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In DOCA-salt hypertension, superoxide anion (O2-·) levels are increased in sympathetic ganglion neurons as compared to normotensive controls; this increase is mediated in part by endothelin-1 (ET-1) via the activation of NADPH oxidase. Increased O2-· reduces norepinephrine uptake via norepinephrine transporter (NET). We tested the hypothesis that ET-1 reduces NET expression in PC12 cells via increases in O2-·, and this effect can be abolished by knockdown of p22phox, an indispensable component of the O2-·-generating NADPH oxidase. Undifferentiated PC12 cells were transfected with short hairpin RNA (shRNA) targeting p22phox. Cells with stable expression of p22phox shRNA (shRNA-p22) had reduced p22phox mRNA (71% knockdown) and protein (55% knockdown). Compared to wild type PC12 cells (WT), O2-· production induced by ET-1 was diminished in shRNA-p22. To assess ET-1 effects mediated by NADPH oxidase on NET, both shRNA-p22 and WT were exposed to ET-1 (100nM) for 30min to 24hrs. ET-1 reduced NET mRNA and protein levels in WT cells; the mRNA levels decreased over time and reached their minimum at 2 hrs (3.41 fold decrease, n=3), while protein levels remained reduced for 24 hrs (68% decrease, n=3). In shRNA-p22 cells, neither NET mRNA nor protein levels were changed by ET-1 treatment. These results indicate that the effects of ET-1 on O2-· production and NET expression in PC12 cells are p22phox -dependent. [ABSTRACT FROM AUTHOR]

Oxidized linoleic acid metabolites (OXLAM) are products of adipocyte lipolysis with the potential to modulate adipose tissue (AT) lipid metabolism and inflammation. In periparturient cows, linoleic acid is preferentially mobilized from AT during lipolysis by hormone-sensitive lipase (HSL) compared with other polyunsaturated fatty acids. Enzymatic and nonenzymatic reactions generate OXLAM from linoleic acid. Among OXLAM, 9-, 10-, and 12-hydroxy-octadecadienoic acids (HODE) are associated with pro-inflammatory responses, whereas 9- and 13-oxo-octadecadienoic acids (oxoODE) and 13-HODE can facilitate inflammation resolution and promote lipogenesis. This study evaluated the effect of HSL activity on OXLAM biosynthesis using subcutaneous AT explants collected from multiparous dairy cows at 10 d before and again at 10 and 24 d after calving. Explants were treated for 3 h without or with the ß-adrenergic agonist isoproterenol (ISO; 1 µM; MilliporeSigma, Burlington, MA) to induce HSL activity. The contribution of HSL to OXLAM biosynthesis was determined by inhibiting its activity with CAY10499 (2 µM; Cayman Chemical, Ann Arbor, MI). After treatments, media and explants were collected for lipidomic analysis using HPLC–tandem mass spectroscopy. Results indicated that ISO increased the biosynthesis of 9-, 12-, and 13-HODE and 9-oxoODE, and this effect was reduced at 24 d after calving. Inhibiting HSL activity partially reversed ISO effects on HODE and 9-oxoODE. Our ex vivo model demonstrated for the first time a direct effect of HSL activity on the biosynthesis of OXLAM in AT, especially at 10 d before and 10 d after calving. The biosynthesis of anti-inflammatory OXLAM is limited during the first weeks after parturition and may promote AT inflammation and lipolytic responses to negative energy balance. These results indicate that HSL activity releases linoleic acid for OXLAM biosynthesis in concentrations of a magnitude that may bypass the need for the activation of phospholipases linked with the inflammatory cascade and thus supports, in part, lipolysis-driven inflammation within AT of periparturient cows. [ABSTRACT FROM AUTHOR]

Oxidative stress contributes to dysfunctional immune responses and predisposes dairy cattle to several metabolic and inflammatory-based diseases. Although the negative effects of oxidative stress on transition cattle are well established, biomarkers that accurately measure oxidative damage to cellular macromolecules are not well defined in veterinary medicine. Measuring 15-F2t-isoprostane, a lipid peroxidation product, is the gold standard biomarker for quantifying oxidative stress in human medicine. The aim of our study was to determine whether changes in 15-F2t-isoprostane concentrations in plasma and milk could accurately reflect changes in oxidant status during different stages of lactation. Using liquid chromatography–tandem mass spectrometry, 15-F2t-isoprostane concentrations were quantified in milk and plasma of 12 multiparous Holstein-Friesian cows that were assigned to 3 different sampling periods, including the periparturient period (1–2 d in milk; n = 4), mid lactation (80–84 d in milk; n = 4), and late lactation (183–215 d in milk; n = 4). Blood samples also were analyzed for indicators of oxidant status, inflammation, and negative energy balance. Our data revealed that 15-F2t-isoprostane concentrations changed at different stages of lactation and coincided with changes in other gauges of oxidant status in both plasma and milk. Interestingly, milk 15-F2t-isoprostane concentrations and other indices of oxidant status did not follow the same trends as plasma values at each stage of lactation. Indeed, during the periparturient period, systemic 15-F2t-isoprostane increased significantly accompanied by an increase in the systemic oxidant status index. Milk 15-F2t-isoprostane was significantly decreased during the periparturient period compared with other lactation stages in conjunction with a milk oxidant status index that trended lower during this period. The results from this study indicate that changes in 15-F2t-isoprostane concentrations in both milk and plasma may be strong indicators of an alteration in redox status both systemically and within the mammary gland. [ABSTRACT FROM AUTHOR]

Modern dairy cattle suffer from increased incidence and severity of mastitis during major physiological transitions of the lactation cycle. Oxidative stress, a condition resulting from inadequate antioxidant defense against reactive oxygen and nitrogen species, is a major underlying component of mastitis pathophysiology. Isoprostanes (IsoP) are molecules derived from cellular lipid membranes upon non-enzymatic interaction with reactive species during inflammation, and are regarded as highly sensitive and specific biomarkers of oxidative stress. Changes in IsoP concentrations have been noted during major physiological transitions and diseases such as coliform mastitis in dairy cattle. However, the biological role of IsoP during oxidative stress in dairy cows has not been well-elucidated. Therefore, this study aimed to characterize the impacts of IsoP on oxidative stress outcomes in a bovine model of acute endothelial inflammation. Bovine aortic endothelial cells (BAEC; n = 4) were stimulated with 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) or lipopolysaccharide (LPS) with or without 15-F 2t -IsoP to determine how IsoP influence oxidative stress outcomes. Our endothelial inflammation model showed relatively decreased reactive metabolites and increased barrier integrity in cells treated with both the agonist and IsoP compared to agonist treatment alone. However, IsoP do not appear to affect oxidative stress outcomes during acute inflammation. Understanding the effect of IsoP on BAEC is an early step in elucidating how IsoP impact dairy cows during times of oxidative stress in the context of acute clinical mastitis. Future studies should define the optimal dosing and treatment timing of IsoP to maximize their cytoprotective potential during acute inflammation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Putman, Sordillo and Contreras.)

Adapting to the lactating state requires metabolic adjustments in multiple tissues, especially in the dairy cow, which must meet glucose demands that can exceed 5 kg/day in the face of negligible gastrointestinal glucose absorption. These challenges are met through the process of homeorhesis, the alteration of metabolic setpoints to adapt to a shift in physiological state. To investigate the role of inflammation-associated pathways in these homeorhetic adaptations, we treated cows with the nonsteroidal anti-inflammatory drug sodium salicylate (SS) for the first 7 days of lactation. Administration of SS decreased liver TNF-α mRNA and marginally decreased plasma TNF-α concentration, but plasma eicosanoids and liver NF-κB activity were unaltered during treatment. Despite the mild impact on these inflammatory markers, SS clearly altered metabolic function. Plasma glucose concentration was decreased by SS, but this was not explained by a shift in hepatic gluconeogenic gene expression or by altered milk lactose secretion. Insulin concentrations decreased in SS-treated cows on day 7 compared with controls, which was consistent with the decline in plasma glucose concentration. The revised quantitative insulin sensitivity check index (RQUICKI) was then used to assess whether altered insulin sensitivity may have influenced glucose utilization rate with SS. The RQUICKI estimate of insulin sensitivity was significantly elevated by SS on day 7, coincident with the decline in plasma glucose concentration. Salicylate prevented postpartum insulin resistance, likely causing excessive glucose utilization in peripheral tissues and hypoglycemia. These results represent the first evidence that inflammation-associated pathways are involved in homeorhetic adaptations to lactation.

Thirteen bovine leukemia virus- (BLV-) negative and 22 BLV-positive Holstein cows were immunized with J5 Escherichia coli bacterin at dry off, three weeks before calving, during the second week after calving, and three weeks after the third immunization. Serum was collected before the initial immunization, immediately before the third and fourth immunizations, and 21 days after the fourth immunization. Anti-J5 E. coli IgM, IgG1, and IgG2 titers were determined by ELISA. Anti-J5 E. coli IgM titers did not differ significantly (P = .98) between groups. Increases in anti-J5 E. coli IgG1 titers were higher in the BLV-negative cows (P = .057). Geometric mean anti-J5 E. coli IgG2 titers increased fourfold in the BLV-negative cows, which was significantly higher (P = .007) than the twofold increase in the BLV-positive cows. Cattle infected with BLV may have impaired serologic responses following immunization with J5 bacterin, and response may differ according to antibody isotype.

The relationship between body condition score (BCS) with measures of oxidative status and TNF-alpha production was examined in 16 mid-lactation dairy cows. Cows were selected based on either a normal (2.5-2.7) or a high (> or =3.5) BCS using the standard five-point scaling system. The metabolic status of all cows was determined by plasma nonesterified fatty acid levels (NEFA). Plasma samples or white blood cell lysates also were analyzed for indices of oxidant stress and for the expression of TNF-alpha. Cows with a high BCS had significantly lower NEFA levels when compared to normal BCS cows and the over-conditioned animals were not in a state of negative energy balance. No significant changes in lipid hydroperoxide levels, glutathione peroxidase activity, or the ratio of reduced (GSH) to oxidized (GSSG) glutathione was detected with respect to BCS. However, high BCS cows did have a significantly lower overall antioxidant potential and reduced TrxR activities when compared with the normal BCS cows. Changes in the oxidative state of over-conditioned cows were accompanied by a significantly higher expression of TNF-alpha. Results from this study suggest that cows with a high BCS can experience oxidant stress in the absence of altered energy status. Increased TNF-alpha expression may be related to the pro-oxidant state of over-conditioned cows and possibly be a contributing factor to the enhanced susceptibility to disease in high BCS dairy cattle.

The inducible isoform of nitric oxide synthase (iNOS) is implicated in atherosclerosis, malignancy, rheumatoid arthritis, tissue and reperfusion injuries. A key determinant of the pro-oxidant versus protective effects of NO is the underlying redox status of the tissue. Selenoproteins, such as glutathione peroxidases (GPxs) and thioredoxin reductases, are key components of cellular defence and promote optimal antioxidant/oxidant balance. In this study, we have investigated the relationship between Se status, iNOS expression and NO production in Se-deficient and Se-supplemented RAW 264.7 macrophage cell lines. The cellular GPx activity, a measure of Se status, was 17-fold lower in Se-deficient RAW 264.7 cells and the total cellular oxidative tone, as assessed by flow cytometry with 2',7'-dichlorodihydrofluorescein diacetate, was higher in the Se-deficient cells than the Se-supplemented cells. Upon lipopolysaccharide (LPS) stimulation of these cells in culture, we found significantly higher iNOS transcript and protein expression levels with an increase in NO production in Se-deficient RAW 264.7 cells than the Se-supplemented cells. Electrophoretic mobility-shift assays, nuclear factor-kappaB (NF-kappaB)-luciferase reporter assays and Western blot analyses indicate that the increased expression of iNOS in Se deficiency could be due to an increased activation and consequent nuclear localization of the redox-sensitive transcription factor NF-kappaB. These results suggest an inverse relationship between cellular Se status and iNOS expression in LPS-stimulated RAW 264.7 cells and provide evidence for the beneficial effects of dietary Se supplementation in the prevention and/or treatment of oxidative-stress-mediated inflammatory diseases.