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ABSTRACTTrueperella pyogenesis an important bacterial pathogen of a wide range of domestic and wild animals. Autophagy plays a key role in eliminating T. pyogenesin a process that is dependent on mechanistic target of rapamycin (mTOR). The endoplasmic reticulum (ER) stress response also is critical for autophagy regulation. However, the relationship between ER stress and T. pyogenesis uncharacterized and the intracellular survival mechanisms of T. pyogeneshave not been investigated adequately. In this study, we show that T. pyogenesinvades goat endometrial epithelial cells (gEECs). Meanwhile, we observed that GRP78 was upregulated significantly, and that unfolded protein response (UPR) also were activated after infection. Additionally, treatment with activators and inhibitors of ER stress downregulated and upregulated, respectively, intracellular survival of T. pyogenes. Blocking the three arms of the UPR pathway separately enhanced T. pyogenessurvival and inflammatory reaction to different levels. We also show that LC3-labeled autophagosomes formed around the invading T. pyogenesand that autolysosome-like vesicles were visible in gEECs using transmission electron microscopy. Moreover, tunicamycin did not inhibit the intracellular survival of T. pyogenesunder conditions in which autophagy was blocked. Finally, severe challenge with T. pyogenesinduced host cell apoptosis which also may indicate a role for ER stress in the infection response. In summary, we demonstrate here that ER stress and UPR are novel modulators of autophagy that inhibit T. pyogenesintracellular survival in gEECs, which has the potential to be developed as an effective therapeutic target in T. pyogenesinfectious disease.

Milk fat is a main nutritional component of milk, and it has become one of the important traits of dairy cow breeding. Recently, there is increasing evidence that microRNAs (miRNA) play significant roles in the process of milk fat synthesis in the mammary gland. Primary bovine mammary epithelial cells (BMEC) were harvested from midlactation cows and cultured in DMEM/F-12 medium with 10% fetal bovine serum, 100 units/mL penicillin, 100 µg/mL streptomycin, 5 µg/mL bovine insulin, 1 µg/mL hydrocortisone, and 2 µg/mL bovine prolactin. We found that miR-34b mimic transfection in BMEC reduced the content of intracellular triacylglycerol (TAG) and lipid droplet accumulation via triacylglycerol assay and Oil Red O staining; meanwhile, overexpression of miR-34b inhibited mRNA expression of lipid metabolism-related genes such as peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FASN), fatty acid binding protein 4 (FABP4), and CCAAT enhancer binding protein alpha (C/EBPα). Whereas miR-34b inhibitor resulted in completely opposite results. Furthermore, q-PCR and western blot analysis revealed the mRNA and protein expression levels of DCP1A were downregulated in miR-34b mimic transfection group and upregulated in miR-34b inhibitor group. Moreover, luciferase reporter assays verified that DCP1A was the direct target of miR-34b and DCP1A gene silencing in BMEC-inhibited TAG accumulation and suppressed lipid droplet formation. In conclusion, these findings revealed a novel miR-34b-DCP1A axis that has a significant role in regulating milk fat synthesis and suggested that miR-34b may be used to improve the beneficial ingredients in milk.

In domestic ruminants, a receptive endometrium is crucial for successful pregnancy. Although many essential molecular modulators and pathways have been identified during early pregnancy, the precise mechanisms regulating goat endometrial function remains largely unknown. Here, we describe a novel regulator during early pregnancy, whereby hormones increased CREB3 regulatory factor (CREBRF) expression and act as a potential activator of autophagy in endometrial epithelial cells (EECs) via the mTOR pathway. Our results showed that knockdown of CREBRF via shCREBRF hampered EECs proliferation by S-phase cell cycle arrest and significantly inhibited endometrial function. We also reported that CREBRF-mTOR-autophagy pathway plays a vital role in regulating endometrial function, with a blockade of the mTOR by rapamycin demonstrating the regulatory function on prostaglandin (PGs) secretion and cell attachment in EECs. Moreover, chloroquine pretreatment also proved the above conclusion. Collectively, our findings provide new insight into the molecular mechanisms of goat endometrial function and indicate that the CREBRF-mTOR-autophagy pathway plays a central role in PGs secretion and cell attachment.The protein CREB3 regulatory factor (CREBRF) acts as a potential activator of autophagy in endometrial epithelial cells via the mTOR pathway.Knockdown of CREBRF via shCREBRF hampered EECs proliferation by S-phase cell cycle arrest and significantly inhibited endometrial function.The CREBRF-mTOR-autophagy pathway plays a vital role in regulating endometrial function, with a blockade of the mTOR by rapamycin demonstrating the regulatory function on prostaglandins (PGs) secretion and cell attachment in EECs.