Explore the vast range of titles available.

In bovine mammary epithelial cells (BMECs), a cascade of inflammatory reactions induced by lipopolysaccharide (LPS) has been shown to result in cell injury and apoptosis. The present study aims to reveal the protective effect of ferulic acid (FA) on LPS-induced BMEC apoptosis and explore its potential molecular mechanisms. First, we showed that FA had low cytotoxicity to BMECs and significantly decreased cell apoptosis and the proinflammatory response induced by LPS. Next, FA blocked LPS-induced oxidative stress by restoring the balance of the redox state and inhibiting mitochondrial dysfunction, the main contributor to LPS-induced apoptosis and ROS generation. Furthermore, the relief of inflammation and redox disturbance in the FA preconditioning group were accompanied by weaker NF-κB activation, enhanced Nrf2 activation and maintained cell viability compared to the LPS group. When BMECs were treated with FA alone, we observed that Nrf2 activation was induced before the inhibition of NF-κB activation and that the Keap1–Nrf2 relationship was disturbed. We concluded that FA prevented LPS-induced BMEC apoptosis by reversing the dominant relationship between NF-κB and Nrf2.

Iron accumulates progressively in the brain with age, and iron-induced oxidative stress has been considered as one of the initial causes for Alzheimer’s disease (AD) and Parkinson’s disease (PD). Based on the role of hepcidin in peripheral organs and its expression in the brain, we hypothesized that this peptide has a role to reduce iron in the brain and hence has the potential to prevent or delay brain iron accumulation in iron-associated neurodegenerative disorders. Here, we investigated the effects of hepcidin expression adenovirus (ad-hepcidin) and hepcidin peptide on brain iron contents, iron transport across the brain–blood barrier, iron uptake and release, and also the expression of transferrin receptor-1 (TfR1), divalent metal transporter 1 (DMT1), and ferroportin 1 (Fpn1) in cultured microvascular endothelial cells and neurons. We demonstrated that hepcidin significantly reduced brain iron in iron-overloaded rats and suppressed transport of transferrin-bound iron (Tf-Fe) from the periphery into the brain. Also, the peptide significantly inhibited expression of TfR1, DMT1, and Fpn1 as well as reduced Tf-Fe and non-transferrin-bound iron uptake and iron release in cultured microvascular endothelial cells and neurons, while downregulation of hepcidin with hepcidin siRNA retrovirus generated opposite results. We concluded that, under iron-overload, hepcidin functions to reduce iron in the brain by downregulating iron transport proteins. Upregulation of brain hepcidin by ad-hepcidin emerges as a new pharmacological treatment and prevention for iron-associated neurodegenerative disorders.

During the perinatal period, the bovine mammary epithelial cells of dairy cows exhibit vigorous metabolism and produce large amounts of reactive oxygen species (ROS). The resulting redox balance disruption leads to oxidative stress, one of the main causes of mastitis. Puerarin (PUE) is a natural flavonoid in the root of PUE that has attracted extensive attention as a potential antioxidant. This study first investigated whether PUE could reduce oxidative damage and mastitis induced by hydrogen peroxide (H2O2) in bovine mammary epithelial cells in vitro and elucidated the molecular mechanism. In vitro, BMECs (Bovine mammary epithelial cells) were divided into four treatment groups: Control group (no treatment), H2O2 group (H2O2 stimulation), PUE + H2O2 group (H2O2 stimulation before PUE rescue) and PUE group (positive control). The growth of BMECs in each group was observed, and oxidative stress-related indices were detected. Fluorescence quantitative PCR (qRT–PCR) was used to detect the expression of tightly linked genes, antioxidant genes, and inflammatory factors. The expression of p65 protein was detected by Western blot. In vivo, twenty cows with an average age of 5 years having given birth three times were divided into the normal dairy cow group, normal dairy cow group fed PUE, mastitis dairy cow group fed PUE, and mastitis dairy cow group fed PUE (n = 5). The contents of TNF-α, IL-6, and IL-1β in milk and serum were detected. In BMECs, the results showed that the PUE treatment increased the activities of glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and total antioxidant capacity (T-AOC); ROS and malondialdehyde (MDA) levels were reduced. Thus, PUE alleviated H2O2-induced oxidative stress in vitro. In addition, the PUE treatment eliminated the inhibition of H2O2 on the expression of oxidation genes and tight junction genes, and the enrichment degree of NRF-2, HO-1, xCT, and tight junctions (claudin4, occludin, ZO-1 and symplekin) increased. The PUE treatment also inhibited the expression of NF-κB-associated inflammatory factors (IL-6 and IL-8) and the chemokine CCL5 in H2O2-induced BMECs. In vivo experiments also confirmed that feeding PUE can reduce the expression of inflammatory factors in the milk and serum of lactating dairy cows. In conclusion, PUE can effectively reduce the oxidative stress of bovine mammary epithelial cells, enhance the tight junctions between cells, and play an anti-inflammatory role. This study provides a theoretical basis for PUE prevention and treatment of mastitis and oxidative stress. The use of PUE should be considered as a feed additive in future dairy farming.

Bovine mastitis is a common infectious disease which causes huge economic losses in dairy cattle. Bovine mammary epithelial cell (BMEC) damage usually directly causes the decrease of milk production, which is one of the most important causes of economic loss. NETs, novel effector mechanisms, are reported to exacerbate the pathogenesis of several inflammatory diseases. NETs formation has also been observed in the milk and mammary glands of sheep. However, the effects and detailed mechanisms of NETs on BMEC damage remain unclear. Thus, we aim to examine the effects of NETs on BMECs , and further to investigate the detail mechanism. In this study, the cytotoxicity of NETs on BMECs was determined using lactic dehydrogenase (LDH) levels in culture supernatants. Histone-induced BMEC damage was examined by flow cytometry and immunofluorescence analysis. The activities of caspase 1, caspase 3, caspase 11, and NLRP3 was detected using western blotting and immunohistochemical analysis. The results showed that NETs and their component histone significantly increased cytotoxicity to BMECs, suggesting the critical role of NETs, and their component histone in BMEC damage. In addition, histone could also induce necrosis, pyroptosis, and apoptosis of BMECs, and the mechanisms by which histone leads to BMEC damage occurred via activating caspase 1, caspase 3, and NLRP3. Altogether, NETs formation regulates inflammation and BMEC damage in mastitis. Inhibiting excess NETs formation may be useful to ameliorate mammary gland damage associated with mastitis.

The blood–brain barrier (BBB) is a dynamic structure that maintains the homeostasis of the brain and thus proper neurological functions. BBB compromise has been found in many pathological conditions, including neuroinflammation. Monocyte chemoattractant protein-1 (MCP1), a chemokine that is transiently and significantly up-regulated during inflammation, is able to disrupt the integrity of BBB and modulate the progression of various diseases, including excitotoxic injury and hemorrhage. In this review, we first introduce the biochemistry and biology of MCP1, and then summarize the effects of MCP1 on BBB integrity as well as individual BBB components.

Agouti signalling protein (ASIP) is a coat colour-related protein and also is a protein-related to lipid metabolism, which had first been found in agoutis. According to our previous study, ASIP is a candidate gene that affects the lipid metabolism in bovine adipocytes. However, its effect on milk lipid has not been reported yet. This study focused on the effect of the ASIP gene on the lipid metabolism of mammary epithelial cells in cattle. The ASIP gene was knocked out in bMECs by using CRISPR/Cas9 technology. The result of transcriptome sequencing showed that the differentially expressed genes associated with lipid metabolism were mainly enriched in the fatty acids metabolism pathways. Furthermore, the contents of intracellular triglycerides were significantly increased (p < 0.05), and cholesterol tended to rise (p > 0.05) in bMECs with the knockout of the ASIP gene. Fatty acid assays showed a significant alteration in medium and long-chain fatty acid content. Saturated and polyunsaturated fatty acids were significantly up-regulated (p < 0.05), and monounsaturated fatty acids were significantly decreased in the ASIP knockout bMECs (p < 0.05). The Q-PCR analysis showed that knockout of ASIP resulted in a significant reduction of gene expressions like PPARγ, FASN, SCD, and a significant up-regulation of genes like FABP4, ELOVL6, ACSL1, HACD4 prompted increased mid-to long-chain fatty acid synthesis. Overall, ASIP plays a pivotal role in regulating lipid metabolism in bMECs, which could further influence the component of lipid in milk.

Background Emerging evidence suggests that chronic stress compromises blood‒brain barrier (BBB) integrity by disrupting brain microvascular endothelial cells (BMECs), contributing to the development of cognitive impairments. Thus, targeting the BBB is expected to be a promising treatment strategy. The biological function of rutin has been investigated in neurological disorders; however, its regulatory role in stress–induced BBB damage and cognitive decline and the underlying mechanisms remain elusive. Methods In a chronic unpredictable mild stress (CUMS) mouse model, a fluorescent dye assay and behavioral tests, including a novel object recognition test and Morris water maze, were performed to evaluate the protective effects of rutin on BBB integrity and cognition. The effects of rutin on BMEC function were also investigated in hCMEC/D3 cells (a human brain microvascular endothelial cell line) in vitro. Furthermore, the molecular mechanisms by which rutin restores BBB endothelium dysfunction were explored via RNA–seq, quantitative real–time PCR, western blotting, immunofluorescence and chromatin immunoprecipitation. Finally, biotinylated tumor necrosis factor–α (TNF–α) was employed to test the influence of rutin on the ability of circulating TNF–α to cross the BBB. Results We identified that rutin attenuated BBB hyperpermeability and cognitive impairment caused by the 8–week CUMS procedure. Moreover, rutin promoted the proliferation, migration and angiogenesis ability of BMECs, and the integrity of the cellular monolayer through positively regulating the expression of genes involved. Furthermore, rutin impeded histone deacetylase 1 (HDAC1) recruitment and stabilized H3K27ac to increase Claudin–5 protein levels. Ultimately, normalization of the hippocampal HDAC1‒Claudin–5 axis by rutin blocked the infiltration of circulating TNF–α into the brain parenchyma and alleviated neuroinflammation. Conclusions This work establishes a protective role of rutin in regulating BMEC function and BBB integrity, and reveals that rutin is a potential drug candidate for curing chronic stress–induced cognitive deficits.

Endothelial cell dysfunction and diabetic vascular complications are intrinsically linked. Although BDNF plays a protective role in cerebral microvascular complications caused by diabetes, the mechanisms of this activity are not fully clear. In this study, we investigated the role of BDNF in the hyperglycemic injury of BMECs and its associated intracellular signal transduction pathways. BMECs were treated with 33 mM glucose to imitate the endothelium under hyperglycemic conditions. The high-glucose treatment caused cell dysfunction, as evaluated by oxidative stress and cell apoptosis, which could be alleviated by BDNF. In addition, BDNF preserved mitochondrial function as assessed by mPTP opening, mitochondrial membrane potential, calcium content, and mitochondrial biogenesis markers. Western blot analysis of LC3-II, p62, and TOMM20 and the detection of mRFP-GFP-LC3 adenovirus for autophagy flux revealed that BDNF enhanced autophagy flux. Furthermore, BDNF activated mitophagy, which was confirmed by the observed colocalization of LC3-II with BNIP3 and from transmission electron microscopy observations. The HIF-1α/BNIP3 signaling pathway was associated with BDNF/TrkB-induced mitophagy. In addition, BDNF-induced mitophagy played a protective role against BMEC damage under hyperglycemia. Thus, the results of this study suggest that BDNF/TrkB/HIF-1α/BNIP3-mediated mitophagy protects BMECs from hyperglycemia.

Background Due to their ability to limitlessly proliferate and specialize into almost any cell type, human induced pluripotent stem cells (iPSCs) offer an unprecedented opportunity to generate human brain microvascular endothelial cells (BMECs), which compose the blood–brain barrier (BBB), for research purposes. Unfortunately, the time, expense, and expertise required to differentiate iPSCs to purified BMECs precludes their widespread use. Here, we report the use of a defined medium that accelerates the differentiation of iPSCs to BMECs while achieving comparable performance to BMECs produced by established methods. Methods Induced pluripotent stem cells were seeded at defined densities and differentiated to BMECs using defined medium termed E6. Resultant purified BMEC phenotypes were assessed through trans-endothelial electrical resistance (TEER), fluorescein permeability, and P-glycoprotein and MRP family efflux transporter activity. Expression of endothelial markers and their signature tight junction proteins were confirmed using immunocytochemistry. The influence of co-culture with astrocytes and pericytes on purified BMECs was assessed via TEER measurements. The robustness of the differentiation method was confirmed across independent iPSC lines. Results The use of E6 medium, coupled with updated culture methods, reduced the differentiation time of iPSCs to BMECs from thirteen to 8 days. E6-derived BMECs expressed GLUT-1, claudin-5, occludin, PECAM-1, and VE-cadherin and consistently achieved TEER values exceeding 2500 Ω × cm 2 across multiple iPSC lines, with a maximum TEER value of 4678 ± 49 Ω × cm 2 and fluorescein permeability below 1.95 × 10 −7 cm/s. E6-derived BMECs maintained TEER above 1000 Ω × cm 2 for a minimum of 8 days and showed no statistical difference in efflux transporter activity compared to BMECs differentiated by conventional means. The method was also found to support long-term stability of BMECs harboring biallelic PARK2 mutations associated with Parkinson’s Disease. Finally, BMECs differentiated using E6 medium responded to inductive cues from astrocytes and pericytes and achieved a maximum TEER value of 6635 ± 315 Ω × cm 2 , which to our knowledge is the highest reported in vitro TEER value to date. Conclusions Given the accelerated differentiation, equivalent performance, and reduced cost to produce BMECs, our updated methods should make iPSC-derived in vitro BBB models more accessible for a wide variety of applications.

miR-126 is closely related to the occurrence of various complications after intracerebral hemorrhage (ICH), but the molecular mechanism is not fully elucidated. This study aimed to explore the mechanism of miR-126-3p in alleviating brain injury after ICH. Serum miR-126-3p levels were compared between patients with IHC and healthy controls. A rat model of ICH was generated by intracerebral injection of Type VII collagenase. The rats were intracerebral injected with miR-126-3p mimics or negative control miRNA. Rat brain microvascular endothelial cells (BMECs) were used as a cell model of blood-brain barrier (BBB), and validated by immunofluorescence staining of Factor VIII. The BBB permeability of BMECs after miR-126-3p antagomir transfection was determined by FITC-dextran 20 through a confluent BMECs layer (measured over 120 min). The binding site of miR-126-3p in the 3'UTR of VCAM-1 was predicated by TargetScan, and verified by dual luciferase reporter assay. The expression levels of miR-126-3p and vascular cell adhesion molecule-1 (VCAM-1) in rat brain tissues and BMECs were measured by real-time PCR or western blotting. Serum miR-126-3p level was markedly down-regulated in patients with ICH. The rats with ICH had decreased miR-126-3p levels in serum and hemorrhagic area, while those changes were reversed by the treatment with miR-126-3p mimic. VCAM-1 is a direct target of miR-126-3p, and VCAM-1 expression in hemorrhagic area was down-regulated by the administration of miR-126-3p mimic in rats. Inhibition of miR-126-3p by anti-miR126 treatment in BMECs resulted in barrier leakage. miR-126-3p attenuates intracerebral hemorrhage-induced blood-brain barrier disruption, which is associated with down-regulated expression of VCAM-1 in hemorrhagic area.