Aflatoxin M1 (AFM1) and ochratoxin A (OTA) are mycotoxins commonly found in milk; however, their effects on intestinal epithelial cells have not been reported. In the present study, we show that AFM1 (0.12 and 12 μM) and OTA (0.2 and 20 μM) individually or collectively increased the paracellular flux of lucifer yellow and fluorescein isothiocyanate (FITC)-dextrans (4 and 40 kDa) and decreased transepithelial electrical resistance values in differentiated Caco-2 cells after 48 h of exposure, indicating increased epithelial permeability. Immunoblotting and immunofluorescent analysis revealed that AFM1, OTA, and their combination decreased the expression levels of tight junction (TJ) proteins and disrupted their structures, namely, claudin-3, claudin-4, occludin, and zonula occludens-1 (ZO-1), and p44/42 mitogen-activated protein kinase (MAPK) partially involved in the mycotoxins-induced disruption of intestinal barrier. The effects of a combination of AFM1 and OTA on intestinal barrier function were more significant (p < 0.05) than those of AFM1 and OTA alone, yielding additive or synergistic effects. The additive or synergistic effects of AFM1 and OTA on intestinal barrier function might affect human health, especially in children, and toxin risks should be considered.

Cardiovascular disease (CVD) is a significant cause of death worldwide. Because of its major individual differences in genetic background, pathogenesis, and disease progression pattern, the mortality risk rate remains high following conventional Western medicine diagnosis under current guidelines. Traditional Chinese medicine (TCM) has important multi-target, multi-pathway, and multi-layer benefits that can effectively address western medicine deficiencies. It was therefore commonly used in CVD diagnosis. Oxidative stress is also one of the main factors of CVD. Likewise, this main reaction regulator is the nuclear factor erythroid-2-related (Nrf2) factor. When activated, it can be transferred to the nucleus and initiated in the downstream pathway, thus playing an anti-oxidant stress role. As one of the most crucial endogenous protection systems in the body, Nrf2-related / heme oxygenase 1 (Nrf2/HO-1) signaling pathway is Nrf2's most classic approach to playing roles. Recently, various advances have been made to research and explain TCM by manipulating this pathway to treat CVD using modern molecular biology and other approaches. This analysis summarized the relationship between Nrf2/HO-1 signaling route, CVD and TCM. Further, Autodock calculation was also conducted to determine the binding amino acid on this TCM to Nrf2 and HO-1.

Suppressor of cytokine signaling 3 (SOCS3) is a cytokine-induced negative feedback-loop regulator of cytokine signaling. More and more evidence has proved it to be an inhibitor of signal transducers and activators of transcription 5 (STAT5). Here, we used dairy cow mammary epithelial cells (DCMECs) to analyze the function of SOCS3 and the interaction between SOCS3 and STAT5a. The expression of SOCS3 was found in cytoplasm and nucleus of DCMECs by fluorescent immunostaining. Overexpression and inhibition of SOCS3 brought a remarkable milk protein synthesis change through the regulation of JAK2/STAT5a pathway activity, and SOCS3 expression also decreased SREBP-1c expression and fatty acid synthesis. Inhibited STAT5a activation correlated with reduced SOCS3 expression, which indicated that SOCS3 gene might be one of the targets of STAT5a activation, DCMECs treated with L-methionine (Met) resulted in a decrease of SOCS3 expression. SOCS3 could also decrease cell proliferation and viability by CASY-TT detection. Together, our findings indicate that SOCS3 acts as an inhibitor of JAK2/STAT5a pathway and disturbs fatty acid synthesis by decreasing SREBP-1c expression, which validates its involvement in both milk protein synthesis and fat synthesis. In aggregate, these results reveal that low SOCS3 expression is required for milk synthesis and proliferation of DCMECs in vitro.

Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase, whose activity is inhibited by AKT phosphorylation. This inhibitory phosphorylation of GSK3β can in turn play a regulatory role through phosphorylation of several proteins (such as mTOR, elF2B) to promote protein synthesis. mTOR is a key regulator in protein synthesis and cell proliferation, and recent studies have shown that both GSK3β and mTORC1 can regulate SREBP1 to promote fat synthesis. Thus far, however, the cross talk between GSK3β and the mTOR pathway in the regulation of milk synthesis and associated cell proliferation is not well understood. In this study the interrelationship between GSK3β and the mTOR/S6K1 signaling pathway leading to milk synthesis and proliferation of dairy cow mammary epithelial cells (DCMECs) was analyzed using techniques including GSK3β overexpression by transfection, GSK3β inhibition, mTOR inhibition and methionine stimulation. The analyses revealed that GSK3β represses the mTOR/S6K1 pathway leading to milk synthesis and cell proliferation of DCMECs, whereas GSK3β phosphorylation enhances this pathway. Conversely, the activated mTOR/S6K1 signaling pathway downregulates GSK3β expression but enhances GSK3β phosphorylation to increase milk synthesis and cell proliferation, whereas inhibition of mTOR leads to upregulation of GSK3β and repression of GSK3β phosphorylation, which in turn decreases milk synthesis, and cell proliferation. These findings indicate that GSK3β and phosphorylated GSK3β regulate milk synthesis and proliferation of DCMECs via the mTOR/S6K1 signaling pathway. These findings provide new insight into the mechanisms of milk synthesis.

Tudor staphylococcal nuclease (Tudor-SN) is a highly conserved and ubiquitously expressed multifunctional protein, related to multiple and diverse cell type- and species-specific cellular processes. Studies have shown that Tudor-SN is mainly expressed in secretory cells, however knowledge of its role is limited. In our previous work, we found that the protein level of Tudor-SN was upregulated in the nucleus of bovine mammary epithelial cells (BMEC). In this study, we assessed the role of Tudor-SN in milk synthesis and cell proliferation of BMEC. We exploited gene overexpression and silencing methods, and found that Tudor-SN positively regulates milk synthesis and proliferation via Stat5a activation. Both amino acids (methionine) and estrogen triggered NFκB1 to bind to the gene promoters of Tudor-SN and Stat5a, and this enhanced the protein level and nuclear localization of Tudor-SN and p-Stat5a. Taken together, these results suggest the key role of Tudor-SN in the transcriptional regulation of milk synthesis and proliferation of BMEC under the stimulation of amino acids and hormones.

Streptococcus agalactiae (GBS) is a major pathogen causing mastitis in dairy cows while causing oxidative stress. Matrine is an alkaloid compound extracted from the roots of Sophora flavescens, a plant used in traditional Chinese medicine. It possesses antioxidant, immunomodulatory, anti-inflammatory, and pro-apoptotic properties. The aim of this study was to investigate the regulatory effects of matrine on the virulence of the ATCC strain (ATCC13813) and clinical GBS strains by transcriptome analysis and qRT-PCR validation. The results showed that the ABC transporter, peptidoglycan biosynthesis, and quorum-sensing pathways were significantly altered in ATCC (4 mg/mL) and GBS (12 mg/mL) strains after matrine treatment at MIC concentrations. Additionally, genes related to invasion and immune escape, including CylE, CAMP, ScpB, and CpsA, and genes related to the expression of adhesion and virulence factors, such as Bac, Lmb, PI2a, and PI2b, were significantly downregulated (p < 0.05). Overall, these data suggest that matrine effectively inhibits the virulence genes of GBS, thereby reducing immune evasion and infection by decreasing the synthesis of capsular polysaccharides and host cell adhesion.