Identification of a broadly neutralizing epitope within Gc protein of Akabane virus using newly prepared neutralizing monoclonal antibodies.

Akabane virus (AKAV) is characterized by abortion, stillbirth, premature birth, and congenital deformities in livestock and is widely distributed throughout Australia, Southeast Asia, East Asia, the Middle East, and Africa. Gc protein is the major neutralizing target of AKAV and is often considered as an immunogen to prepare neutralizing antibodies. In this study, we prepared and characterized three monoclonal antibodies (mAbs), 4D1, 4E6, and 4F12, against the Gc protein of AKAV (TJ2016 strain). Western blot (WB) and indirect immunofluorescence assay (IFA) analysis proved that the mAbs can react with both the truncated recombinant AKAV Gc protein and the natural Gc protein produced in the AKAV-infected cells. Further research demonstrated that these mAbs possess neutralizing activity. We next defined a neutralizing epitope 1134SVQSFDGKL1142 by screening a panel of overlapping peptides spanning the truncated Gc protein (aa991∼1232) using the generated neutralizing mAbs. Bioinformatic analysis shows that the neutralizing epitope is highly conserved across different genotypes of AKAV. The newly produced neutralizing mAbs and the identified neutralizing epitope in this study enrich the antigenic epitope information of the AKAV Gc protein and could have potential applications in the development of antigen and antibody detection systems that are specific to AKAV.

Kaempferol protects gut-vascular barrier from high glucose-induced disorder via NF-κB pathway.

Kaempferol is a natural edible flavonoid reported to treat high-fat diet-induced intestinal inflammation; however, the underlying molecular mechanisms remain unclear. This research aims to investigate the protective effect of kaempferol on the gut-vascular barrier (GVB) induced by high glucose and elucidate the underlying mechanism. Evans blue albumin efflux assay was used to test endothelial cell permeability. The results showed that kaempferol (50 µM) significantly reversed the high glucose-induced monolayer barrier permeability of rat intestinal microvascular endothelial cells (RIMVECs), while kaempferol significantly alleviated the high glucose-induced rarefication of the tight junction protein Claudin-5. Moreover, kaempferol also reduced high glucose-induced angiogenesis and cell migration via inhibiting the VEGFR2/p38 pathway. Kaempferol also protected against high glucose-induced overproduction of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 by inhibiting NF-κB p65 nuclear translocation. In addition, kaempferol had similar effects to the NF-κB inhibitor SN50 in reducing high glucose-induced ICAM-1 expression and endothelial barrier permeabilization. Our findings in part reveal the pathological mechanism of hyperglycemia-related gastrointestinal diseases and underlie the molecular mechanism of kaempferol in inhibiting bowel inflammation from a novel perspective.

Current Attitudes of Chinese Dairy Practitioners to Pain and Its Management in Intensively Raised Dairy Cattle.

Pain in dairy cattle is gaining attention globally. This study investigated the current attitudes of Chinese dairy practitioners to pain and its management in intensively raised dairy cattle. A total of 465 valid questionnaires with 26 painful conditions scored on numerical rating scales were collected from dairy practitioners. Data were analysed by descriptive statistics, analysis of variance, principal component analysis, and multivariate regression models. Dystocia was perceived as the most painful, while mild mastitis with milk changes only was perceived as the least painful. Respondents who agreed with the statement "pain management is worthwhile" tended to give a higher pain score. Young respondents (≤23 years old) and those from farms with ≤1000 cattle had lower pain scores for conditions with severe pain and low variability but higher pain scores for conditions with less severe pain and high variability, whereas highly educated respondents had consistently lower pain scores. As for pain management, older respondents (≥24 years old) tended to choose non-steroidal anti-inflammatory drugs, and farms with >1000 cattle were more likely to use analgesics. Training in pain perception and management should be emphasised with the hope of promoting animal welfare and reducing unnecessary production losses.

Kaempferol prevents angiogenesis of rat intestinal microvascular endothelial cells induced by LPS and TNF-α via inhibiting VEGF/Akt/p38 signaling pathways and maintaining gut-vascular barrier integrity.

Kaempferol is a major flavonoid found in natural plant extracts; it shows great potential in anti-inflammatory and anti-cancer medicine. However, the underlying mechanism of the protective action of kaempferol on the gut-vascular barrier (GVB) and the active sites preventing intestinal micro-angiogenesis has not been reported. The purpose of our study is to investigate the protective effect of kaempferol on the barrier damage induced by lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF-α), and its mechanism of protective action on intestinal micro-angiogenesis. Our data showed that the combination of LPS and TNF-α activates the inflammatory response of the rat intestinal microvascular endothelial cells (RIMVECs), leading to overexpression of vascular endothelial growth factors (VEGFs). Also, the permeability of GVB and transepithelial electrical resistance (TEER) constructed by Transwell and the tubular structure of RIMVEC were significantly affected. Kaempferol (25, 50, and 100 µM) decreased the inflammatory factor secretion and GVB permeability, down-regulated the expression of VEGFs, p-Akt, and hypoxia-inducible factor-1alpha (HIF-1α). It also alleviated the abnormal expression of tight junction proteins (TJs). Moreover, kaempferol may prevents intestinal angiogenesis in the presence of Akt inhibitor (MK-2206 2HCl) by regulating tube formation and downstream signaling of the VEGF/Akt pathways. In addition, the wound healing test showed that kaempferol had a similar effect in the presence of p38 inhibitor (SB203580), which intuitively restrained the migration of RIMVECs and reduced the p38 MAPK signaling. Our results demonstrated that kaempferol exhibits significant anti-inflammatory effects in LPS and TNF-α induced inflammatory environments. Kaempferol prevents intestinal angiogenesis by impeding the tube formation and migration of RIMVECs. It also suppresses the expression of angiogenesis-related signals, thereby protecting the GVB.

Esculetin inhibits the pyroptosis of microvascular endothelial cells through NF-κB /NLRP3 signaling pathway.

The effect of Esculetin on pyroptosis and its possible mechanism in endothelium were explored. 10 µg/mL LPS and 0.5 mM ATP were used to stimulate the rat intestinal microvascular endothelial cells. Then add different concentrations of Esculetin (20µM, 40 µM) to the culture medium containing LPS and ATP culturing for 24 h. The expression of p-NF-κB p65, NF-κB p65, I-κB, p-I-κB, NLRP3, ASC, caspase-1, and gasdermin-D were detected by Western blot, and the release level of IL-18 and IL-1ß were measured by ELISA. The NLRP3 inhibitor MCC950 was used at the concentration of 10 µM for 4 h to disentangle the potential mechanism of the influence of Esculetin on pyroptosis. In our experiments, the expression of gasdermin-d and important proteins of NF-κB and NLRP3 signaling pathways were inhibited by Esculetin. Besides, Esculetin also attenuated the morphological changes like swelling rupture and pores on the membrane caused by pyroptosis thereby protecting cells from being damaged by pyroptosis. Combining with the effect of Esculetin on proteins above and its protective effect on cell morphology, we believe that Esculetin has an anti-pyroptosis effect. The inhibiting pyroptosis effects mentioned above are similar to MCC950, which means the anti-pyroptosis effects of Esculetin are associated with the NLRP3 signaling pathway. In conclusion, Esculetin inhibits the pyroptosis of microvascular endothelial cells through the NF-κB/NLFP3 signaling pathway and is expected to be conducive in treating pyroptosis-related diseases.

Naringenin prevents TNF-α-induced gut-vascular barrier disruption associated with inhibiting the NF-κB-mediated MLCK/p-MLC and NLRP3 pathways.

The microvasculature endothelium accurately regulates the passage of molecules across the gut-vascular barrier (GVB), which plays an essential role in intestinal immunity. Naringenin is reported to have therapeutic potential against several intestinal disorders. However, the effect of naringenin on GVB disruption has been rarely studied. This study aims to investigate the effect of naringenin on GVB function and the potential mechanism. In the present study, the in vitro GVB disruption of rat intestinal microvascular endothelial cells (RIMVEC) was induced by 50 ng mL-1 of tumor necrosis factor-α (TNF-α). The integrity of the in vitro GVB was determined by Evans blue (EB)-albumin efflux assay and trans-endothelial electrical resistance (TER). Meanwhile, the expression of tight junction proteins and the related NF-κB, MLCK/p-MLC and NLRP3 pathways were determined using enzyme-linked immunosorbent assay (ELISA), real-time quantitative polymerase chain reaction (RT-qPCR), western blot analysis and immunofluorescence. The results show that naringenin (100 µM) inhibits TNF-α-induced interleukin (IL)-6 hypersecretion, alleviates GVB disruption and mitigates the change in the tight junction protein expression pattern. Naringenin inhibits the GVB-disruption-associated activation of the MLCK/p-MLC system and TLR4/NF-κB/NLRP3 pathways. Furthermore, naringenin shows a similar effect to that of NF-κB inhibitor Bay 11-7082 in reducing the TNF-α-induced activation of NLRP3, p-MLC and secondary GVB disruption. The results suggest that naringenin evidently alleviates TNF-α-induced in vitro GVB disruption via the maintenance of a tight junction protein pattern, partly with the inhibition of the NF-κB-mediated MLCK/p-MLC and NLRP3 pathway activation.

Coix Seed Diet Ameliorates Immune Function Disorders in Experimental Colitis Mice.

Coix seed is a functional food in the Chinese diet that possesses the ability to alleviate ulcerative colitis clinically. However, the underlying mechanisms remain ambiguous. In this study, we investigated the protective effect of the Coix seed diet on experimental colitis mice. The mice were randomly divided into four groups: control group, model group, Coix seed feed group, and positive control group. The maintenance feed of the mice was replaced with Coix seed feed 10 days before orally administering the mice 5% (w/v) dextran sulfate sodium drink. As a result, the Coix seed feed alleviated colitis symptoms, maintained the complete blood count at a normal level, reduced the pathological score, relieved inflammatory cytokine secretion, and alleviated oxidative stress. Network pharmacology analysis was used for further exploration of the targets of Coix seed feed. The results showed that T-cell regulation is one of the targets of Coix seed feed, and the analysis of the T-lymphocyte subset and innate immune cell distribution of the colon tissue supported the network pharmacology results. In conclusion, Coix seed, as a staple food, can alleviate experimental colitis, and the mechanism may be related to the immune regulation effect of Coix seeds.

Rhein from Rheum rhabarbarum Inhibits Hydrogen-Peroxide-Induced Oxidative Stress in Intestinal Epithelial Cells Partly through PI3K/Akt-Mediated Nrf2/HO-1 Pathways.

Rheum rhabarbarum has been widely used as a herbal medicine and food in China. The objective of this study was to investigate the cytoprotective action and underlying mechanisms of rhein, one active ingredient isolated from R. rhabarbarum, on H2O2-challenged rat small intestine epithelial cells (IEC-6 cells). H2O2-challenged IEC-6 cells were incubated in the pretreatment with or without rhein or LY294002, a PI3K/Akt inhibitor. The cell viability, apoptosis, intracellular reactive oxygen species (ROS), and antioxidants were measured. The expressions of heme oxygenase 1 (HO-1), nuclear factor erythroid 2-related factor (Nrf2), Akt, and p-Akt were evaluated by western blotting. Meanwhile, LY294002 was also used to investigate the role of PI3K/Akt in the rhein-induced cytoprotective role. The results showed that pretreatment of rhein could reverse the inhibition of cell viability and suppress the apoptosis, caspase-3 activity, and intracellular ROS induced by H2O2. Rhein also supported SOD activity catalase activity, glutathione S-transferase activity, and glutathione content. Furthermore, rhein induced the protein expression of HO-1 together with its upstream mediator Nrf2 and activated the phosphorylation of Akt in IEC-6 cells. LY294002 inhibited increased cell viability, upregulated the lowered apoptotic rate, and enhanced the weakened ROS levels. Although the inhibition of PI3K/Akt did not inhibit the Nrf2 nuclear level under 4 µM rhein, LY294002 inhibited the Nrf2 nuclear level under 2 µM rhein and blocked HO-1 expression. These data demonstrated that rhein protected IEC-6 cells against oxidative damage partly via PI3K/Akt and Nrf2/HO-1 pathways.