Joint Damage and Neuropathic Pain in Rats Treated With Lysophosphatidic Acid.

Joint pain is a complex phenomenon that involves multiple endogenous mediators and pathophysiological events. In addition to nociceptive and inflammatory pain, some patients report neuropathic-like pain symptoms. Examination of arthritic joints from humans and preclinical animal models have revealed axonal damage which is likely the source of the neuropathic pain. The mediators responsible for joint peripheral neuropathy are obscure, but lysophosphatidic acid (LPA) has emerged as a leading candidate target. In the present study, male and female Wistar rats received an intra-articular injection of LPA into the right knee and allowed to recover for 28 days. Joint pain was measured by von Frey hair algesiometry, while joint pathology was determined by scoring of histological sections. Both male and female rats showed comparable degenerative changes to the LPA-treated knee including chondrocyte death, focal bone erosion, and synovitis. Mechanical withdrawal thresholds decreased by 20-30% indicative of secondary allodynia in the affected limb; however, there was no significant difference in pain sensitivity between the sexes. Treatment of LPA animals with the neuropathic pain drug amitriptyline reduced joint pain for over 2 hours with no sex differences being observed. In summary, intra-articular injection of LPA causes joint degeneration and neuropathic pain thereby mimicking some of the characteristics of neuropathic osteoarthritis.

Blockade of GPR55 in dorsal periaqueductal gray produces anxiety-like behaviors and evocates defensive aggressive responses in alcohol-pre-exposed rats.

GPR55 is a receptor expressed in several central nervous system areas, including the periaqueductal gray (PAG). Current knowledge of GPR55 physiology in PAG only covers pain integration, but it is involved in other actions such as anxiety, panic, motivated behaviors, and alcohol intake. In the present study, juvenile male Wistar rats were unexposed (alcohol-naïve group; A-naïve) or exposed to alcohol for 5 weeks (alcohol-pre-exposed group; A-pre-exposed). Posteriorly, animals received intra dorsal-PAG (D-PAG) injections of vehicle (10% DMSO), LPI (1 nmol/0.5 µl) and ML-193 (1 nmol/0.5 µl, a selective GPR55 antagonist). Finally, defensive burying behavior (DBB) paradigm and alcohol preference were evaluated. Compared to the A-naïve group, the A-pre-exposed vehicle group had higher (p < 0.05): (i) time of immobility; (ii) latency to and duration of burying; and (iii) alcohol consumption. In both groups (i.e., A-naïve and A-pre-exposed) treatment with LPI: (i) decreased duration of burying (p < 0.05); (ii) suppressed time of immobility; and (iii) increased alcohol intake (p < 0.05). On the other hand, treatment with ML-193: (i) decreased duration of immobility in A-pre-exposed (but not in A-naïve rats); (ii) promoted an aggressive response against the shock-probe in A-pre-exposed rats (p < 0.05); and (iii) increased alcohol intake (p < 0.05). Our results suggest that blockade of GPR55 in D-PAG is associated with anxiety-like behaviors, defensive aggressive behaviors, and higher alcohol intake, whereas LPI in D-PAG produced anxiolytic-like effects (probably GPR55-mediated), but not prevention of alcohol intake.

Permissive Modulation of Sphingosine-1-Phosphate-Enhanced Intracellular Calcium on BKCa Channel of Chromaffin Cells.

Sphingosine-1-phosphate (S1P), is a signaling sphingolipid which acts as a bioactive lipid mediator. We assessed whether S1P had multiplex effects in regulating the large-conductance Ca2+-activated K+ channel (BKCa) in catecholamine-secreting chromaffin cells. Using multiple patch-clamp modes, Ca2+ imaging, and computational modeling, we evaluated the effects of S1P on the Ca2+-activated K+ currents (IK(Ca)) in bovine adrenal chromaffin cells and in a pheochromocytoma cell line (PC12). In outside-out patches, the open probability of BKCa channel was reduced with a mean-closed time increment, but without a conductance change in response to a low-concentration S1P (1 µM). The intracellular Ca2+ concentration (Cai) was elevated in response to a high-dose (10 µM) but not low-dose of S1P. The single-channel activity of BKCa was also enhanced by S1P (10 µM) in the cell-attached recording of chromaffin cells. In the whole-cell voltage-clamp, a low-dose S1P (1 µM) suppressed IK(Ca), whereas a high-dose S1P (10 µM) produced a biphasic response in the amplitude of IK(Ca), i.e., an initial decrease followed by a sustained increase. The S1P-induced IK(Ca) enhancement was abolished by BAPTA. Current-clamp studies showed that S1P (1 µM) increased the action potential (AP) firing. Simulation data revealed that the decreased BKCa conductance leads to increased AP firings in a modeling chromaffin cell. Over a similar dosage range, S1P (1 µM) inhibited IK(Ca) and the permissive role of S1P on the BKCa activity was also effectively observed in the PC12 cell system. The S1P-mediated IK(Ca) stimulation may result from the elevated Cai, whereas the inhibition of BKCa activity by S1P appears to be direct. By the differentiated tailoring BKCa channel function, S1P can modulate stimulus-secretion coupling in chromaffin cells.

Chronic central modulation of LPA/LPA receptors-signaling pathway in the mouse brain regulates cognition, emotion, and hippocampal neurogenesis.

Several studies have demonstrated that lysophosphatidic acid (LPA) acts through its LPA receptors in multiple biological and behavioral processes, including adult hippocampal neurogenesis, hippocampal-dependent memory, and emotional regulation. However, analyses of the effects have typically involved acute treatments, and there is no information available regarding the effect of the chronic pharmacological modulation of the LPA/LPA receptors-signaling pathway. Thus, we analyzed the effect of the chronic (21 days) and continuous intracerebroventricular (ICV) infusion of C18:1 LPA and the LPA1-3 receptor antagonist Ki16425 in behavior and adult hippocampal neurogenesis. Twenty-one days after continuous ICV infusions, mouse behaviors in the open field test, Y-maze test and forced swimming test were assessed. In addition, the hippocampus was examined for c-Fos expression and α-CaMKII and phospho-α-CaMKII levels. The current study demonstrates that chronic C18:1 LPA produced antidepressant effects, improved spatial working memory, and enhanced adult hippocampal neurogenesis. In contrast, chronic LPA1-3 receptor antagonism disrupted exploratory activity and spatial working memory, induced anxiety and depression-like behaviors and produced an impairment of hippocampal neurogenesis. While these effects were accompanied by an increase in neuronal activation in the DG of C18:1 LPA-treated mice, Ki16425-treated mice showed reduced neuronal activation in CA3 and CA1 hippocampal subfields. Treatment with the antagonist also induced an imbalance in the expression of basal/activated α-CaMKII protein forms. These outcomes indicate that the chronic central modulation of the LPA receptors-signaling pathway in the brain regulates cognition and emotion, likely comprising hippocampal-dependent mechanisms. The use of pharmacological modulation of this pathway in the brain may potentially be targeted for the treatment of several neuropsychiatric conditions.

The effect of intra-striatal administration of GPR55 agonist (LPI) and antagonist (ML193) on sensorimotor and motor functions in a Parkinson's disease rat model.

OBJECTIVE: G protein-coupled receptor 55 (GPR55) is an orphan G protein-coupled receptor with various physiological functions. Recent evidence suggests that this receptor may be involved in the control of motor functions. Therefore, in the present study, we evaluated the effects of intra-striatal administration of GPR55 selective ligands in a rat model of Parkinson's disease. METHODS: Experimental Parkinson was induced by unilateral intra-striatal administration of 6-hydroxydopamine (6-OHDA, 10 µg/rat). L-α-lysophosphatidylinositol (LPI, 1 and 5 µg/rat), an endogenous GPR55 agonist, and ML193 (1 and 5 µg/rat), a selective GPR55 antagonist, were injected into the striatum of 6-OHDA-lesioned rats. Motor performance and balance skills were evaluated using the accelerating rotating rod and the ledged beam tests. The sensorimotor function of the forelimbs and locomotor activity were assessed by the adhesive removal and open field tests, respectively. RESULTS: 6-OHDA-lesioned rats had impaired behaviours in all tests. Intra-striatal administration of LPI in 6-OHDA-lesioned rats increased time on the rotarod, decreased latency to remove the label, with no significant effect on slip steps, and locomotor activity. Intra-striatal administration of ML193 also increased time on the rotarod, decreased latency to remove the label and slip steps in 6-OHDA-lesioned rats mostly at the dose of 1 µg/rat. CONCLUSIONS: This study suggests that the striatal GPR55 is involved in the control of motor functions. However, considering the similar effects of GPR55 agonist and antagonist, it may be concluded that this receptor has a modulatory role in the control of motor deficits in an experimental model of Parkinson.

Supplemental effects of dietary lysophospholipids in lactation diets on sow performance, milk composition, gut health, and gut-associated microbiome of offspring.

Dietary lysophospholipids (LPL) would influence milk composition of sows, thus positively affect intestinal health of offspring. The objective of this study was to determine effects of dietary LPL fed to lactating sows on performance, milk characteristics, gut health, and gut-associated microbiome of offspring. Sixty pregnant sows were allotted to 2 treatments in a randomized complete block design with parity and BW as blocks on day 110 of gestation. Treatments were CON (no added LPL) and LPL (0.05% LPL; Lipidol-Ultra, Pathway Intermediates, Shrewsbury, UK). Sows were fed 2 kg/d from day 110 of gestation until farrowing and ad libitum after farrowing. Diets were formulated to meet NRC requirement for lactating sows. Colostrum and milk samples from 12 sows per treatment were collected to measure nutrients and immunoglobulins on days 1 and 18 of lactation, respectively. Twelve piglets per treatment (1 piglet per litter) were euthanized on day 18 to collect tissues to measure tumor necrosis factor-α, interleukin-8 (IL-8), malondialdehyde, protein carbonyl, IgA, histomorphology, crypt cell proliferation rate, and microbiota in the jejunum and colon. Data were analyzed using the MIXED procedure of SAS, and the mortality was analyzed using the GLIMMIX procedure of SAS. There was no difference in sow BW, parity, and litter size between treatments on day 0 of lactation. Sows fed LPL had increased (P < 0.05) litter BW gain (53.9 vs. 59.4 kg) and decreased piglet mortality (13.9% vs. 10.6%) on day 18 of lactation. Sows fed LPL had increased (P < 0.05) omega-6:omega-3 (22.1 vs. 23.7) and unsaturated:saturated (1.4 vs. 1.6) fatty acids ratios with increased oleic acid (29.1% vs. 31.4%) and tended to have increased (P = 0.092) IgG (1.14 vs. 1.94 g/L) and linoleic acid (17.7% vs. 18.7%) in the milk on day 18 of lactation. Piglets from sows fed LPL had increased (P < 0.05) IL-8 (184 vs. 245 pg/mg) and crypt cell proliferation rate (39.4% vs. 40.9%) and tended to have increased (P = 0.095) Firmicutes:Bacteroidetes ratio (1.0 vs. 3.5) in the jejunum. In conclusion, sows fed with LPL had milk with increased IgG, oleic acids, and linoleic acids without changes in BW and backfat during lactation. These changes could contribute to improved survivability and intestinal health of piglets by increasing IL-8 concentration, enhancing balance among gut-associated microbiome, and increasing enterocyte proliferation in the jejunum.

Declining BRCA-Mediated DNA Repair in Sperm Aging and its Prevention by Sphingosine-1-Phosphate.

Recent data suggest that paternal age can have major impact on reproductive outcomes, and with increased age, there is increased likelihood of chromosomal abnormalities in the sperm. Here, we studied DNA damage and repair as a function of male aging and assessed whether sphingosine-1-phosphate (S1P), a ceramide-induced death inhibitor, can prevent sperm aging by enhancing DNA double-strand breaks (DSB) repair. We observed a significant increase in DNA damage with age and this increase was associated with a decline in the expression of key DNA DSB repair genes in mouse sperm. The haploinsufficiency of BRCA1 male mice sperm showed significantly increased DNA damage and apoptosis, along with decreased chromatin integrity when compared to similar age wild type (WT) mice. Furthermore, haploinsufficiency of BRCA1 male mice had lower sperm count and smaller litter size when crossed with WT females. The resulting embryos had a higher probability of growth arrest and reduced implantation. S1P treatment decreased genotoxic-stress-induced DNA damage in sperm and enhanced the expressions of key DNA repair genes such as BRCA1. Co-treatment with an ATM inhibitor reversed the effects of S1P, implying that the impact of S1P on DNA repair is via the ATM-mediated pathway. Our findings indicate a key role for DNA damage repair mechanism in the maintenance of sperm integrity and suggest that S1P can improve DNA repair in sperm. Further translational studies are warranted to determine the clinical significance of these findings and whether S1P can delay male reproductive aging. There is mounting evidence that sperm quality declines with age, similar to that of the oocyte. However, the reasons behind this decline are poorly understood and there is no medical intervention to improve sperm quality. Our study suggests a strong role for DNA damage repair in maintenance of sperm quality, and for the first time, a potential pharmaceutical approach to prevent sperm aging.

Preventive preclinical efficacy of intravenously administered sphingosine-1-phosphate (S1P) in strengthening hypoxia adaptive responses to acute and sub-chronic hypobaric hypoxia.

Sphingosine-1-phosphate (S1P) is emerging as a hypoxia responsive bio-lipid; systemically raised levels of S1P are proposed to have potential hypoxia pre-conditioning effects. The study aims to evaluate the hypoxia pre-conditioning efficacy of exogenously administered S1P in rats exposed to acute (24-48 hs (h)) and sub-chronic (7 days) hypobaric hypoxia. Sprague-Dawley rats (200 ± 20 g) were preconditioned with 1 µg/kg body weight S1P intravenously for three consecutive days. On the third day, control and S1P preconditioned animals were exposed to hypobaric hypoxia equivalent to 7620 m for 24 h, 48 h and 7 days. Post exposure analysis included body weight quantitation, blood gas/chemistry analysis, vascular permeability assays, evaluation of oxidative stress/inflammation parameters, and estimation of hypoxia responsive molecules. S1P preconditioned rats exposed to acute HH display a significant reduction in body weight loss, as a culmination of improved oxygen carrying capacity, increased 2,3- diphosphoglycerate levels and recuperation from energy deficit. Pathological disturbances such as vascular leakage in the lungs and brain, oxidative stress, pro-inflammatory milieu and raised level of endothelin-1 were also reined. The adaptive and protective advantage conferred by S1P in the acute phase of hypobaric hypoxia exposure, is observed to precipitate into an improved sustenance even after sub-chronic (7d) hypobaric hypoxia exposure as indicated by decreased body weight loss, lower edema index and improvement in general pathology biomarkers. Conclusively, administration of 1 µg/kg body weight S1P, in the aforementioned schedule, confer hypoxia pre-conditioning benefits, sustained up to 7 days of hypobaric hypoxia exposure.

Effects of lysophospholipid supplementation to reduced energy, crude protein, and amino acid diets on growth performance, nutrient digestibility, and blood profiles in broiler chickens.

Two experiments investigated the effects of lysophospholipid (LPL) supplementation on low-energy and low-nitrogenous diets for broilers. A total of 300 one-day-old male chicks (Ross 308) was allotted to 5 treatments in a completely randomized design. Each group consisted of 6 replicates with 10 birds each. Experimental diet I included positive control (PC) having 3,025 (starter), 3,150 (grower), and 3,200 kcal/kg (finisher) of ME; negative control (NC) was 150 kcal/kg of ME lower than PC, and LPL-05, LPL-10, and LPL-15 treatments were NC + 0.05%, 0.10%, and 0.15% of LPL supplementation, respectively. Experimental diet II included positive control (PC) having a formulated amount of crude protein including Lys and Met + Cys that met the Ross 308 standards; negative control (NC) was 4% lower CP and AA than PC; other treatments were supplemented with LPL at 0.05% (LPL-05), 0.10% (LPL-10), and 0.15% (LPL-15) into the NC, respectively. Experiment I showed that growth performance linearly increased as the LPL inclusion increased (P < 0.001). Broilers fed LPL-10 and LPL-15 increased digestibility of DM (P < 0.05), crude protein (P < 0.01), and total amino acids (P < 0.01) compared to NC. Serum glucose (P < 0.01) and high-density lipoprotein (P < 0.05) concentrations were greater in groups fed LPL-10 than those fed PC. Furthermore, leg muscle increased in birds fed LPL-10 compared with NC (P < 0.05). Experiment II observed a linear response to LPL supplementation in the whole period, in terms of body weight gain (P = 0.015) and feed conversion ratio (P = 0.027). Feeding of 0.15% LPL had promising effects on digestibility of crude protein and ether extract compared with NC (P < 0.01 and P < 0.05, respectively). Overall, LPL could be considered as a feed additive to reduced energy (-150 kcal/kg) or nitrogenous diets (-5%) in order to improve growth performance and nutrient digestibility without adverse effects on lymphoid organs and hepatic enzyme of broilers.

Investigation of sphingosin-1-phosphate-triggered matriptase activation using a rat primary hepatocyte model.

Sphingosine-1-phosphate (S1P) has been reported as a matriptase activator. The aim of this study was to reveal if S1P can influence hepcidin production. Furthermore, we investigated how S1P can affect the viability and the redox status of primary hepatocytes. Rat primary hepatocytes were cultivated for 72 h and were treated with 50, 200, 1000 ng/ml S1P. Cell-free supernatants were collected every 24 h. Cell viability was tested by a colorimetric method using tetrazolium compound (MTS). The hepcidin levels in the cell-free supernatants were examined with hepcidin sandwich ELISA to determine the effect of S1P on the hepcidin-modulating ability of matriptase. In order to estimate the extent of S1P-generated oxidative stress, extracellular H2O2 measurements were performed by the use of fluorescent dye. Based on the findings, S1P treatment did not cause cell death for 72 h at concentrations up to 1000 ng/ml. S1P did not influence the extracellular H2O2 production for 72 h. The hepcidin levels were significantly suppressed in hepatocytes exposed to S1P treatment. Further studies would be needed to explore the exact mechanism of action of S1P.

Effects of lysophospholipid on growth performance, carcass yield, intestinal development, and bone quality in broilers.

A study was conducted to evaluate the effects of supplementing different levels of lysophospholipid (LPL) to normal or reduced energy diets on growth performance, carcass yield, intestinal morphology, and skeletal development in broilers. A total of 960 one-day-old Cobb 500 male birds were allocated using a 2 × 4 factorial arrangement with 2 energy levels (NE: normal and RE: 100 kcal/kg metabolizable energy reduction) and 4 LPL supplement levels (0, 0.025, 0.050, and 0.075%). Three diet phases were fed throughout the trial: starter (days 0 to 7), grower (days 8 to 21), and finisher (days 22 to 42) phases. Body weight (BW), feed intake (FI), and feed conversion ratio were calculated at the end of each phase. At day 7 and 21, duodenum and jejunum samples were collected for intestinal morphology and claudin-3 expression analyses, and tibia were sampled for bone quality analyses. At day 42, 4 birds per replicate were selected to measure carcass yield. The results showed low metabolizable energy diets impaired bird's growth performance, intestine development, and bone quality. The 0.075% LPL supplement in NE improved BW, BW gain, and FI in the finisher and overall period compared with no LPL supplement in NE (P < 0.05). In RE, the 0.025% LPL supplement significantly improved growth performance compared to the other treatments in RE (P < 0.05). The interactions on processing parameters were detected with LPL supplement in NE diets; 0.025, 0.05, and 0.075% LPL supplements significantly increased pectoral major percentages compared to the one without LPL supplement in NE (P < 0.05). The 0.075% LPL supplement increased dressing percentage (cold carcass weight/live BW) compared with the others (P < 0.05). The intestine morphology results showed LPL had positive effects on intestine development mainly during the early age (day 7) and claudin-3 expression at both day 7 and 21. Furthermore, LPL supplement significantly increased the total Ca and P deposition and positively affected the bone structure development. In summary, dietary LPL supplementation promoted growth performance, carcass yield, intestinal development, intestinal health, and bone quality.

Sequential Bone-Marrow Cell Delivery of VEGFA/S1P Improves Vascularization and Limits Adverse Cardiac Remodeling After Myocardial Infarction in Mice.

Microvascular dysfunction and resulting tissue hypoxia is a major contributor to the pathogenesis and evolution of cardiovascular diseases (CVD). Diverse gene and cell therapies have been proposed to preserve the microvasculature or boost angiogenesis in CVD, with moderate benefit. This study tested in vivo the impact of sequential delivery by bone-marrow (BM) cells of the pro-angiogenic factors vascular endothelial growth factor (VEGFA) and sphingosine-1-phosphate (S1P) in a myocardial infarction model. For that, mouse BM cells were transduced with lentiviral vectors coding for VEGFA or sphingosine kinase (SPHK1), which catalyzes S1P production, and injected them intravenously 4 and 7 days after cardiac ischemia-reperfusion in mice. Sequential delivery by transduced BM cells of VEGFA and S1P led to increased endothelial cell numbers and shorter extravascular distances in the infarct zone, which support better oxygen diffusion 28 days post myocardial infarction, as shown by automated 3D image analysis of the microvasculature. Milder effects were observed in the remote zone, together with increased proportion of capillaries. BM cells delivering VEGFA and S1P also decreased myofibroblast abundance and restricted adverse cardiac remodeling without major impact on cardiac contractility. The results indicate that BM cells engineered to deliver VEGFA/S1P angiogenic factors sequentially may constitute a promising strategy to improve micro-vascularization and oxygen diffusion, thus limiting the adverse consequences of cardiac ischemia.

Sphingosine 1-phosphate promotes mesenchymal stem cell-mediated cardioprotection against myocardial infarction via ERK1/2-MMP-9 and Akt signaling axis.

AIMS: The sphingolipid metabolite sphingosine 1­phosphate (S1P) has emerged as a potential cardioprotective molecule against ischemic heart disease. Moreover, S1P triggers mobilization and homing of bone marrow-derived stem/progenitor cells into the damaged heart. However, it remains elusive whether S1P promotes mesenchymal stem cells (MSCs)-mediated cardioprotection against ischemic heart diseases. MAIN METHODS: Adipose tissue-derived MSCs (AT-MSCs) were obtained from GFP transgenic mice or C57BL/6J. Myocardial infarction (MI) was induced in C57BL/6J mice by ligation of the left anterior descending coronary artery (LAD). Subsequently, S1P-treated AT-MSCs or vehicle-treated AT-MSCs were intravenously administered for 24 h after induction of MI or sham procedure. KEY FINDINGS: Pre-conditioning with S1P significantly enhanced the migratory and anti-apoptotic efficacies of AT-MSCs. In MI-induced mice, intravenous administration of S1P-treated AT-MSCs significantly augmented their homing and engraftment in ischemic area. Besides, AT-MSCs with S1P pre-treatment exhibited enhanced potencies to inhibit cardiomyocyte apoptosis and fibrosis, and stimulate angiogenesis and preserve cardiac function. Mechanistic studies revealed that S1P promoted AT-MSCs migration through activation of ERK1/2-MMP-9, and protected AT-MSCs against apoptosis via Akt activation. Further, S1P activated the ERK1/2 and Akt via S1P receptor 2 (S1PR2), but not through S1PR1. S1PR2 knockdown by siRNA, however, significantly attenuated S1P-mediated AT-MSCs migration and anti-apoptosis. SIGNIFICANCE: The findings of the present study revealed the protective efficacies of S1P pretreatment on the survival/retention and cardioprotection of engrafted MSCs. Pre-conditioning of donor MSCs with S1P is an effective strategy to promote the therapeutic potential of MSCs for ischemic heart diseases.

Addition of high load of lysophosphatidic acid to standard and high-fat chows causes no significant changes of its circulating and peripheral tissue levels but affects body weight and visceral fat mass of mice.

Oral administration of lysophosphatidic acid (LPA), a critical intercellular lipid mediator, exerts wound healing and antiulcer effects on gastrointestinal system. To evaluate effects of food-derived LPA on body homeostasis, we measured LPA levels by liquid chromatography-tandem mass spectrometry in chows, feces, plasma, liver, and visceral fat of mice fed a normal or high-fat chow supplemented with or without LPA-rich soybean phospholipids for 30 days. Reductions in daily body weight gains and visceral fat mass were mainly related to lower chow intake by mice fed the LPA-rich high-fat chow, whereas reduced body weight gains and fat mass were mainly related to decreased intestinal triacylglycerol absorption in mice fed LPA-rich chow. Our results showed no significant increase in plasma, liver, or adipose LPA levels, even if a quite high LPA concentration (2.0%) in chows was ingested daily, suggesting limited effects of food-derived LPA on the lumen side of the digestive tract. © 2018 BioFactors, 44(6):548-557, 2018.

Effects of dietary supplemental lysophospholipids and vitamin C on performance, antioxidant enzymes, lipid peroxidation, thyroid hormones and serum metabolites of broiler chickens reared under thermoneutral and high ambient temperature.

The effects of dietary supplemental lysophospholipids (LPLs) and vitamin C (VC) on performance, activity of antioxidant enzymes, and thyroid hormones of broiler chickens reared under thermoneutral and high ambient temperatures were evaluated. A total of 1,680 broiler chicks (Cobb 500) in finishing rearing period (days 21-38 of age) were allotted to two groups: thermoneutral (TN) and heat stress (HS). In the TN group, 480 chicks were subjected to four treatments with four replicates (n = 30) and maintained in usual ambient temperature (24 ± 1°C). In HS group, the remaining 1,200 chicks were subjected to four treatments with 10 replicates (n = 30) and exposed to high ambient temperature (34 ± 1°C for 8 hr daily). In both groups, four iso-caloric and iso-nitrogenous experimental diets based on a 2 × 2 factorial arrangements including supplemental LPLs (0 or 1,000 mg/kg) and VC (0 or 500 mg/kg) were formulated and used. Supplemental LPLs decreased (p < 0.05) body weight gain and increased FCR in the TN and HS groups. In the TN group, increased (p < 0.05) serum glucose was observed in chickens fed with dietary supplemental VC. In the HS group, decreased (p < 0.05) total protein concentration was detected in birds fed with supplemental LPLs. In both TN and HS groups, decreased (p < 0.05) uric acid concentration was detected in chicks fed with the VC-supplemented diets. A significant (p < 0.05) interaction between LPLs and VC on lactate concentration in the TN group was observed. In the HS group, decreased breast malondialdehyde concentration was detected in birds fed with the VC-supplemented diet. In the TN group, increased serum total antioxidant status was detected in birds fed with the LPLs-supplemented diet. In conclusion, LPLs improved oxidative stability and increased the antioxidant capacity of the serum. In addition, vitamin C modified heat stress and reduced lipid peroxidation.

Lysophosphatidic acid induces neuronal cell death via activation of asparagine endopeptidase in cerebral ischemia-reperfusion injury.

Lysophosphatidic acid (LPA), an extracellular signaling molecule, influences diverse biological events, including the pathophysiological process induced after ischemic brain injury. However, the molecular mechanisms mediating the pathological change after ischemic stroke remain elusive. Here we report that asparagine endopeptidase (AEP), a lysosomal cysteine proteinase, is regulated by LPA during stroke. AEP proteolytically cleaves tau and generates tauN368 fragments, triggering neuronal death. Inhibiting the generation of LPA reduces the expression of AEP and tauN368, and alleviates neuronal cell death. Together, this evidence indicates that the LPA-AEP pathway plays a key role in the pathophysiological process induced after ischemic stroke. Inhibition of LPA could be a useful therapeutic for treating neuronal injury after stroke.

Lysophosphatidic acid propagates post-injury Schwann cell dedifferentiation through LPA1 signaling.

Lysophosphatidic acid (LPA) is a pleiotropic signaling lipid that acts as ligand for at least six specific G-protein coupled receptors. Schwann cells (SC) are known to mainly express the LPA1 receptor subtype. An emerging body of evidence has linked LPA with injury-induced peripheral nerve demyelination as well as neuropathic pain. However, the molecular mechanisms underlying its demyelinating effect have not been conclusively elucidated. We aimed to decipher the demyelinating effect in vitro as well as in vivo by studying markers of SC differentiation and dedifferentiation: Myelinated dorsal root ganglia (DRG) cultures were treated either with LPA, LPA plus AM095 (LPA1 antagonist) or vehicle. Myelin content was subsequently investigated by Sudan Black staining and immunocytochemistry. In vivo, we performed sciatic nerve crush in C57BL/6 mice treated with AM095 at 10mg/kg. In DRG cultures, LPA caused a significant reduction of myelin as demonstrated by both Sudan Black staining and immunocytochemical analysis of myelin basic protein. Demyelination was paralleled by an upregulation of TNF-alpha as well as downregulation of Sox10, a marker for SC differentiation. LPA mediated effects were largely blocked by the addition of the LPA1 receptor antagonist AM095. In the in vivo model, AM095 treatment prior to crush injury increased Sox10 expression in SCs in the distal nerve stump while reducing the number of cells expressing the SC dedifferentiation marker Sox2. Additionally, TNF-alpha immunofluorescence was reduced in CD11b-positive cells. These data indicate that LPA may be a critical factor that shifts SCs towards a post-injury phenotype and contributes to the onset of Wallerian degeneration.

2-O-Carba-oleoyl cyclic phosphatidic acid induces glial proliferation through the activation of lysophosphatidic acid receptor.

Lysophosphatidic acid (LPA) and cyclic phosphatidic acid (cPA) are one of the lipid mediators regulating cell proliferation and differentiation through the activation of LPA receptors. An LPA receptor-mediated signal is important for the development of the central nervous system, while it has been demonstrated that LPA caused microglial activation and astroglial dysfunction. Previously, we have reported that cPA and carba analog of cPA, 2-O-carba-cPA (2ccPA), protected neural damage caused by transient ischemia. However, little is known about the target cell of cPA/2ccPA in the central nervous systems. Here, we examined the effect of 2ccPA on glial proliferation and differentiation using the primary astrocytes and oligodendrocyte precursor cells (OPCs) cultures. 2ccPA increased the DNA synthesis of astrocytes and OPCs, but it did not reduce the formazan production in the mitochondria. Further, 2ccPA increased the cell number and cell survival against oxidative stress. The inhibition of LPA receptors by ki16425 abolished 2ccPA-induced DNA synthesis. Extracellular signal-regulated kinase (ERK) was activated by 2ccPA, which contributed to the astroglial DNA synthesis. These results suggest that 2ccPA is a beneficial regulator of glial population through the activation of LPA receptor without reduction of mitochondrial activity.

Sphingosine-1-phosphate ameliorates the cardiac hypertrophic response through inhibiting the activity of histone deacetylase-2.

Inhibition of histone deacetylase-2 (HDAC2), which is a prohypertrophic factor in the heart, can functionally attenuate cardiac hypertrophy. The present study aimed to investigate whether sphingosine­1­phosphate (S1P), which has recently been reported to suppress HDAC2 activity, could ameliorate the cardiac hypertrophic response and improve cardiac function in mice with transverse aortic constriction (TAC), as well as to determine the underlying mechanisms. Briefly, 8­week­old male C57BL/6 mice were randomly divided into sham, TAC and TAC + S1P groups; the results indicated that S1P treatment attenuated TAC­induced cardiac dysfunction. In addition, heart size and the expression levels of fetal cardiac genes were reduced in the TAC + S1P group compared with in the TAC group. Furthermore, in cultured H9c2 cells exposed to phenylephrine, S1P was revealed to decrease cardiomyocyte size and the exaggerated expression of fetal cardiac genes. The present study also demonstrated that S1P had no effect on HDAC2 expression, but it did suppress its activity and increase acetylation of histone H3 in vivo and in vitro. Krüppel­like factor 4 (KLF4) is an antihypertrophic transcriptional regulator, which mediates HDAC inhibitor­induced prevention of cardiac hypertrophy; in the present study, KLF4 was upregulated by S1P. Finally, the results indicated that S1P receptor 2 (S1PR2) may be involved in the antihypertrophic effects, whereas the suppressive effects of S1P on HDAC2 activity were independent of S1PR2. In conclusion, the present study demonstrated that S1P treatment may ameliorate the cardiac hypertrophic response, which may be partly mediated by the suppression of HDAC2 activity and the upregulation of KLF4; it was suggested that S1PR2 may also be involved. Therefore, S1P may be considered a potential therapy for the treatment of heart diseases caused by cardiac hypertrophy.

Lysophosphatidic acid protects against acetaminophen-induced acute liver injury.

We investigated the effect of lysophosphatidic acid (LPA) in experimental acetaminophen (APAP)-induced acute liver injury. LPA administration significantly reduced APAP-challenged acute liver injury, showing attenuated liver damage, liver cell death and aspartate aminotransferase and alanine aminotransferase levels. APAP overdose-induced mortality was also significantly decreased by LPA administration. Regarding the mechanism involved in LPA-induced protection against acute liver injury, LPA administration significantly increased the glutathione level, which was markedly decreased in APAP challenge-induced acute liver injury. LPA administration also strongly blocked the APAP challenge-elicited phosphorylation of JNK, ERK and GSK3ß, which are involved in the pathogenesis of acute liver injury. Furthermore, LPA administration decreased the production of TNF-α and IL-1ß in an experimental drug-induced liver injury animal model. Mouse primary hepatocytes express LPA1,3-6, and injection of the LPA receptor antagonist KI16425 (an LPA1,3-selective inhibitor) or H2L 5765834 (an LPA1,3,5-selective inhibitor) did not reverse the LPA-induced protective effects against acute liver injury. The therapeutic administration of LPA also blocked APAP-induced liver damage, leading to an increased survival rate. Collectively, these results indicate that the well-known bioactive lipid LPA can block the pathogenesis of APAP-induced acute liver injury by increasing the glutathione level but decreasing inflammatory cytokines in an LPA1,3,5-independent manner. Our results suggest that LPA might be an important therapeutic agent for drug-induced liver injury.