT5) has multiple enzymatic activities and regulates NH3-induced autophagy in tumor cells. In order to determine whether SIRT5 regulates NH3-induced bovine mammary epithelial cell apoptosis and autophagy, cells with SIRT5 overexpression or knockdown were generated and in addition, bovine mammary epithelial cells were treated with SIRT5 inhibitors. The results showed that SIRT5 overexpression reduced the content of NH3 and glutamate in cells by inhibiting glutaminase activity in glutamine metabolism, and reduced the ratio of ADP/ATP. The results in the SIRT5 knockdown and inhibitor groups were comparable, including increased content of NH3 and glutamate in cells by activating glutaminase activity, and an elevated ratio of ADP/ATP. It was further confirmed that SIRT5 inhibited the apoptosis and autophagy of bovine mammary epithelial cells through reverse transcription-quantitative PCR, western blot, flow cytometry with Annexin V FITC/PI staining and transmission electron microscopy. In addition, it was also found that the addition of LY294002 or Rapamycin inhibited the PI3K/Akt or mTOR kinase signal, decreasing the apoptosis and autophagy activities of bovine mammary epithelial cells induced by SIRT5-inhibited NH3. In summary, the PI3K/Akt/mTOR signal involved in NH3-induced cell autophagy and apoptosis relies on the regulation of SIRT5. This study provides a new theory for the use of NH3 to regulate bovine mammary epithelial cell apoptosis and autophagy, and provides guidance for improving the health and production performance of dairy cows.

The anti-apoptotic and anti-autophagic effects of EPO through PI3K/Akt/mTOR signaling pathway in MAC-T cells.

Lipopolysaccharide (LPS) is an important inflammatory and infected factor of bacterial mastitis, which treated bovine mammary epithelial cells (MAC-T) in our previous studies, as mastitis cells model in vitro. Erythropoietin (EPO) is a well-known hematopoietic hormone with antioxidative, anti-apoptotic, and anti-inflammatory roles. We hypothesized that EPO might regulate the apoptosis and autophagy to attenuate the inflammation of mastitis. Western blot, RT-PCR, transmission electron microscope analysis and Annexin V-FITC/PI were used to evaluate the regulation of EPO on apoptosis and autophagy in inflammatory MAC-T cells. These results demonstrated that EPO promoted the proliferation of MAC-T cells. Meanwhile, EPO had a better anti-inflammatory effect in MAC-T cells with LPS treatment. Certainly, EPO also showed anti-apoptotic and anti-autophagic effects. Interestingly, we found that the beneficial effect of EPO on inflammatory MAC-T cells depended on the PI3K/Akt/mTOR signaling pathway, which was involved in the regulation of apoptosis and autophagy. Generally, this study provides an insight for EPO to inhibit apoptosis and autophagy of inflammatory MAC-T cells via PI3K/Akt/mTOR signaling pathway.

Extracellular production of recombinant sus scrofa trefoil factor 3 by Brevibacillus choshinensis.

Trefoil factor 3 (TFF3) is involved in cell adhesion, motility and apoptosis, regulates mucosal immunity and maintains the functional integrity of intestinal epithelia. The upregulation of TFF3 expression in the weaning rat intestine attracted our interest. The present study hypothesized that TFF3 may serve a role in preventing diarrhea in weaning piglets, which is an important consideration in the pig farming industry. Previous recombinant TFF3 protein expression yields obtained from Escherichia coli were too low and the bioactivity of the protein was poor. Hence, this expression system was unsuitable for industrial applications. The present study explored the production of recombinant sus scrofa TFF3 in a Brevibacillus choshinensis (B. choshinensis) expression system, aiming to enhance the expression level of bioactive protein. To achieve this, the sus scrofa TFF3-encoding gene fragment was fused into an E. coli-Brevibacillus shuttle vector pNCMO2. High levels of TFF3 (30 mg/l) were produced and secreted into the B. choshinensis culture medium in soluble form with a molecular mass of 13.6 kDa and high immunoreactivity in western blotting. Thus, Brevibacillus may be used to produce useful mucosal factors for biochemical analyses and mucosal protection, and in industrial applications to produce novel inhibitors of diarrhea.

S100 calcium-binding protein A12 as a diagnostic index for subclinical mastitis in cows.

Diagnosis of subclinical mastitis is very important in management of the dairy industry and improvement of dairy cow productivity. S100A12, that is found in related tissues of mammals, is considered as an index for diagnosing inflammatory reaction. To evaluate whether S100A12 is involved in subclinical mastitis, milk somatic cell mRNA from 276 dairy cows was used to detect the transcriptional level of S100A12 by real-time quantitative polymerase chain reaction. A predictive analysis for mastitis was performed, and the correlation between S100A12 and other subclinical mastitis indicators was also assessed. The transcriptional levels of S100A12 in the milk of cows with mastitis were significantly higher than those in the milk of healthy cows (p < 0.05). The correlation analysis showed that S100A12 was positively associated with the somatic cell count and the sodium and chloride concentrations of milk. In contrast, a negative correlation was found between S100A12 and the potassium concentration and pH of milk. However, no significant correlation was detected between S100A12 and the other parameters, such as protein, lactose, ash, fat, density, Ca2+ and SNF. These results suggested that the S100A12 level in milk may serve as a diagnostic tool for subclinical mastitis in cows without obvious clinical signs.

GABA regulates the proliferation and apoptosis of MAC-T cells through the LPS-induced TLR4 signaling pathway.

Subacute ruminal acidosis (SARA) can cause rapid lipopolysaccharide (LPS) elevation and milk yield decline in lactating ruminants. LPS has been shown to promote apoptosis and reduce the proliferation of mammary epithelial cells. Previous studies have shown that γ- amino butyric acid (GABA) can enhance production performance, regulating ß-cell apoptosis and proliferation. Whether GABA can regulate apoptosis and proliferation induced by LPS in mammary epithelial cells is unknown. In this paper, we detected the role of GABA on proliferation and apoptosis as well as inflammation induced by LPS in bovine mammary epithelial cells (MAC-T cell line). In addition, we explored the role mechanism of GABA in LPS-induced MAC-T cells response through detecting the NFκB signaling pathway key molecules. The results suggested that GABA reduced the effects of cell apoptosis induced by LPS. Furthermore, GABA inhibited the expression of inflammatory cytokines activated by LPS. More importantly, blocking GABA receptors with its antagonist, GABA could not reduce the expression of inflammatory and pro-apoptotic factors activated by LPS. Notably, GABA significantly decreased the TLR4, NFκB p65, and MyD88 mRNA expression levels that were elevated by LPS. Our data indicated that GABA can improve cell viability and decrease apoptosis induced by LPS, while exerting an anti-inflammatory effect through the NFκB signaling pathway.

Characterization and anti-inflammation role of swine IFITM3 gene.

IFITM3 is involved in cell adhesion, apoptosis, immune, and antivirus activity. Furthermore, IFITM3 gene has been considered as a preferential marker for inflammatory diseases, and positive correlation to pathological grades. Therefore, we assumed that IFITM3 was regulated by different signal pathways. To better understand IFITM3 function in inflammatory response, we cloned swine IFITM3 gene, and detected IFITM3 distribution in tissues, as well as characterized this gene. Results indicated that the length of swine IFITM3 gene was 438 bp, encoding 145 amino acids. IFITM3 gene expression abundance was higher in spleen and lungs. Moreover, we next constructed the eukaryotic expression vector PBIFM3 and transfected into PK15 cells, finally obtained swine IFITM3 gene stable expression cell line. Meanwhile, we explored the effects of LPS on swine IFITM3 expression. Results showed that LPS increased IFITM3 mRNA abundance and exhibited time-dependent effect for LPS treatment. To further demonstrate the mechanism that IFITM3 regulated type I IFNs production, we also detected the important molecules expression of TLR4 signaling pathway. In transfected and non-transfected IFITM3 PK15 cells, LPS exacerbated the relative expression of TLR4-NFκB signaling molecules. However, the IFITM3 overexpression suppressed the inflammatory development of PK15 cells. In conclusion, these data indicated that the overexpression of swine IFITM3 could decrease the inflammatory response through TLR4 signaling pathway, and participate in type I interferon production. These findings may lead to an improved understanding of the biological function of IFITM3 in inflammation.

Swine IRF3/IRF7 attenuates inflammatory responses through TLR4 signaling pathway.

To explore the role of IRF3/IRF7 during inflammatory responses, we investigated the effects of swine IRF3/IRF7 on TLR4 signaling pathway and inflammatory factors expression in porcine kidney epithelial PK15 cell lines. We successfully constructed eukaryotic vectors PB-IRF3 and PB-IRF7, transfected these vectors into PK15 cells and observed GFP under a fluorescence microscope. In addition, RT-PCR was also used to detect transfection efficiency. We found that IRF3/IRF7 was efficiently overexpressed in PK15 cells. Moreover, we evaluated the effects of IRF3/IRF7 on the TLR4 signaling pathway and inflammatory factors by RT-PCR. Transfected cells were treated with lipopolysaccharide (LPS) alone, or in combination with a TBK1 inhibitor (LiCl). We revealed that IRF3/IRF7 enhanced IFNα production, and decreased IL-6 mRNA expression. Blocking the TBK1 pathway, inhibited the changes in IFNα, but not IL-6 mRNA. This illustrated that IRF3/IRF7 enhanced IFNα production through TLR4/TBK1 signaling pathway and played an anti-inflammatory role, while IRF3/IRF7 decreased IL-6 expression independent of the TBK1 pathway. Trends in MyD88, TRAF6, TBK1 and NFκB mRNA variation were similar in all treatments. LPS increased MyD88, TRAF6, TBK1 and NFκB mRNA abundance in PBR3/PBR7 and PBv cells, while LiCl blocked the LPS-mediated effects. The levels of these four factors in PBR3/PBR7 cells were higher than those in PBv. These results demonstrated that IRF3/IRF7 regulated the inflammatory response through the TLR4 signaling pathway. Overexpression of swine IRF3/IRF7 in PK15 cells induced type I interferons production, and attenuated inflammatory responses through TLR4 signaling pathway.

Comparation of the effects of different 5'-untranslated regions (UTRs) on gene expression in HEK293 cells.

OBJECTIVES: To evaluate four 5'-UTRs on GFP expression in HEK293T cells. RESULTS: The recombinant plasmids were constructed by restriction enzyme digestion, digestion and DNA sequencing. Quantitative real-time PCR and western blotting results showed that the transcription and translation level of PPRV-GFP mRNA was significantly lower than that of the other reporters. The transcription and translation level of ChEF1-GFP was the highest in HEK293T cells. CONCLUSIONS: Different UTRs can significantly affect protein expression. Additionally, the findings also will be useful in biological applications that require tuning of gene expression and system optimization.