Inducible Nitric Oxide Synthase Inhibitor Research Articles

Bioactive α-Pyrone Derivatives from the Endophytic Fungus Diaporthe sp. CB10100 as Inducible Nitric Oxide Synthase Inhibitors

Inducible nitric oxide synthase (iNOS) produces NO from l-arginine and plays critical roles in inflammation and immune activation. Selective and potent iNOS inhibitors may be potentially used in many indications, such as rheumatoid arthritis, pain, and neurodegeration. In the current study, five new compounds, including a dibenzo-α- pyrone derivative ellagic acid B (5) and four α-pyrones diaporpyrone A–D (9–12), together with three known compounds (6–8), were isolated from the endophytic fungus Diaporthe sp. CB10100. The structures of these new natural products were unambiguously elucidated using NMR, HRESIMS or electronic circular dichroism calculations. Ellagic acid B (5) features a tetracyclic 6/6/6/6 ring system with a fused 2H-chromene, which is different from ellagic acid (4) with a fused 2H-chromen-2-one. Both 2-hydroxy-alternariol (6) and alternariol (7) reduced the expression of iNOS at protein levels in a dose-dependent manner, using a lipopolysaccharide (LPS)-induced RAW264.7 cell models. Also, they decreased the protein expression levels of pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin-6 and monocyte chemotactic protein 1. Importantly, 6 and 7 significantly reduced the production of NO as low as 10 μM in LPS-induced RAW264.7 cells. Molecular docking of 6 and 7 to iNOS further suggests that both of them may interact with iNOS. Our study suggests that 6 and 7, as well as the alternariol scaffold may be further developed as potential iNOS inhibitors.

Nitric Oxide in the Spinal Cord Is Involved in the Hyperalgesia Induced by Tetrahydrobiopterin in Chronic Restraint Stress Rats.

It has been well recognized that exposure to chronic stress could increase pain responding and exacerbate pain symptoms, resulting in stress-induced hyperalgesia. However, the mechanisms underlying stress-induced hyperalgesia are not yet fully elucidated. To this end, we observed that restraint as a stressful event exacerbated mechanical and thermal hyperalgesia, accompanied with up-regulation of nitric oxide (NO) (P < 0.001), GTP cyclohydrolase 1 (GCH1) (GCH1 mRNA: P = 0.001; GCH1 protein: P = 0.001), and tetrahydrobiopterin (BH4) concentration (plasma BH4: P < 0.001; spinal BH4: P < 0.001) on Day 7 in restraint stress (RS) rats. Intrathecal injection of Nω-nitro-L-arginine methyl ester (L-NAME), a non-specific NO synthase inhibitor, or N-([3-(aminomethyl)phenyl]methyl) ethanimidamide, a special inhibitor of inducible NO synthase (iNOS), for seven consecutive days attenuated stress-induced hyperalgesia and decreased the production of NO (P < 0.001). Interestingly, 7-nitro indazole, a special inhibitor of neuronal NO synthase, alleviated stress-induced hyperalgesia but did not affect spinal NO synthesis. Furthermore, intrathecal injection of BH4 not only aggravated stress-induced hyperalgesia but also up-regulated the expression of spinal iNOS (iNOS mRNA: P = 0.015; iNOS protein: P < 0.001) and NO production (P < 0.001). These findings suggest that hyperalgesia induced by RS is associated with the modulation of the GCH1–BH4 system and constitutively expressed spinal iNOS. Thus, the GCH1–BH4–iNOS signaling pathway may be a new novel therapeutic target for pain relief in the spinal cord.

Distinct phenotypic expression levels of macrophages in neonatal lungs.

Alveolar macrophages are the front-line defense against environmental pathogens. However, to the best of our knowledge, differences in function and phenotypic expression levels of macrophages between neonatal and adult lungs have not previously been determined. The present study investigated lung tissues and analyzed blood samples to find cell markers of M1 and M2 macrophages in neonatal and adult rats. Pulmonary sepsis was induced by intrapleural instillation of lipopolysaccharide (LPS; 20 mg/kg) and survival time after administration of LPS was measured. In certain neonates, a selective inducible nitric oxide synthase (iNOS) inhibitor, 1400w, was administered prior to induction of pulmonary sepsis. Compared with adults, fetal and neonatal lung tissues had significantly higher levels of iNOS and CD86 (M1 markers), whereas the expression levels of CD206 and arginase-1 (M2 markers) were lower in the neonatal lung. The circulating cells that co-expressed CD68 (monocytes and macrophages) and CD86 in the blood were also significantly higher in neonates than in adults (25.9±6.6 vs. 11.6±2.2%; P=0.007. At basal unstimulated conditions, lung tissue concentrations of nitrite and nitrate (NOx) were significantly lower in the neonates than in adults (112.1±55.9 vs. 340.9±124.9 µM/g; P<0.001). However, NOx was increased following administration of LPS. Administration of 1400w suppressed lung tissue levels of NOx and improved the survival time in neonatal rats treated with LPS. The present study demonstrated that M1 is the primary macrophage phenotype in the neonatal lung and that higher iNOS expression levels do not have a protective effect against pulmonary endotoxins in neonates. Overproduction of NO by iNOS in neonatal alveolar macrophages may result in detrimental effects during pulmonary inflammation.

Biomaterial-Facilitated Immunotherapy for Established Oral Cancers.

We evaluated a peptide-based immunotherapy termed SynerGel: an injectable, biomaterial-based platform for intratumoral drug delivery. A drug-mimicking peptide hydrogel named L-NIL-MDP was loaded with an antitumor cyclic dinucleotide (CDN) immunotherapy agonist. The biomaterial combines inducible nitric oxide synthase (iNOS) inhibition with controlled delivery of CDNs, demonstrating between 4- and 20-fold slower drug release than commercially available hydrogels. SynerGel allowed for immune-mediated elimination of established treatment-resistant oral tumors in a murine model, with a median survival of 67.5 days compared with 44 days in no-treatment control. This report details findings for a promising therapy showing improved efficacy over previous hydrogel systems.

Co-culture of pro-inflammatory macrophages and myofibroblasts: Evaluating morphological phenotypes and screening the effects of signaling pathway inhibitors.

Skeletal muscle regeneration is a complex process influenced by non‐myogenic macrophages and fibroblasts, which acquire different phenotypes in response to changes in the injury milieu or changes in experimental conditions. In vitro, serum stimulates the differentiation of fibroblasts into myofibroblasts, while lipopolysaccharide (LPS) stimulates the polarization of unstimulated (M0) macrophages to acquire an M1 pro‐inflammatory phenotype. We characterized these phenotypes using morphology (with circularity as shape descriptor; perfect circularity = 1.0) and phenotype‐specific markers. Myofibroblasts (high α‐smooth muscle actin [SMA] expression) had high circularity (mean 0.60 ± 0.03). Their de‐differentiation to fibroblasts (low α‐SMA expression) significantly lessened circularity (0.47 ± 0.01 and 0.35 ± 0.02 in 2% or 0% serum culture media respectively (p < 0.05). Unstimulated (M0) macrophages (no CD86 expression) had high circularity (0.72 ± 0.02) which decreased when stimulated to M1 macrophages (CD86 expression) (LPS; 0.61 ± 0.02; p < 0.05). Utilizing these established conditions, we then co‐cultured M1 macrophages with myofibroblasts or myoblasts. M1 macrophages significantly decreased relative myofibroblast numbers (from 223 ± 22% to 64 ± 7%), but not myoblast numbers. This pro‐inflammatory co‐culture model was used to rapidly screen the following four compounds for ability to prevent M1 macrophage‐mediated decrease in myofibroblast numbers: L‐NAME (inducible nitric oxide synthase inhibitor), SB203580 (p38 mitogen‐activated protein kinase inhibitor), SP600125 (c‐Jun N‐terminal kinase inhibitor) and LY294002 (phosphoinositide 3‐kinase [PI3K] inhibitor). We found that LY294002 rescued myofibroblasts and decreased macrophage numbers. Myofibroblast rescue did not occur with L‐NAME, SB203580 or SP600125 incubation. In conclusion, these data suggest a PI3K‐associated mechanism whereby myofibroblasts can be rescued, despite simulated pro‐inflammatory conditions.

Effect of NOS Inhibitors and Anticoagulants on Nitric Oxide Production in a Tissue-factor Induced Rat DIC Model.

We examined the mechanism of nitric oxide (NO) production in a tissue-factor (TF)-induced disseminated intravascular coagulation (DIC) model in rats, using inducible nitric oxide synthase (iNOS) inhibitor (L-NIL), endothelial nitric oxide synthase (eNOS) inhibitor (L-NAME), Factor Xa inhibitor (DX-9065a), and thrombin inhibitor argatroban. Experimental DIC was induced by sustained infusion of 3.75 U/kg TF for 4 h via the tail vein. We then investigated the effect of these four agents on TF-induced DIC. Administration of L-NIL or L-NAME during induction of TF-induced DIC did not affect hemostatic markers, whereas elevated plasma levels of NO metabolites (NOX) were significantly suppressed by co-administration of L-NAME. A significant increase in eNOS-mRNA expression was observed in the TF-induced DIC model. Argatroban almost completely suppressed eNOS-mRNA expression. eNOS plays an important role in the NO production in the TF-induced DIC, and thrombin is a key stimulant of eNOS-mRNA expression in this model.

Histological assessment of the local effect of different concentrations of aminoguanidine hydrochloride on bone healing in rats

Nitric oxide (NO) has several functions in bone healing and affects bone metabolism. Selective inducible NO synthase (iNOS) inhibitors can be used to assess the efficacy of NO for healing of bone defects. This study sought to assess the local effect of different concentrations of aminoguanidine hydrochloride (AG), a selective iNOS inhibitor, on bone healing in rats. In this animal experimental study, 72 rats were divided into six groups of control, placebo, 5% AG, 10% AG, 15% AG, and 20% AG. A bone defect measuring 5 mm × 5 mm was created in the femur. The defect remained empty in the control group. In the placebo group, neutral gel was placed in the bone defect, and in the remaining four AG groups, different concentrations of AG were applied to the defects. Bone healing was assessed histologically. The healing score in the six groups was analyzed by the Kruskal-Wallis test. A P < 0.05 was considered statistically significant. The healing score in 20%, 15%, 10%, and 5% AG groups was significantly higher than that in the neutral gel and control groups (P < 0.01). Among the four groups of AG, 20% concentration showed better results, but the difference was not significant. Four concentrations of AG caused greater bone healing compared to the other two groups. Selective iNOS inhibitors such as AG can be used to promote local bone healing.

A Review on Newer Ocular Drug Delivery Systems with an Emphasis on Glaucoma.

Glaucoma is an irreversible condition resulting from the increase in intraocular pressure (IOP); which leads to permanent loss of vision with the destruction of retinal ganglion cells (RGCs). The IOP elevations are controlled in normal by the physiological flow of aqueous humour. A population with age above 40 is more susceptible to glaucoma. Other factors like gender, genetics, race etc. plays major roles in the development of the disease. Current treatment methods available for the disease includes drugs come under the classes of beta receptor blockers, carbonic anhydrase inhibitors, cholinergic agonists, prostaglandins etc. N-methyl-D-aspartate (NMDA) antagonists, inducible nitric oxide synthase (iNOS) inhibition, cytoskeletal agents, Rho-kinase inhibitors etc are few novel targets sites which are in research focus for the treatment of the disease. Developments in nanomedicine are also being evaluated for their potential in treating the growing glaucomatous population. Nanosystems are suggested to avoid the difficulties in tackling the various ocular barriers to a limit, help to decrease the instillation frequency of topical medication and can provide drug delivery in a sustained or controlled manner. This review focuses on the current and emerging treatment methods for glaucoma along with some of the nanoformulations for ocular drug delivery.

Involvement of nitric oxide pathway in the anti-inflammatory effect of modafinil on indomethacin-, stress-, and ethanol -induced gastric mucosal injury in rat

Gastric ulcer is a prevalent disease with various etiologies, including non-steroidal anti-inflammatory drugs (NSAIDs), stress conditions, and alcohol, resulting in an inflammatory condition in the gastric mucosa. The aim of this study was to explore the protective effects of modafinil on gastric erosions induced by indomethacin, water-immersion stress, and alcohol in rats and to evaluate the role of nitric oxide (NO) pathway. Animals were allocated to the three experimental models of gastric ulcer – indomethacin (30 mg/kg PO), water-immersion stress, and ethanol (5 ml/kg PO). Induction of gastric ulcer in all models caused an increase in J-score (macroscopic assessment), biochemical markers, including tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), and myeloperoxidase (MPO), and microscopic destructions. Administration of modafinil (50 and 100 mg/kg i. p) significantly improved J-score in the indomethacin (P < 0.05) and stress models (P < 0.001). Moreover, the level of TNF-α IL-1β, and MPO was deceased after modafinil administration (P < 0.001). However, modafinil did not have any effects on gastric injury induced by ethanol. In addition, co-administration of L-NAME (a non-specific NO synthase inhibitor) and aminoguanidine (an inducible NO synthase inhibitor) with modafinil significantly neutralized the gastroprotective effect of modafinil in the indomethacin and water-immersion stress groups (P < 0.05, and P < 0.01; respectively), while 7-nitroindazole (a neuronal NO synthase inhibitor) did not show such reversing effects. In conclusion, modafinil possesses gastroprotective effects on the gastric lesions induced by indomethacin and stress, which are probably mediated via the inflammation inhibition and NO pathway modulation.

HIGH-MOBILITY GROUP BOX 1 PROTEIN ANTAGONIZES THE IMMUNOSUPPRESSIVE CAPACITY AND THERAPEUTIC EFFECT OF MESENCHYMAL STEM CELLS IN ACUTE KIDNEY INJURY

Purpose: Kidney ischemia reperfusion injury (IRI) is a common cause of acute kidney injury and an unavoidable consequence of kidney transplantation and still lacks specific therapeutics. In recent years, mesenchymal stem cell (MSC) emerges as a promising cell-based therapy. The reparative effects of MSC largely dependent on its unique immunosuppressive property, which is primed and regulated by the microenvironments. Methods: We employed mice kidney IRI model and MSC cell line to monitor the IRI related checkpoints. siRNAs were utilized to knock down the potential key factors for function. Statistical analysis was performed by using one-way ANOVA with Tukey’s post hoc procedure by SPSS. Results: The expression of high-mobility group box 1 protein (HMGB1) is increased not only in the acute phase of IRI, but also during the recovery stage, and the HMGB1 upregulation is correlated with the injury severity. We found that HMGB1 could diminish the immunosuppressive capacity of MSC in the presence of pro-inflammatory cytokines in vitro and in vivo. Toll like receptor 4 (TLR4)-mediated inducible nitric oxide synthase (iNOS) inhibition might contribute to the effect of HMGB1 on MSCs. Importantly, we further demonstrated that HMGB1 blocking strategies would augment the therapeutic efficacy of MSCs on renal IRI. Conclusions: These findings demonstrate that HMGB1 plays a crucial role in shaping the immunoregulatory property of MSCs within the microenvironments, providing novel insights into the crosstalk between MSCs and microenvironment components, suggesting HMGB1 signals as a promising target to improve MSC-based therapy.

IL-17F induces inflammation, dysfunction and cell death in mouse islets

Type 17 immune responses, typified by the production of the cytokines IL-17A and IL-17F, have been implicated in the development of type 1 diabetes in animal models and human patients, however the underlying pathogenic mechanisms have not been clearly elucidated. While previous studies show that IL-17A enhances inflammatory gene expression and cell death in mouse β-cells and human islets, the function of IL-17F in pancreatic β-cells is completely untested to date. Here we show that IL-17F exhibits potent pathogenic effects in mouse β-cell lines and islets. IL-17F signals via the IL-17RA and -RC subunits in β-cells and in combination with other inflammatory cytokines induces expression of chemokine transcripts, suppresses the expression of β-cell identity genes and impairs glucose stimulated insulin secretion. Further IL-17F induces cell death in primary mouse islets. This occurs via Jnk, p38 and NF-κB dependent induction of Nos2 and is completely ablated in the presence of an inducible nitric oxide synthase (iNOS) inhibitor. Together these data indicate that IL-17F possesses similar pathogenic activities to IL-17A in mouse β-cell lines and islets and is likely to be a type 17 associated pathogenic factor in type 1 diabetes.

Interaction of morphine tolerance with pentylenetetrazole-induced seizure threshold in mice: The role of NMDA-receptor/NO pathway

N-methyl-d-aspartate receptor (NMDA-R)/nitric oxide (NO) pathway is involved in the intensification of the analgesic effect of opioids and the reduction of the intensity of opioids tolerance and dependence. In the current study, we investigated the involvement of NMDA-R/NO pathway in chronic morphine-treated mice in both the development of tolerance to the analgesic effect of morphine and in pentylenetetrazole (PTZ)-induced seizure threshold. Chronic treatment with morphine (30 mg/kg) exhibited increased seizure resistance in morphine-induced tolerant mice. The development of morphine tolerance was withdrawn when used concomitantly with NOS inhibitors and NMDA-R antagonist, suggesting that the development of tolerance to the anticonvulsant effect of morphine (30 mg/kg) is mediated through the NMDA-R/NO pathway. A dose-dependent biphasic seizure modulation of morphine was demonstrated in the acute treatment with morphine; acute treatment at a dose of 0.5 mg/kg shows the anticonvulsant effect and at a dose of 30 mg/kg shows proconvulsant effect. However, a different pattern was observed in the mice treated chronically with morphine: they demonstrated tolerance in the tail-flick test; five consecutive days of chronic treatment with a high dose of morphine (30 mg/kg) showed anticonvulsant effect while a low dose of morphine (0.5 mg/kg) showed a proconvulsant effect. The anticonvulsant effect of morphine was inhibited completely by the concomitant administration of NO synthase (NOS) inhibitors including nonspecific NOS inhibitor (L-NAME, 10 mg/kg), inducible NOS inhibitor (aminoguanidine, 50 mg/kg), and neuronal NOS inhibitor (7-nitroindazole (7-NI), 15 mg/kg) for five consecutive days. Besides, five days injection of NMDA-R antagonist (MK-801, 0.05 mg/kg) significantly inhibited the anticonvulsant effect of morphine on the PTZ-induced clonic seizures.The results revealed that chronic treatment with morphine leads to the development of tolerance in mice, which in turn may cause an anticonvulsant effect in a high dose of morphine via the NMDA-R/NO pathway.

Inducible nitric oxide synthase inhibitor, aminoguanidine improved Ki67 as a marker of neurogenesis and learning and memory in juvenile hypothyroid rats.

In the present study, the effect of inducible nitric oxide (NO) synthase inhibitor, aminoguanidine (AG) on neurogenesis indicators, learning and memory, and oxidative stress status in juvenile hypothyroid (Hypo) rats was evaluated. The studied groups were including: (a) Control, (b) Hypo, (c-e) Hypo-AG 10, Hypo-AG 20, and Hypo-AG 30. Hypothyroidism was induced in the groups 2-5 by adding propylthiouracil in drinking water (0.05%). AG (10, 20, or 30mg/kg) was daily injected intraperitoneally in the groups 3-5. The rats of the groups 1 and 2 were injected by saline instead of AG. After 6weeks treatment, Morris water maze (MMW) and passive avoidance (PA) tests were done. Deep anesthesia was then induced and the brain tissue was excised for biochemical parameters measuring. Ki67 as a maker of neurogenesis and thiol, superoxide dismutase (SOD), and catalase (CAT) as oxidative stress indicators were decreased in the brain of Hypo group, whereas malondialdehyde (MDA) and NO metabolites were enhanced. AG improved Ki67, thiol, CAT, and SOD while decreased MDA and NO metabolites. The escape latency in the MWM test increased in the Hypo group. The spending time in the target quadrant in the probe test of MWM and step-through latency in the PA test in the Hypo group was lower than Control group. AG reversed all the negative behavioral effects of hypothyroidism. These results revealed that AG improved neurogenesis, learning and memory impairments, and oxidative imbalance in the brain juvenile Hypo rats.

Antiglioma Activity of Aryl and Amido-Aryl Acetamidine Derivatives Targeting iNOS: Synthesis and Biological Evaluation.

Nitric oxide is an important inflammation mediator with a recognized role in the development of different cancers. Gliomas are primary tumors of the central nervous system with poor prognosis, and the expression of the inducible nitric oxide synthase correlates with the degree of malignancy, changes in vascular reactivity, and neo-angiogenesis. Therefore, targeting the nitric oxide biosynthesis appears as a potential strategy to impair glioma progression. In the present work a set of aryl and amido-aryl acetamidine derivatives were synthesized to obtain new potent and selective inducible nitric oxide synthase inhibitors with improved physicochemical parameters with respect to the previously published molecules. Compound 17 emerged as the most promising inhibitor and was evaluated on C6 rat glioma cell line, showing antiproliferative effects and high selectivity over astrocytes.

The Characterization of Ground Raspberry Seeds and the Physiological Response to Supplementation in Hypertensive and Normotensive Rats.

This study aimed to evaluate the protective role of ground raspberry seeds (RBS) as a source of polyphenols and essential fatty acids on blood plasma enzymatic antioxidant status, lipid profile, and endothelium-intact vasodilation during physiological and pathological conditions. Young normotensive Wistar–Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs) at ten weeks of age were fed with either a control diet or were supplemented with added 7% RBS for six weeks (n = 6). The main component of RBS was dietary fiber (64%) and the main polyphenols were ellagitannins (1.2%) and flavan-3-ols (0.45%). Irrespective of the rat model, ground RBS decreased liver enzyme aspartate aminotransferase (0.9-fold) and hydrogen peroxide scavenging capacity (Catalase, 0.9-fold). In supplemented SHRs, preincubation with inducible nitric oxide synthase (iNOS) inhibitor 1400W, nonselective cyclooxygenase (COX) inhibitor indomethacin, selective COX-2 inhibitor NS-398, prostacyclin (PGI2) synthesis inhibitor tranylcypromine (TCP), thromboxane receptor (TP) antagonist SQ-29548, thromboxane synthesis inhibitor furegrelate, and 20-HETE synthesis inhibitor HET0016 induced the same relaxant response to acetylcholine as in the nonsupplemented control group. In supplemented WKYs, atherogenic index was decreased (0.8-fold), while iNOS and COX-2-derived PGI2 increased acetylcholine-induced vasodilation. These effects of ground RBS may constitute a potential mechanism for preventing cardiovascular diseases.

Intravenous Arginine Administration Downregulates NLRP3 Inflammasome Activity and Attenuates Acute Kidney Injury in Mice with Polymicrobial Sepsis.

Acute kidney injury (AKI) is a major complication of sepsis. Nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasomes are multiprotein complexes that mediate septic AKI. L-arginine (Arg) is a conditionally essential amino acid in catabolic conditions and a substrate for nitric oxide (NO) production; however, its use in sepsis is controversial. This study investigated the effect of intravenous Arg supplementation on modulating NLRP3 inflammasome activity in relation to septic AKI. Mice were divided into normal control (NC), sham, sepsis saline (SS), and sepsis Arg (SA) groups. In order to investigate the role of NO, L-N6-(1-iminoethyl)-lysine hydrochloride (L-NIL), an inducible NO synthase inhibitor, was administered to the sepsis groups. Sepsis was induced using cecal ligation and puncture (CLP). The SS and SA groups received saline or Arg via tail vein 1 h after CLP. Mice were sacrificed at 6, 12, and 24 h after sepsis. The results showed that compared to the NC group, septic mice had higher plasma kidney function parameters and lower Arg levels. Also, renal NLRP3 inflammasome protein expression and tubular injury score increased. After Arg treatment, plasma Arg and NO levels increased, kidney function improved, and expressions of renal NLRP3 inflammasome-related proteins were downregulated. Changes in plasma NO and renal NLRP3 inflammasome-related protein expression were abrogated when L-NIL was given to the Arg sepsis groups. Arg plus L-NIL administration also attenuated kidney injury after CLP. The findings suggest that intravenous Arg supplementation immediately after sepsis restores plasma Arg levels and is beneficial for attenuating septic AKI, partly via NO-mediated NLRP3 inflammasome inhibition.

High-mobility group box 1 protein antagonizes the immunosuppressive capacity and therapeutic effect of mesenchymal stem cells in acute kidney injury

BackgroundKidney ischemia reperfusion injury (IRI) is a common cause of acute kidney injury and an unavoidable consequence of kidney transplantation and still lacks specific therapeutics. Recently, mesenchymal stem cell (MSC) has been emerging as a promising cell-based therapy for IRI in the context of transplantation. MSC negatively regulates the secretion of pro-inflammatory as well as the activation of immune cells during IRI through its unique immunosuppressive property.MethodsWe employed mice kidney IRI model and MSC cell line to monitor the IRI related checkpoints. siRNAs were utilized to knock down the potential key factors for mechanistic analysis. Statistical analysis was performed by using one-way ANOVA with Tukey’s post hoc procedure by SPSS.ResultsThe expression of high-mobility group box 1 protein (HMGB1) is increased in the acute phase as well as the recovery stage of IRI. Importantly, the HMGB1 upregulation is correlated with the injury severity. HMGB1 diminishes the MSC induced immunosuppressive capacity in the presence of pro-inflammatory cytokines in vitro. Toll like receptor 4 (TLR4)-mediated inducible nitric oxide synthase (iNOS) inhibition contributes to the negative effect of HMGB1 on MSCs. HMGB1-TLR4 signaling inhibition augments the therapeutic efficacy of MSCs in mice renal IRI model.ConclusionsThese findings demonstrate that HMGB1 plays a crucial role in shaping the immunoregulatory property of MSCs within the microenvironments, providing novel insights into the crosstalk between MSCs and microenvironment components, suggesting HMGB1 signals as a promising target to improve MSC-based therapy.

Intravenous Arginine Administration Benefits CD4+ T-Cell Homeostasis and Attenuates Liver Inflammation in Mice with Polymicrobial Sepsis.

This study investigated the effects of a single dose of arginine (Arg) administration at the beginning of sepsis on CD4+ T-cell regulation and liver inflammation in C57BL/6J mice. Mice were divided into normal control (NC), sham (SH), sepsis saline (SS), and sepsis Arg (SA) groups. An inducible nitric oxide (NO) synthase (iNOS) inhibitor was administered to additional sepsis groups to evaluate the role of NO during sepsis. Sepsis was induced using cecal ligation and puncture (CLP). The SS and SA groups received saline or Arg (300 mg/kg body weight) via tail vein 1 h after CLP. Mice were euthanized at 12 and 24 h post-CLP. Blood, para-aortic lymph nodes, and liver tissues were collected for further measurement. The findings showed that sepsis resulted in decreases in blood and para-aortic lymph node CD4+ T-cell percentages, whereas percentages of interleukin (IL)-4- and IL-17-expressing CD4+ T cells were upregulated. Compared to the SS group, Arg administration resulted in maintained circulating and para-aortic lymph node CD4+ T cells, an increased Th1/Th2 ratio, and a reduced Th17/Treg ratio post-CLP. In addition, levels of plasma liver injury markers and expression of inflammatory genes in liver decreased. These results suggest that a single dose of Arg administered after CLP increased Arg availability, sustained CD4+ T-cell populations, elicited more-balanced Th1/Th2/Th17/Treg polarization in the circulation and the para-aortic lymph nodes, and attenuated liver inflammation in sepsis. The favorable effects of Arg were abrogated when an iNOS inhibitor was administered, which indicated that NO may be participated in regulating the homeostasis of Th/Treg cells and subsequent liver inflammation during sepsis.

The Cardioprotective Effects of Aminoguanidine on Lipopolysaccharide Induced Inflammation in Rats.

Myocardial dysfunction, a major component of sepsis-induced multiorgan failure, contributes to the production of massive amounts of pro-inflammatory cytokines. Nitric oxide (NO) is known to act as a precursor of free radicals in inflammation. This research was conducted to assess the effect of aminoguanidine (AG) on lipopolysaccharide (LPS)-induced heart injury. 50 male rats were categorized into five groups (n = 10): (1) control, (2) LPS, (3) LPS-AG50, (4) LPS-AG100, and (5) LPS-AG150. LPS (1mg/kg) was injected for 5weeks, and AG (50, 100 and 150mg/kg) was injected 30min prior to LPS administration. All drugs were injected intraperitoneally. LPS-evolved cardiovascular toxicity was indicated by the augmentation in the level of nitric oxide (NO) metabolites, interleukin (IL)-6 and malondialdehyde (MDA), as well as reduced contents of total thiol groups, catalase (CAT), and superoxide dismutase (SOD) activity in serum, heart, and aortic tissues. In AG treated groups, noxious effects of LPS were not observed in the serum and harvested tissues. AG reduced MDA, NO metabolites, and IL- 6 and increased total thiol, CAT, and SOD activity in the heart, aorta and serum. As an inhibitor of inducible NO synthase (iNOS), AG further reduced LPS-induced oxidative stress and inflammation, hence considered as cardioprotective.

Rat bone mesenchymal stem cells exert antiproliferative effects on nicotine‑exposed T cells via iNOS production.

Adoptive transfer of bone marrow‑derived mesenchymal stem cells (BMSCs) significantly alleviates smoking‑induced chronic obstructive pulmonary disease (COPD) in rats. Considering the critical roles of T cells during COPD development, the present study aimed to further identify the molecular mechanisms underlying the antiproliferative effect of BMSCs on splenic T cells isolated from rats following chronic exposure to nicotine. Splenic T cells were co‑cultured with rat BMSCs at various ratios and subsequently, T‑cell proliferation was measured using the Cell Counting Kit‑8 assay. The effects of the inducible nitric oxide synthase (iNOS) inhibitor N‑nitro‑L‑arginine methylester (L‑NAME) and short hairpin (sh)RNA‑lentivirus‑mediated knockdown of iNOS in BMSCs on T‑cell proliferation were evaluated. The expression levels of iNOS and STAT5 phosphorylation in BMSCs and T cells, respectively, were assessed by reverse transcription‑quantitative PCR and western blotting. A higher ratio of BMSCs to T cells resulted in increased inhibition of T‑cell proliferation; therefore, the ratio of 1:20 was selected for further invitro experiments. At a dose of 5µM, L‑NAME displayed the strongest ability to reverse the antiproliferative effects of BMSCs in the co‑culture system. Both L‑NAME treatment and shRNA‑mediated knockdown of iNOS expression significantly decreased the suppressive effects of BMSCs, downregulated iNOS expression at the mRNA and protein levels in BMSCs, and enhanced STAT5 phosphorylation in Tcells. BMSCs inhibited the proliferation of nicotine‑exposed Tcells, which was associated with iNOS expression in BMSCs and decreased STAT5 phosphorylation in T cells. The present study indicated the potential mechanisms underlying the beneficial effects of BMSC infusion in patients with chronic smoking‑induced COPD and emphysema.