Moringa oleifera Leaves’ Extract Enhances Nonspecific Immune Responses, Resistance against Vibrio alginolyticus, and Growth in Whiteleg Shrimp (Penaeus vannamei)

Moringa is widely known as a plant with high medicinal properties. Therefore, moringa has a high potential for use as an immunostimulant in shrimp. This study investigated the effect of a moringa water extract on the immune response, resistance against V. alginolyticus, and growth performance of whiteleg shrimp. To perform the in vitro assay, hemocytes were incubated with different concentrations of the moringa extract. Furthermore, the moringa extract was incorporated at 0 (control), 1.25 g (ME1.25), 2.5 g (ME2.5), and 5.0 g (ME5.0) per kg of diet for the in vivo assay. During the rearing period, immune responses, namely the total hemocyte count (THC), phenoloxidase (PO) activity, phagocytosis activity, superoxide anion production, and immune-related gene expression were examined on days 0, 1, 2, 4, 7, 14, 21, and 28. Growth performance was measured 60 days after the feeding period. Furthermore, the shrimp were challenged with V. alginolyticus after being fed for different feeding durations. The results of the in vitro assay revealed that 100–250 ppm of the moringa extract enhanced the PO activity, phagocytic rate (PR), and superoxide anion production. The findings of the in vivo assay demonstrated that the THC, PO activity, PR, and immune-related gene expression, including alpha-2-macroglobulin, prophenoloxidase II, penaeidin2, penaeidin3, anti-lipopolysaccharide factor, crustin, lysozyme, superoxide dismutase, and clotting protein, were higher in the group of ME.25 and ME5.0 than in the control and ME1.25 at several time points. Growth performance was significantly increased (p < 0.05) in the ME2.5 group compared to the control group. Furthermore, the dietary ME2.5 resulted in a higher survival rate compared to that of the control group after challenging with V. alginolyticus, especially at ME2.5 administered for 4 and 7 days. This study indicated that the incorporation of the moringa extract at 2.5 g per kg of diet enhanced the immune response, the growth performance of the whiteleg shrimp, and the resistance against V. alginolyticus infection.

Salusin-β in Intermediate Dorsal Motor Nucleus of the Vagus Regulates Sympathetic-Parasympathetic Balance and Blood Pressure

The dorsal motor nucleus of the vagus (DMV) is known to control vagal activity. It is unknown whether the DMV regulates sympathetic activity and whether salusin-β in the DMV contributes to autonomic nervous activity. We investigated the roles of salusin-β in DMV in regulating sympathetic-parasympathetic balance and its underline mechanisms. Microinjections were carried out in the DMV and hypothalamic paraventricular nucleus (PVN) in male adult anesthetized rats. Renal sympathetic nerve activity (RSNA), blood pressure and heart rate were recorded. Immunohistochemistry for salusin-β and reactive oxidative species (ROS) production in the DMV were examined. Salusin-β was expressed in the intermediate DMV (iDMV). Salusin-β in the iDMV not only inhibited RSNA but also enhanced vagal activity and thereby reduced blood pressure and heart rate. The roles of salusin-β in causing vagal activation were mediated by NAD(P)H oxidase-dependent superoxide anion production in the iDMV. The roles of salusin-β in inhibiting RSNA were mediated by not only the NAD(P)H oxidase-originated superoxide anion production in the iDMV but also the γ-aminobutyric acid (GABA)A receptor activation in PVN. Moreover, endogenous salusin-β and ROS production in the iDMV play a tonic role in inhibiting RSNA. These results indicate that salusin-β in the iDMV inhibits sympathetic activity and enhances vagal activity, and thereby reduces blood pressure and heart rate, which are mediated by NAD(P)H oxidase-dependent ROS production in the iDMV. Moreover, GABAA receptor in the PVN mediates the effect of salusin-β on sympathetic inhibition. Endogenous salusin-β and ROS production in the iDMV play a tonic role in inhibiting sympathetic activity.

First Evidence of In Vitro Effects of C6O4—A Substitute of PFOA—On Haemocytes of the Clam Ruditapes philippinarum

Alternative chemicals to per- and poly-fluoroalkyl substances have recently been introduced in various industrial processes. C6O4 (difluoro{[2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetic acid) is a new surfactant and emulsifier used as a replacement for perfluorooctanoic acid (PFOA). From an ecotoxicological point of view, in vitro assays are useful tools for assessing the negative effects and understanding the mechanisms of action of chemicals at the cellular level. Here, we present the results of an in vitro study in which the effects of C6O4 were evaluated—for the first time—on haemocytes of the clam Ruditapes philippinarum. Cells were exposed to three concentrations of C6O4 (0.05, 0.5, 5 μg/mL) and the effects on haemocyte viability, haemocyte morphology, differential haemocyte count, lysosomal membrane stability, superoxide anion production, acid phosphatase, and β-glucuronidase activities, as well as on the percentage of micronuclei and chromosomal aberrations were evaluated. The results demonstrated that C6O4 significantly affected haemocyte morphology, lysosomal membrane stability, hydrolytic enzyme activity, and superoxide anion production, and promoted chromosomal aberrations. To the best of our knowledge, this is the first study revealing the in vitro effects of C6O4, a substitute for PFOA, on haemocytes from a bivalve species.

Therapeutic hypothermia augments the restorative effects of PKC-β and Nox2 inhibition on an in vitro model of human blood–brain barrier

To investigate whether therapeutic hypothermia augments the restorative impact of protein kinase C-β (PKC-β) and Nox2 inhibition on an in vitro model of human blood–brain barrier (BBB). Cells cultured in normoglycaemic (5.5 mM) or hyperglycaemic (25 mM, 6 to 120 h) conditions were treated with therapeutic hypothermia (35 °C) in the absence or presence of a PKC-β inhibitor (LY333531, 0.05 μM) or a Nox2 inhibitor (gp91ds-tat, 50 μM). BBB was established by co-culture of human brain microvascular endothelial cells (HBMECs) with astrocytes (HAs) and pericytes. BBB integrity and function were assessed via transendothelial electrical resistance (TEER) and paracellular flux of sodium fluorescein (NaF, 376 Da). Nox activity (lucigenin assay), superoxide anion production (cytochrome-C reduction assay), cellular proliferative capacity (wound scratch assay) and actin cytoskeletal formation (rhodamine-phalloidin staining) were assessed both in HBMECs and HAs using the specific methodologies indicated in brackets. Therapeutic hypothermia augmented the protective effects of PKC-β or Nox2 inhibition on BBB integrity and function in experimental setting of hyperglycaemia, as evidenced by increases in TEER and concomitant decreases in paracellular flux of NaF. The combinatory approaches were more effective in repairing physical damage exerted on HBMEC and HA monolayers by wound scratch and in decreasing Nox activity and superoxide anion production compared to sole treatment regimen with either agent. Similarly, the combinatory approaches were more effective in suppressing actin stress fibre formation and maintaining normal cytoskeletal structure. Therapeutic hypothermia augments the cerebral barrier-restorative capacity of agents specifically targeting PKC-β or Nox2 pathways.

Chitosan-Induced Activation of the Antioxidant Defense System Counteracts the Adverse Effects of Salinity in Durum Wheat

The present study was carried out with the aim of (i) evaluating the effect of chitosan (CTS) on the growth of durum wheat under salinity and (ii) examining CTS-regulated mechanisms of salinity tolerance associated with the antioxidant defense system. To achieve these goals, durum wheat seedlings were treated with CTS at different molecular weight, low (L-CTS, 50–190 kDa), medium (M-CTS, 190–310 kDa) and high (H-CTS, 310–375 kDa). The results obtained show that exposure to 200 mM NaCl reduced the shoot and the root dried biomass by 38% and 59%, respectively. The growth impairment induced by salinity was strongly correlated with an increase in the superoxide anion production (5-fold), hydrogen peroxide content (2-fold) and malondialdehyde (MDA) content (4-fold). Seedlings responded to the oxidative stress triggered by salinity with an increase in the total phenolic content (TPC), total flavonoid content (TFC) and total antioxidant activity (TAA) by 67%, 51% and 32%, respectively. A salt-induced increase in the activity of the antioxidant enzymes superoxide dismutase and catalase (CAT) of 89% and 86%, respectively, was also observed. Treatment of salt-stressed seedlings with exogenous CTS significantly promoted seedling growth, with the strongest effects observed for L-CTS and M-CTS, which increased the shoot biomass of stressed seedlings by 32% and 44%, respectively, whereas the root dried biomass increased by 87% and 64%, respectively. L-CTS and M-CTS treatments also decreased the superoxide anion production (57% and 59%, respectively), the hydrogen peroxide content (35% and 38%, respectively) and the MDA content (48% and 56%, respectively) and increased the TPC (23% and 14%, respectively), the TFC (19% and 10%, respectively), the TAA (up to 10% and 7%, respectively) and the CAT activity (29% and 20%, respectively). Overall, our findings indicate that CTS exerts its protective role against the oxidative damages induced by salinity by enhancing the antioxidant defense system. L-CTS and M-CTS were the most effective in alleviating the adverse effect of NaCl, thus demonstrating that the CTS action is strictly related to its molecular weight.

Regulation of Superoxide by BAP31 through Its Effect on p22phox and Keap1/Nrf2/HO-1 Signaling Pathway in Microglia

Reactive oxygen species (ROS) production by activation of microglia is considered to be a major cause of neuronal dysfunction, which can lead to damage and death through direct oxidative damage to neuronal macromolecules or derangement of neuronal redox signaling circuits. BAP31, an integral ER membrane protein, has been defined as a regulatory molecule in the CNS. Our latest studies have found that BAP31 deficiency leads to activation of microglia. In this study, we discovered that BAP31 deficiency upregulated LPS-induced superoxide anion production in BV2 cells and mice by upregulating the expression level of p22phox and by inhibiting the activation of Nrf2-HO-1 signaling. Knockdown of p22phox/keap1 or use of an NADPH oxidase inhibitor (apocynin) reversed the production of superoxide anion and inflammatory cytokines, which then reduced neuronal damage and death in vitro and in vivo. These results suggest that BAP31 deficiency contributes to microglia-related superoxide anion production and neuroinflammation through p22phox and keap1. Furthermore, the excess superoxide anion cooperated with inflammatory cytokines to induce the damage and death of neurons. Thus, we determined that BAP31 is an important regulator in superoxide anion production and neuroinflammation, and the downstream regulators or agonists of BAP31 could therefore be considered as potential therapeutic targets in microglial-related superoxide anion production and neuroinflammation.

Role of TSPO/VDAC1 Upregulation and Matrix Metalloproteinase-2 Localization in the Dysfunctional Myocardium of Hyperglycaemic Rats

Clinical management of diabetic cardiomyopathy represents an unmet need owing to insufficient knowledge about the molecular mechanisms underlying the dysfunctional heart. The aim of this work is to better clarify the role of matrix metalloproteinase 2 (MMP-2) isoforms and of translocator protein (TSPO)/voltage-dependent anion-selective channel 1 (VDAC1) modulation in the development of hyperglycaemia-induced myocardial injury. Hyperglycaemia was induced in Sprague-Dawley rats through a streptozocin injection (35 mg/Kg, i.p.). After 60 days, cardiac function was analysed by echocardiography. Nicotinamide Adenine Dinucleotide Phosphate NADPH oxidase and TSPO expression was assessed by immunohistochemistry. MMP-2 activity was detected by zymography. Superoxide anion production was estimated by MitoSOX™ staining. Voltage-dependent anion-selective channel 1 (VDAC-1), B-cell lymphoma 2 (Bcl-2), and cytochrome C expression was assessed by Western blot. Hyperglycaemic rats displayed cardiac dysfunction; this response was characterized by an overexpression of NADPH oxidase, accompanied by an increase of superoxide anion production. Under hyperglycaemia, increased expression of TSPO and VDAC1 was detected. MMP-2 downregulated activity occurred under hyperglycemia and this profile of activation was accompanied by the translocation of intracellular N-terminal truncated isoform of MMP-2 (NT-MMP-2) from mitochondria-associated membrane (MAM) into mitochondria. In the onset of diabetic cardiomyopathy, mitochondrial impairment in cardiomyocytes is characterized by the dysregulation of the different MMP-2 isoforms. This can imply the generation of a “frail” myocardial tissue unable to adapt itself to stress.

Polysaccharides from the green alga Caulerpa racemosa (Agardh, 1873) improve the immune response and antioxidant status in the white shrimp Litopenaeus vannamei (Boone, 1931) (Dendrobranchiata, Penaeidae)

The objective of this study was to investigate the effects of Caulerpa racemosa (Agardh, 1873) polysaccharides (CRP) on non-specific immunity of the white shrimp, Litopenaeus vannamei (Boone, 1931). The extracted polysaccharide consists of galactose, glucose, and mannose. Total polyphenolic compound and total flavonoids content in CRP were determined. In vitro CRP treatment did not alter cell viability of haemocytes and significantly activated phenoloxidase (PO) activity, superoxide anion production capacity, phagocytic rate, and index in haemocytes. Immune parameters, such as total haemocyte count, PO activity, phagocytosis, and superoxide anion production, were significantly elevated in CRP-fed shrimp during the rearing period. Malondialdehyde levels in the hepatopancreas of CRP-fed shrimp were lower than in control shrimp from day 7 to day 28. These results show that CRP exhibits antioxidant activity and can activate immune responses of white shrimp in vitro and in vivo. Therefore, CRP can be considered a functional feed additive to improve white shrimp immunity.