Arginase-induced cell death pathways and metabolic changes in cancer cells are not altered by insulin.

Arginine, a semi-essential amino acid, is critical for cell growth. Typically, de novo synthesis of arginine is sufficient to support cellular processes, however, it becomes vital for cancer cells that are unable to synthesise arginine due to enzyme deficiencies. Targeting this need, arginine depletion with enzymes such as arginase (ARG) has emerged as a potential cancer therapeutic strategy. Studies have proposed using high dose insulin to induce a state of hypoaminoacidaemia in the body, thereby further reducing circulating arginine levels. However, the mitogenic and metabolic properties of insulin could potentially counteract the therapeutic effects of ARG. Our study examined the combined impact of insulin and ARG on breast, lung, and ovarian cell lines, focusing on cell proliferation, metabolism, apoptosis, and autophagy. Our results showed that the influence of insulin on ARG uptake varied between cell lines but failed to promote the proliferation of ARG-treated cells or aid recovery post-ARG treatment. Moreover, insulin was largely ineffective in altering ARG-induced metabolic changes and did not prevent apoptosis. In vitro, at least, these findings imply that insulin does not offer a growth or survival benefit to cancer cells being treated with ARG.

Triptolide inhibits the proinflammatory potential of myeloid-derived suppressor cells via reducing Arginase-1 in rheumatoid arthritis.

Triptolide (TPT) is widely used in the treatment of rheumatoid arthritis (RA). However, its regulatory mechanisms are not fully understood. This study demonstrated that Myeloid-derived suppressor cells (MDSCs) were expanded in both RA patients and arthritic mice. The frequency of MDSCs was correlated with RA disease severity and T helper 17 (Th17) responses. MDSCs from RA patients promoted the polarization of Th17 cells in vitro, which could be substantially attenuated by blocking arginase-1 (Arg-1). TPT inhibited the differentiation of MDSCs, particularly the monocytic MDSCs (M-MDSCs) subsets, as well as the expression of Arg-1 in a dose dependent manner. Alongside, TPT treatment reduced the potential of MDSCs to promote the polarization of IL-17+ T cell in vitro. Consistently, TPT immunotherapy alleviated adjuvant-induced arthritis (AIA) in a mice model, and reduced the frequency of MDSCs, M-MDSCs and IL-17+ T cells simultaneously. The presented data suggest a pathogenic role of MDSCs in RA and may function as a novel and effective therapeutic target for TPT in RA.

Ginseng-derived nanoparticles reprogram macrophages to regulate arginase-1 release for ameliorating T cell exhaustion in tumor microenvironment.

BACKGROUND: Lines of evidence indicated that, immune checkpoints (ICs) inhibitors enhanced T cell immune response to exert anti-tumor effects. However, T cell exhaustion has been so far a major obstacle to antitumor immunotherapy in colorectal cancer patients. Our previous studies showed that ginseng-derived nanoparticles (GDNPs) inhibited the growth of various tumors by reprograming tumor-associated macrophages (TAMs) and downregulated the ICs expression on T cells in tumor microenvironment (TME), but the underlying effector mechanisms remained unclear. METHODS: The correlation between arginase-1 (ARG1) and T cells was computed based on the colorectal cancer patients in TCGA database. In vitro, we observed that GDNPs reprogrammed TAMs inhibited ARG1 release and ultimately ameliorated T cell exhaustion according to several techniques including WB, PCR, ELISA and flow cytometry. We also used an in vivo MC38 tumor-bearing model and administered GDNPs to assess their anti-tumor effects through multiple indices. The mechanism that GDNPs improved T cell exhaustion was further clarified using the bioinformatics tools and flow cytometry. RESULTS: GDNPs reprogramed TAMs via reducing ARG1 production. Moreover, normalized arginine metabolism ameliorated T cell exhaustion through mTOR-T-bet axis, resulting in reduced ICs expression and enhanced CD8+ T cells expansion. CONCLUSIONS: By regulating the mTOR-T-bet axis, GDNPs reprogramed macrophages to regulate ARG1 release, which further ameliorated T cell exhaustion in TME. These findings provided new insights into comprehending the mechanisms underlying the mitigation of T cell exhaustion, which may facilitate the development of innovative therapeutic strategies in the field of cancer treatment.

The effects of a comparatively higher dose of 1000 mg/kg/d of oral L- or D-arginine on the L-arginine metabolic pathways in male Sprague-Dawley rats.

Oral L-arginine supplements are popular mainly for their nitric oxide mediated vasodilation, but their physiological impact is not fully known. L-arginine is a substrate of several enzymes including arginase, nitric oxide synthase, arginine decarboxylase, and arginine: glycine amidinotransferase (AGAT). We have published a study on the physiological impact of oral L- and D-arginine at 500 mg/kg/day for 4 wks in male Sprague-Dawley rats. We investigated the effects of oral L-arginine and D-arginine at a higher dose of 1000 mg/kg/d for a longer treatment duration of 16 wks in 9-week-old male Sprague-Dawley rats. We measured the expression and activity of L-arginine metabolizing enzymes, and levels of their metabolites in the plasma and various organs. L-arginine did not affect the levels of L-arginine and L-lysine in the plasma and various organs. L-arginine decreased arginase protein expression in the upper small intestine, and arginase activity in the plasma. It also decreased AGAT protein expression in the liver, and creatinine levels in the urine. L-arginine altered arginine decarboxylase protein expression in the upper small intestine and liver, with increased total polyamines plasma levels. Endothelial nitric oxide synthase protein was increased with D-arginine, the presumed metabolically inert isomer, but not L-arginine. In conclusion, oral L-arginine and D-arginine at a higher dose and longer treatment duration significantly altered various enzymes and metabolites in the arginine metabolic pathways, which differed from alterations produced by a lower dose shorter duration treatment published earlier. Further studies with differing doses and duration would allow for a better understanding of oral L-arginine uses, and evidence based safe and effective dose range and duration.

Tetramerization of STAT5 regulates monocyte differentiation and the dextran sulfate sodium-induced colitis in mice.

In response to external stimuli during immune responses, monocytes can have multifaceted roles such as pathogen clearance and tissue repair. However, aberrant control of monocyte activation can result in chronic inflammation and subsequent tissue damage. Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces monocyte differentiation into a heterogenous population of monocyte-derived dendritic cells (moDCs) and macrophages. However, the downstream molecular signals that dictate the differentiation of monocytes under pathological conditions is incompletely understood. We report here that the GM-CSF-induced STAT5 tetramerization is a critical determinate of monocyte fate and function. Monocytes require STAT5 tetramers to differentiate into moDCs. Conversely, the absence of STAT5 tetramers results in a switch to a functionally distinct monocyte-derived macrophage population. In the dextran sulfate sodium (DSS) model of colitis, STAT5 tetramer-deficient monocytes exacerbate disease severity. Mechanistically, GM-CSF signaling in STAT5 tetramer-deficient monocytes results in the overexpression of arginase I and a reduction in nitric oxide synthesis following stimulation with lipopolysaccharide. Correspondingly, the inhibition of arginase I activity and sustained supplementation of nitric oxide ameliorates the worsened colitis in STAT5 tetramer-deficient mice. This study suggests that STAT5 tetramers protect against severe intestinal inflammation through the regulation of arginine metabolism.

Blueberry Leaf Extract Prevents Lacrimal Hyposecretion in Sjögren's Syndrome-like Model of Non-obese Diabetic Mice.

BACKGROUND/AIM: This study evaluated the effect of blueberry leaf hot water extract (BLEx) on Sjögren's syndrome (SS)-like lacrimal hyposecretion in male non-obese diabetic (NOD) mice. MATERIALS AND METHODS: NOD or BALB/c mice were fed 1% BLEx or control (AIN-93G) for 2 weeks from the age of 4 to 6 weeks. Pilocarpine-induced tear volume was measured using a phenol red-impregnated thread. The lacrimal glands were evaluated histologically by H&E staining. The IL-1ß and TNF-α levels in the lacrimal gland tissue were measured by ELISA. The mRNA expression levels of secretion-related proteins were measured by real-time PCR. LC3 I/II and arginase 1 expression levels were measured by western blot. RESULTS: After feeding with BLEx, pilocarpine-induced tear secretion in NOD mice was increased. In contrast, the mRNA expression levels of the cholinergic muscarinic M3 receptor, aquaporin 5, and ion channels related to lacrimal secretion were not changed by BLEx administration. In addition, the protein expression of arginase 1, which was recently reported to be involved in tear hyposecretion in NOD mice, was also not improved by BLEx administration. Although infiltration in the lacrimal gland of NOD mice was not decreased, the levels of TNF-α and the autophagy-related protein LC3 were significantly suppressed by BLEx treatment. CONCLUSION: BLEx treatment may ameliorate lacrimal hyposecretion in NOD mice by delaying the progression of autoimmune disease by suppressing autophagy in lacrimal glands.

Fadogia agrestis (Schweinf. Ex Hiern) Stem Extract Restores Selected Biomolecules of Erectile Dysfunction in the Testicular and Penile Tissues of Paroxetine-Treated Wistar Rats.

Inadequate release of nitric oxide (NO) by the penile tissue impacts negatively on penile erection causing erectile dysfunction (ED). Fadogia agrestis has been implicated in the management of ED without information on key biomolecules associated with ED in male rats. Therefore, this study evaluated the influence of aqueous extract of Fadogia agrestis stem (AEFAS) on key biomolecules associated with ED in the penile and testicular tissues of male Wistar rats induced with ED by paroxetine. Thirty male rats were assigned into 6 groups (I, II, III, IV, V and VI) of 5. Group I (sham control, without ED) was administered distilled water orally. Paroxetine-induced ED rats in groups II (negative control), III (positive control), IV, V and VI received distilled water, sildenafil citrate (SC, 50 mg/kg body weight) and AEFAS at 18, 50 and 100 mg/kg body weight respectively. Paroxetine lowered/reduced (p < 0.05) the MF, IF, EF, NO, cGMP, catalase, SOD, T-SH, GSH and GST whilst it prolonged/increased ML, IL, EL, PEI, AChE, PDE5, arginase, ACE, TBARS and H2O2. Contrastingly, AEFAS like sildenafil citrate increased (p < 0.05) the penile and testicular NO, cGMP, catalase, SOD, T-SH, GSH and GST and reduced AChE, PDE5, arginase, ACE, TBARS and H2O2 to levels that compared favourably (p > 0.05) with those of sham control. The study concluded that AEFAS restored the NO/cGMP pathway and ED-associated key enzymes in the penile and testicular tissues of male rats via antioxidant means. The study recommended the use of aqueous extract of Fadogia agrestis stem in managing ED after clinical trials.

Capparis spinosa inhibits Leishmania major growth through nitric oxide production in vitro and arginase inhibition in silico.

Cutaneous leishmaniasis is an infectious disease, considered as a major public health problem in different regions of the world. The current treatments are limited due to their toxicity and treatment failures, which have increased the search for new substances of natural origin to control this infection. Capparis spinosa is an important medicinal plant, rich in biochemical compounds with a broad range of activities including antimicrobial effects. Nevertheless, more investigations are still needed to determine its effect on Leishmania parasites. This study aimed to evaluate the effect of C. spinosa' extracts on Leishmania major promastigotes and amastigotes growth as well as on L-arginine metabolic pathways, especially the production of leishmanicidal molecules such as nitric oxide. Our results showed that C. spinosa' methanolic and aqueous extracts contained polyphenols and flavonoids at different concentrations. The methanolic extract of C. spinosa, compared to the aqueous extract, showed significantly higher amounts of total polyphenols (21.23 ± 1.08) mg GAE/g of dw (P < 0.05), as well as a higher antioxidant activity evaluated respectively by Reducing Power and DPPH (EC50: 0.31 ± 0.02 and 7.69 ± 1.28) mg/ml. Both extracts significantly inhibited L. major promastigotes and intra-macrophagic amastigotes growth in vitro in a dose-dependent manner (P < 0.001) and induced NO production not only in Leishmania-infected macrophages but also in uninfected macrophages, without showing any cytotoxicity in vitro. Furthermore, in silico docking studies showed that C. spinosa compounds identified by RP-HPLC exhibited inhibitory activity against the arginase enzyme. The leishmanicidal effect of C. spinosa may be due to its phenolic content and its mechanism of action may be mediated by an increase in NO production and by the inhibition of arginase enzyme in silico. These findings support the hypothesis that C. spinosa might be a valuable source of new biomolecules for leishmaniasis treatment.

Anti-inflammatory effect of polyherbal composition with hepatoprotective and choleretic properties on LPS-stimulated murine macrophages.

OBJECTIVES: A polyherbal formulation with hepatoprotective and choleretic properties combining pharmacological potential of eight medicinal plants was developed in Nargiz Medical center (Republic of Azerbaijan) for the use as herbal tea. To explore the effect of polyherbal composition on the metabolism of LPS-stimulated macrophages in vitro. METHODS: The qualitative and quantitative phytochemical analysis was conducted using specific color reactions and gas chromatography-mass spectrometry (GC-MS). Nitric oxide (NO) assay was determined using the Griess reaction. Reactive oxygen species (ROS) generation was measured using ROS-sensitive fluorescence indicator, H2DCFDA, by flow cytometry. Arginase activity was examined by colorimetric method. RESULTS: The studied polyherbal formulation exerted anti-inflammatory activity in LPS-stimulated macrophages which was evidenced by dose-dependent decrease of ROS generation and by shift of arginine metabolism to the increase of arginase activity and decrease of NO release. CONCLUSIONS: Our findings suggest that the herbal tea reduces macrophage inflammatory activity, that provide an important rationale to utilize it for the attenuation of chronic inflammation typical of hepatobiliary disorders.

Targeting arginase-1 exerts antitumor effects in multiple myeloma and mitigates bortezomib-induced cardiotoxicity.

Multiple myeloma (MM) remains an incurable malignancy of plasma cells despite constantly evolving therapeutic approaches including various types of immunotherapy. Increased arginase activity has been associated with potent suppression of T-cell immune responses in different types of cancer. Here, we investigated the role of arginase 1 (ARG1) in Vκ*MYC model of MM in mice. ARG1 expression in myeloid cells correlated with tumor progression and was accompanied by a systemic drop in ʟ-arginine levels. In MM-bearing mice antigen-induced proliferation of adoptively transferred T-cells was strongly suppressed and T-cell proliferation was restored by pharmacological arginase inhibition. Progression of Vκ*MYC tumors was significantly delayed in mice with myeloid-specific ARG1 deletion. Arginase inhibition effectively inhibited tumor progression although it failed to augment anti-myeloma effects of bortezomib. However, arginase inhibitor completely prevented development of bortezomib-induced cardiotoxicity in mice. Altogether, these findings indicate that arginase inhibitors could be further tested as a complementary strategy in multiple myeloma to mitigate adverse cardiac events without compromising antitumor efficacy of proteasome inhibitors.

Tao-Hong-Si-Wu decoction improves depressive symptoms in model rats via amelioration of BDNF-CREB-arginase I axis disorders.

CONTEXT: The traditional Chinese medicine formula Tao-Hong-Si-Wu decoction (TSD), used for treating ischaemic stroke, has the potential to treat depressive disorder (DD). OBJECTIVE: To explore the effective targets of TSD on DD animal models. MATERIALS AND METHODS: Sprague-Dawley (SD) rats were modelled by inducing chronic unpredictable mild stress (CUMS) during 35 days and treated with three dosages of TSD (2.5, 5 and 10 g/kg) or fluoxetine (10 mg/kg) by oral gavage for 14 days. Bodyweight measurements and behavioural tests were performed to observe the effect of TSD on the CUMS animals. A gas chromatography coupled with mass spectrometry (GC-MS)-based metabolomic analysis was conducted to reveal the metabolic characteristics related to the curative effect of TSD. Levels of the proteins associated with the feature metabolites were analysed. RESULTS: Reduced immobile duration and crossed squares in the behavioural tests were raised by 48.6% and 32.9%, on average, respectively, by TSD treatment (ED50=3.2 g/kg). Antidepressant effects of TSD were associated with 13 decreased metabolites and the restorations of ornithine and urea in the serum. TSD (5 g/kg) raised serum serotonin by 54.1 mg/dL but suppressed arginase I (Arg I) by 47.8 mg/dL in the CUMS rats. Proteins on the brain-derived neurotrophic factor (BDNF)-cAMP response element-binding protein (CREB) axis that modulate the inhibition of Arg I were suppressed in the CUMS rats but reversed by the TSD intervention. DISCUSSION AND CONCLUSIONS: TSD improves depression-like symptoms in CUMS rats. Further study will focus on the antidepressant-like effects of effective compounds contained in TSD.

Moringa seed-supplemented diets modulate ACE activity but not its gene expression in L-NAME-induced hypertensive rats.

INTRODUCTION: We investigated the effect of dietary inclusions of Moringa seed (5% and 10%) on blood pressure, angiotensin-1 converting enzyme (ACE) activity, and gene expression, as well as redox status in hypertensive rats. MATERIAL AND METHODS: Wistar strain albino rats were fed moringa seed-based diets for two weeks prior L-NAME (40 mg/kg/day, p.o.) administration for another ten days. Subsequently, the blood pressure was monitored. Furthermore, the kidney homogenates were assayed for ACE activity and gene expression, as well as oxidative stress markers. RESULTS: The increased (systolic = 297 ± 59.30 mmHg; diastolic= 242 ± 51.96 mmHg) blood pressure, arginase activity, and reduced nitric oxide level were significantly ameliorated in hypertensive rats treated with the seed. However, the elevated ACE activity was significantly reduced but not the upregulated ACE1 gene. Also, the reduced antioxidant enzyme activities were ameliorated with a significant downregulation in their regulator-Nrf2. Rutin (4.07 ± 0.02 mg/g) and quercitrin (4.06 ± 0.01 mg/g), among others, were found in the seed. DISCUSSION: This study suggests that moringa seed offers its antihypertensive properties by acting as an ACE inhibitor but not its gene modulator, and also modulates the antioxidant system through interaction with Nrf2. CONCLUSION: Moringa seed could act as an ACE inhibitor and not its gene modulator.

Folic acid oversupplementation during pregnancy disorders lipid metabolism in male offspring via regulating arginase 1-associated NOS3-AMPKα pathway.

BACKGROUND & AIMS: Folic acid supplementation is widely accepted during pregnancy, as it exerts a protective effect on neural tube defects. However, the long-term underlying effects of folic acid supplementation during pregnancy (FASDP) on offspring remain unclear. METHODS: Thirty pregnant female rats were randomly divided into normal control group, folic acid appropriate supplementation group (2.5 × FA group) and folic acid oversupplementation group (5 × FA group) and fed with corresponding folic acid concentration AIN93G diet. UPLC-Q-TOF-MS, UPLC-TQ-MS and GC-MS were performed to detect the serum metabolites profiles in adult male offspring and explore the effects of FASDP. Moreover, molecular biology technologies were used to clarify the underlying mechanism. RESULTS: We demonstrate that 2.5-folds folic acid leads to dyslipidemic-diabetic slightly in male offspring, while 5-folds folic acid aggravates the disorder and prominent hepatic lipid accumulations. Using untargeted and targeted metabolomics, total 63 differential metabolites and 12 significantly differential KEGG pathways are identified. Of note, arginine biosynthesis, arginine and proline metabolism are the two most significant pathways. Mechanistic investigations reveal that the increased levels of arginase-1 (Arg1) causes the lipid metabolism disorder by regulating nitric oxide synthase-3 (NOS3)-adenosine monophosphate activated protein kinase-α (AMPKα) pathway, resulting in lipid accumulation in hepatocytes. CONCLUSIONS: Our data suggest that maternal folic acid oversupplementation during pregnancy contributes to lipid metabolism disorder in male offspring by regulating Arg1-NOS3-AMPKα pathway.

Review of arginase as a promising biocatalyst: characteristics, preparation, applications and future challenges.

As a committed step in the urea cycle, arginase cleaves l-arginine to form l-ornithine and urea. l-Ornithine is essential to: cell proliferation, collagen formation and other physiological functions, while the urea cycle itself converts highly toxic ammonia to urea for excretion. Recently, arginase was exploited as an efficient catalyst for the environmentally friendly synthesis of l-ornithine, an abundant nonprotein amino acid that is widely employed as a food supplement and nutrition product. It was also proposed as an arginine-reducing agent in order to treat arginase deficiency and to be a means of depleting arginine to treat arginine auxotrophic tumors. Targeting arginase inhibitors of the arginase/ornithine pathway offers great promise as a therapy for: cardiovascular, central nervous system diseases and cancers with high arginase expression. In this review, recent advances in the characteristics, structure, catalytic mechanism and preparation of arginase were summarized, with a focus being placed on the biotechnical and medical applications of arginase. In particular, perspectives have been presented on the challenges and opportunities for the environmentally friendly utilization of arginase during l-ornithine production and in therapies.

The Influence of Water-Soluble Polysaccharides of Crataegus sanguinea Pall. on Nitric Oxide Production by Macrophages.

The in vitro addition of water-soluble polysaccharides isolated from the leaves of Crataegus sanguinea Pall. to culture of mouse peritoneal macrophages induced classical activation of antigen-presenting cells by increasing NO synthase activity and reducing arginase expression.

Arginine Supplementation Promotes Extracellular Matrix and Metabolic Changes in Keratoconus.

Keratoconus (KC) is a common corneal ectatic disease that affects 1:500-1:2000 people worldwide and is associated with a progressive thinning of the corneal stroma that may lead to severe astigmatism and visual deficits. Riboflavin-mediated collagen crosslinking currently remains the only approved treatment to halt progressive corneal thinning associated with KC by improving the biomechanical properties of the stroma. Treatments designed to increase collagen deposition by resident corneal stromal keratocytes remain elusive. In this study, we evaluated the effects of arginine supplementation on steady-state levels of arginine and arginine-related metabolites (e.g., ornithine, proline, hydroxyproline, spermidine, and putrescine) and collagen protein expression by primary human corneal fibroblasts isolated from KC and non-KC (healthy) corneas and cultured in an established 3D in vitro model. We identified lower cytoplasmic arginine and spermidine levels in KC-derived constructs compared to healthy controls, which corresponded with overall higher gene expression of arginase. Arginine supplementation led to a robust increase in cytoplasmic arginine, ornithine, and spermidine levels in controls only and a significant increase in collagen type I secretion in KC-derived constructs. Further studies evaluating safety and efficacy of arginine supplementation are required to elucidate the potential therapeutic applications of modulating collagen deposition in the context of KC.

Neuroblastoma Formation Requires Unconventional CD4 T Cells and Arginase-1-Dependent Myeloid Cells.

Immune cells regulate tumor growth by mirroring their function as tissue repair organizers in normal tissues. To understand the different facets of immune-tumor collaboration through genetics, spatial transcriptomics, and immunologic manipulation with noninvasive, longitudinal imaging, we generated a penetrant double oncogene-driven autochthonous model of neuroblastoma. Spatial transcriptomic analysis showed that CD4+ and myeloid populations colocalized within the tumor parenchyma, while CD8+ T cells and B cells were peripherally dispersed. Depletion of CD4+ T cells or CCR2+ macrophages, but not B cells, CD8+ T cells, or natural killer (NK) cells, prevented tumor formation. Tumor CD4+ T cells displayed unconventional phenotypes and were clonotypically diverse and antigen independent. Within the myeloid fraction, tumor growth required myeloid cells expressing arginase-1. Overall, these results demonstrate how arginine-metabolizing myeloid cells conspire with pathogenic CD4+ T cells to create permissive conditions for tumor formation, suggesting that these protumorigenic pathways could be disabled by targeting myeloid arginine metabolism. SIGNIFICANCE: A new model of human neuroblastoma provides ways to track tumor formation and expansion in living animals, allowing identification of CD4+ T-cell and macrophage functions required for oncogenesis.

M-CSF and prostratin induced Mregs promote immune tolerance in transplanted mice through Arg-1 pathway.

OBJECTIVE: Regulatory macrophages (Mregs) are a group of heterogeneous macrophages. These cells could induce immunosuppressive effects through the expression of immune regulatory molecules and cytokines. METHODS: The differentiation of Mregs was induced by treating bone marrow cells with M-CSF and prostratin in vitro. The cell-phenotypes and immunosuppressive function were determined by flow cytometry. Rt-PCR was employed to assess the mechanisms of Mregs. Skin grafted mouse model was used for in vivo validation. RESULTS: Mregs induced by M-CSF + prostratin had a strong inhibitory effect on T cell proliferation and cytokines production. The phenotype of induced bone marrow cells changed towards Mregs. These Mregs could induce the differentiation of Tregs in vivo. Arg-1 expression in these cells were significantly upregulated. Inhibition of arginase (Arg) or arginine supplement significantly reversed the immunosuppressive function. In mice skin-grafted models, adoptive transfer of these Mregs significantly prolonged allograft survival. In mice models, Arg-1 expression significantly elevated on skin grafts cells and Tregs increased in graft tissues. CONCLUSIONS: We successfully developed a Mregs-inducing protocol with the combination of M-CSF and prostratin in vitro. M-CSF + prostratin induced Mregs prevented mice skin graft rejection through upregulating the expression Arg-1.

Arginase plays an important role in ammonia detoxification of yellow catfish Pelteobagrus fulvidraco.

A two-stage study was carried out to test the mechanism of arginase in ammonia detoxification of yellow catfish. At stage 1, fish was injected lethal half concentration ammonium acetate and 0.9% sodium chloride respectively every 12 h in six replicates for 72 h. The result found that no significant different in serum ammonia contents of fish in ammonium acetate group at hours 12, 24, 36, 48, 60 and 72. At stage 2, ammonium acetate group was split in two, one continued to injected with ammonium acetate (NH3 group) and the other with ammonium acetate and valine (an inhibitor of arginase; Val group); Sodium chloride group also was split in two, one continued to injected with sodium chloride (NaCl group) and the other with sodium chloride and valine (NaCl + Val group). The experiment continued for 12 h. Serum ammonia and liver arginine contents of fish in Val group were higher than those of fish in NH3 group; Compared with NaCl group, arginase activity and ARG 1 expression in liver of fish in Val group were lower; Fish in NaCl and NaCl + Val groups had the lowest serum superoxide dismutase activities, malondialdehyde, tumor necrosis factor-α, interleukin 1 and 8 contents, TNF-α, IL-1 and IL-8 expressions than fish in NH3 and Val groups, and had the higher lysozyme activities, complement 3 and 4 contents. This study indicates that ammonia poisoning would lead to oxidative damage, immunosuppression and inflammation in yellow catfish; Arginase may be an important target of ammonia toxicity in yellow catfish; Exogenous arginine supplementation might alleviate the symptoms of ammonia poisoning in yellow catfish.

Arginase inhibition by rhaponticin increases L-arginine concentration that contributes to Ca2+-dependent eNOS activation.

Although arginase primarily participates in the last reaction of the urea cycle, we have previously demonstrated that arginase II is an important cytosolic calcium regulator through spermine production in a p32-dependent manner. Here, we demonstrated that rhaponticin (RPT) is a novel medicinal-plant arginase inhibitor and investigated its mechanism of action on Ca2+-dependent endothelial nitric oxide synthase (eNOS) activation. RPT was uncompetitively inhibited for both arginases I and II prepared from mouse liver and kidney. It also inhibited arginase activity in both aorta and human umbilical vein endothelial cells (HUVECs). Using both microscope and FACS analyses, RPT treatments induced increases in cytosolic Ca2+ levels using Fluo-4 AM as a calcium indicator. Increased cytosolic Ca2+ elicited the phosphorylations of both CaMKII and eNOS Ser1177 in a time-dependent manner. RPT incubations also increased intracellular L-arginine (L-Arg) levels and activated the CaMKII/AMPK/Akt/eNOS signaling cascade in HUVECs. Treatment of L-Arg and ABH, arginase inhibitor, increased intracellular Ca2+ concentrations and activated CaMKII-dependent eNOS activation in ECs of WT mice, but, the effects were not observed in ECs of inositol triphosphate receptor type 1 knockout (IP3R1-/-) mice. In the aortic endothelium of WT mice, RPT also augmented nitric oxide (NO) production and attenuated reactive oxygen species (ROS) generation. In a vascular tension assay using RPT-treated aortic tissue, cumulative vasorelaxant responses to acetylcholine (Ach) were enhanced, and phenylephrine (PE)-dependent vasoconstrictive responses were retarded, although sodium nitroprusside and KCl responses were not different. In this study, we present a novel mechanism for RPT, as an arginase inhibitor, to increase cytosolic Ca2+ concentration in a L-Arg-dependent manner and enhance endothelial function through eNOS activation. [BMB Reports 2021; 54(10): 516-521].