iNOS Pathway Research Articles

Luteolin alleviates vascular dysfunctions in CLP-induced polymicrobial sepsis in mice.

Luteolin, a naturally occurring flavonoid, is thought to have health-promoting properties as a part of human diet and has been reported to possess a wide range of pharmacological activities. Therefore, the present study was undertaken to evaluate the effect of luteolin pre-treatment on vascular dysfunctions in sepsis induced by caecal ligation and puncture (CLP) in the mouse model. Mice were divided into four groups: sham, luteolin plus sham, CLP, and luteolin plus CLP. Luteolin was administered (0.2mg/kg body weight) intraperitoneally one hour (h) before CLP surgery in mice. 20 ± 2h post CLP surgery, the isolated thoracic aorta of mice was assessed for its vascular reactivity to noradrenaline (NA) and acetylcholine (ACh). To explore the underlying mechanism, aortic mRNA expressions of α1D adrenoceptors, eNOS and iNOS were investigated. In mice with CLP-induced sepsis luteolin pre-treatment markedly increased the survival time and attenuated serum lactate level. The CLP group manifested the reduced vascular reactivity to NA and this deficit was restored by luteolin pre-treatment. However, luteolin pre-treatment did not improve α1D adrenoceptors down-regulation observed in septic mice aorta. In the presence of 1400W, the NA contractile response was significantly restored in CLP mice aortic tissue in comparison with the respective control of septic mice and further enhanced in the presence of luteolin. Luteolin reduced the iNOS mRNA expression and iNOS-derived nitrite production. Pre-treatment with luteolin restored the endothelial dysfunction in septic mice aorta by improving eNOS mRNA expression and enhanced eNOS-derived nitric oxide (NO) production in septic mice aorta and aortic iNOS gene expression and inducible NO production. The present study suggests that the vasoplegic state to NA in aorta was restored through the iNOS pathway and endothelial dysfunction was reversed via eNOS and NO production pathway.

Monocytic myeloid-derived suppressive cells mitigate over-adipogenesis of bone marrow microenvironment in aplastic anemia by inhibiting CD8+ T cells

Aplastic anemia (AA) is a blood disorder resulted from over-activated T-cell related hematopoietic failure, with the characterization of hypocellularity and enhanced adipogenic differentiation of mesenchymal stroma cells (MSCs) in bone marrow (BM). However, little is known about the relationship between immune imbalance and polarized adipogenic abnormity of BM microenvironment in this disease entity. In the present study, we differentiated BM-MSCs into osteoblastic or adipogenic lineages to mimic the osteo-adipogenic differentiation. Activated CD8+ T cells and interferon-γ (IFN-γ) were found to stimulate adipogenesis of BM-MSCs either in vitro or in vivo of AA mouse model. Interestingly, myeloid-derived suppressive cells (MDSCs), one of the immune-regulating populations, were decreased within BM of AA mice. We found that it was not CD11b+Ly6G+Ly6C- granulocytic-MDSCs (gMDSCs) but CD11b+Ly6G-Ly6C+ monocytic-MDSCs (mMDSCs) inhibiting both T cell proliferation and IFN-γ production via inducible nitric oxide synthetase (iNOS) pathway. Single-cell RNA-sequencing (scRNA-seq) of AA- and mMDSCs-treated murine BM cells revealed that mMDSCs transfusion could reconstitute BM hematopoietic progenitors by inhibiting T cells population and signature cytokines and decreasing immature Adipo-Cxcl12-abundant reticular cells within BM. Multi-injection of mMDSCs into AA mice reduced intra-BM T cells infiltration and suppressed BM adipogenesis, which subsequently restored the intra-BM immune balance and eventually prevented pancytopenia and hypo-hematopoiesis. In conclusion, adoptive transfusion of mMDSCs might be a novel immune-regulating strategy to treat AA, accounting for not only restoring the intra-BM immune balance but also improving stroma’s multi-differentiating microenvironment.

Xiongzhi Dilong decoction interferes with calcitonin gene-related peptide (CGRP)-induced migraine in rats through the CGRP/iNOS pathway

To evaluate the effects of Xiongzhi Dilong decoction (XZDLD) and its wind medicine on calcitonin gene-related peptide (CGRP)-induced migraine and explore the mechanism through the CGRP/inducible nitric oxide synthase (iNOS) pathway. Rats were divided into control, model, XZDLD, XZDLD (external wind), XZDLD (internal wind), and olcegepant groups. CGRP was injected into the dura mater to induce a migraine. The frequency of head scratching, cage climbing, and facial grooming was observed. The pain threshold, the levels of CGRP, pituitary adenylate cyclase activating polypeptide (PACAP), substance P (SP), and the plasma protein extravasation (PPE) ratio were measured. The phosphorylation levels of p38, extracellular signal-regulated kinase 1/2 (ERK1/2), and expression of iNOS were detected by western blot. Compared with the model group, the three modified XZDLD groups showed reduced frequency of head scratching and cage climbing in the first 30 min (all P < .05). Facial grooming frequency was reduced in XZDLD and XZDLD (external wind) groups ( P = .0003 and P = .0131, respectively). External wind medicine played a more important role in increasing mechanical pain threshold than internal wind medicine. Moreover, compared with the model group, the three modified XZDLD groups demonstrated reduced plasma levels of CGRP and PACAP (all P < .05). No difference in the SP level was observed among the six groups. XZDLD reduced PPE ratio. XZDLD and XZDLD (external wind) groups suppressed the CGRP/iNOS pathway by inhibiting the p-p38/p38 ratio and the expression of iNOS. No difference in pERK1/2/ERK1/2 ratio was detected among the six groups. XZDLD increases pain threshold, downregulates the expression of CGRP and PACAP, and reduces PPE ratio by inhibiting the CGRP/iNOS pathway. External wind medicine is more effective than internal one on improving facial grooming and head scratching, increasing the mechanical pain threshold, and inhibiting the expression of iNOS.

Chronically Elevated O-GlcNAcylation Limits Nitric Oxide Production and Deregulates Specific Pro-Inflammatory Cytokines

Inflammation is the immune response to harmful stimuli, including pathogens, damaged cells and toxic compounds. However, uncontrolled inflammation can be detrimental and contribute to numerous chronic inflammatory diseases, such as insulin resistance. At the forefront of this response are macrophages, which sense the local microenvironment to respond with a pro-inflammatory, M1-polarized phenotype, or anti-inflammatory, M2-polarized phenotype. M1 macrophages upregulate factors like pro-inflammatory cytokines, to promote inflammatory signaling, and inducible Nitric Oxide Synthase (iNOS), to produce nitric oxide (NO). The generated NO can kill microorganisms to protect the body, but also signal back to the macrophage to limit pro-inflammatory cytokine production to maintain macrophage homeostasis. Thus, the tight regulation of iNOS in macrophages is critical for the immune system. Here, we investigated how elevation of the nutrient-sensitive posttranslational modification, O-GlcNAc, impacts M1 polarized macrophages. We identified increased gene expression of specific pro-inflammatory cytokines (Il-6, Il-1β, Il-12) when O-GlcNAc cycling was blocked. We further uncovered an interaction between O-GlcNAc and iNOS, with iNOS being an OGT target in vitro. Analysis of M1 polarized bone marrow derived macrophages deficient in the enzyme that removes O-GlcNAc, O-GlcNAcase (OGA), revealed decreased iNOS activity as measured by a reduction in NO release. Further, elevated O-GlcNAc acted on Il-6 expression through the iNOS pathway, as iNOS inhibitior L-NIL raised wildtype Il-6 expression similar to OGA deficient cells but had no further effect on the hyper-O-GlcNAcylated cells. Thus O-GlcNAc contributes to macrophage homeostasis through modulation of iNOS activity.

The endometrial transcriptomic response to pregnancy is altered in cows after uterine infection.

Pregnancy induces changes in the transcriptome of the bovine endometrium from 15 days after insemination. However, pregnancy is less likely to occur if cows had a postpartum bacterial infection of the uterus, even after the resolution of disease. We hypothesized that uterine bacterial infection alters the endometrial transcriptomic signature of pregnancy after the resolution of disease. To examine the endometrial transcriptomic signature of pregnancy, cows were inseminated 130 days after intrauterine infusion of pathogenic Escherichia coli and Trueperella pyogenes, subsequently endometrium was collected 16 days after insemination for RNA sequencing. We found 171 pregnancy regulated genes in cows 146 days after bacterial infection. When comparing our findings with previous studies that described the endometrial transcriptomic signature of pregnancy in healthy cows, 24 genes were consistently differentially expressed in pregnancy, including MX1, MX2 and STAT1. However, 12 pregnancy regulated genes were found only in the endometrium of healthy cows, including ISG15 and TRANK1. Furthermore, 28 pregnancy regulated genes were found only in the endometrium of cows following bacterial infection and these were associated with altered iNOS, TLR, and IL-7 signaling pathways. Although 94 predicted upstream regulators were conserved amongst the studies, 14 were found only in the endometrium of pregnant healthy cows, and 5 were found only in cows following bacterial infection, including AIRE, NFKBIA, and DUSP1. In conclusion, there were both consistent and discordant features of the endometrial transcriptomic signature of pregnancy 146 days after intrauterine bacterial infusion. These findings imply that there is an essential transcriptomic signature of pregnancy, but that infection induces long-term changes in the endometrium that affect the transcriptomic response to pregnancy.

Neurobehavioral Toxicity Induced by Carbendazim in Rats and the Role of iNOS, Cox-2, and NF-κB Signalling Pathway.

Carbendazim (CBZ) is one of the most common fungicides used to fight plant fungal diseases, otherwise, it leaves residue on fruits, vegetables, and soil that contaminate the environment, water, animal, and human causing serious health problems. Several studies have reported the reproductive and endocrine pathological disorders induced by CBZ in several animal models, but little is known about its neurotoxicity. So that, the present study aimed to explain the possible mechanisms of CBZ induced neurotoxicity in rats. Sixty male Wistar rats were divided into 4 groups (n = 15). Group (1) received normal saline and was kept as the negative control group, whereas groups (2, 3, 4) received CBZ at 100, 300, 600mg/kg b.wt respectively. All rats received the aforementioned materials daily via oral gavage. Brain tissue samples were collected at 7, 14, 28days from the beginning of the experiment. CBZ induced oxidative stress damage manifested by increasing MDA levels and reducing the levels of TAC, GSH, CAT in some brain areas at 14 and 28days. There were extensive neuropathological alterations in the cerebrum, hippocampus, and cerebellum with strong caspase-3, iNOS, Cox-2 protein expressions mainly in rats receiving 600mg/kg CBZ at each time point. Moreover, upregulation of mRNA levels of NF-κB, TNF-α, IL-1B genes and downregulation of the transcript levels of both AchE and MAO genes were recorded in all CBZ receiving groups at 14 and 28days especially those receiving 600mg/kg CBZ. Our results concluded that CBZ induced dose- and time-dependent neurotoxicity via disturbance of oxidant/antioxidant balance and activation of NF-κB signaling pathway. We recommend reducing the uses of CBZ in agricultural and veterinary fields or finding other novel formulations to reduce its toxicity on non-target organisms and enhance its efficacy on the target organisms.

New insights into arsenic, lead, and iron neurotoxicity: Activation of MAPK signaling pathway and oxidative stress.

Toxic metals cause neurodegeneration via formation of toxic complexes with the cellular compounds and production of highly reactive oxygen species. The present study aimed to investigate the role of mitogen-activated protein kinase (MAPK) signaling pathway in iron, lead, and arsenic induced neurotoxicity. Also, to explore their effect on brain enzymes, inducible nitric oxide synthase (iNOS) and nuclear factor κB (NF-κB) in rat brains. Rats were divided into four groups (n = 8): Control group, lead group (30 mg/kg lead acetate), arsenic group (5 mg/kg sodium arsenite), and iron group (100 mg/kg ferric hydroxide). Treatments were given three times/week orally for 2 months. Brain tissues were assessed for reduced glutathione and malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), alkaline phosphatase (ALP), acid phosphatase (ACP), Na+ /K+ activated adenosine 5'-triphosphatase (Na+ /K+ -ATPase)and acetylcholinesterase (AChE) activities, expression of iNOS and NF-κB, and Western blot analysis of c-Jun NH(2)-terminal kinase (JNK) and extracellular signal-regulated protein kinase (ERK) proteins. Levels of arsenic, iron, and lead were significantly (p = 0.000) increased in blood and brain tissues. Levels of MDA, SOD, CAT, iNOS, and NF-κB gene expression, phosphorylated JNK and phosphorylated ERK proteins were increased significantly in lead, arsenic, and iron treated rat groups compared to control. ALP, ACP, AChE, and ATPase activities in brain were significantly altered in metal-treated rat groups compared to control. Iron, lead, and arsenic induced neurotoxicity activated the pro-inflammatory mediators NF-κB, iNOS, and MAPK pathway and altered the activity of brain ALP, ACP, Na+ /K+ -ATPase, CAT, SOD, and AChE.

Predicting Blood Parasite Load and Influence of Expression of iNOS on the Effect Size of Clinical Laboratory Parameters in Acute Trypanosoma cruzi Infection With Different Inoculum Concentrations in C57BL/6 Mice.

In Chagas disease, the initial responses of phagocyte-mediated innate immunity are strongly associated with the control of Trypanosoma cruzi and are mediated by various signaling pathways, including the inducible nitric oxide synthetase (iNOS) pathway. The clinical and laboratory manifestations of Chagas disease depend on the parasite–host relationship, i.e., the responsive capacity of the host immune system and the immunogenicity of the parasite. Here, we evaluated effect sizes in clinical and laboratory parameters mediated by acute infection with different concentrations of T. cruzi inoculum in mice immunosuppressed via iNOS pathway inactivation. Infection was induced in C57BL/6 wild-type and iNOS-/- mice with the “Y” strain of T. cruzi at three inoculum concentrations (3 × 102, 3 × 103, and 3 × 104). Parasitemia and mortality in both mouse strains were monitored. Immunohistochemistry was performed to quantify amastigotes in cardiac tissues and cardiac musculature cells. Biochemical parameters, such as blood urea nitrogen, sodium, albumin, and globulin concentrations, among others, were measured, and cytokine concentrations were also measured. Effect sizes were determined by the eta squared formula. Compared with that in wild-type animals, mice with an absence of iNOS expression demonstrated a greater parasite load, with earlier infection and a delayed parasitemia peak. Inoculum concentration was positively related to death in the immunosuppressed subgroup. Nineteen parameters (hematological, biochemical, cytokine-related, and histopathological) in the immunocompetent subgroup and four in the immunosuppressed subgroup were associated with parasitemia. Parasitemia, biochemical parameters, and hematological parameters were found to be predictors in the knockout group. The impact of effect sizes on the markers evaluated based on T. cruzi inoculum concentration was notably high in the immunocompetent group (Cohen’s d = 88.50%; p <.001). These findings contribute to the understanding of physiopathogenic mechanisms underlying T. cruzi infection and also indicate the influence of the concentration of T. cruzi during infection and the immunosuppression through the iNOS pathway in clinical laboratory heterogeneity reported in acute Chagas disease.

Chronic rapamycin pretreatment modulates arginase/inducible nitric oxide synthase balance attenuating aging-dependent susceptibility to Trypanosoma cruzi infection and acute myocarditis.

Considering the efficacy of rapamycin in increasing lifespan and healthspan, attenuating the aging-dependent immunological decline, we compared the evolution of Trypanosoma cruzi infection and acute myocarditis in young and elderly mice untreated and chronically treated with this drug. Five groups were investigated: young uninfected and infected, elderly uninfected and infected with Trypanosoma cruzi untreated and treated with rapamycin (4mg/kg every 3days) from the 8th to the 96th week of age. Seven days after the last treatment, elderly mice were inoculated with T. cruzi. Young animals were infected at 8-weeks-old. Untreated elderly mice exhibited increase parasitemia, parasite load and myocarditis, which were associated to down-regulation in IL-2, IL-6, IFN-γ, TNF, anti-T. cruzi immunoglobulin G (IgG) total, IgG1 and IgG2a plasma levels, inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) cardiac production, as well as upregulation in Arginase-1 gene expression and arginase activity compared to young animals. These parameters were improved in rapamycin-pretreated elderly mice, which exhibited a better parasitological control, reduced heart inflammation and microstructural damage. These responses were associated with a better balance between Th1 and Th2 effectors similar to that observed in young animals, including an improved activation of Th1 cytokines and the iNOS pathway that positively regulates NO biosynthesis, contradicting the predominant activation of the arginase pathway in untreated elderly animals. Thus, our findings suggest that chronic pretreatment with rapamycin can attenuate immunosenescence in mice, contributing to prolong parasite resistance and attenuate acute myocarditis in elderly host challenged by T. cruzi.

Abstract P5-17-07: Phase 1B/2 clinical trial targeting nitric oxide in the treatment of chemo-refractory metaplastic triple-negative breast cancer patients

Abstract BACKGROUND Metaplastic breast cancer (MpBC) is an extremely rare, therapeutically recalcitrant and aggressive variant of triple negative breast cancer (TNBC). We have previously shown molecular alterations in inducible nitric oxide (iNOS) signaling in MpBC is associated with worse overall survival. Preclinical models have shown pan-NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) reduces tumor growth and epithelial to mesenchymal transition in mesenchymal cell lines. We have also previously shown in TNBC patients treated with LNMMA and docetaxel, the non-responders have a higher expression of M2 macrophage vs the responders have decreased pro-tumor N2 neutrophils at the end of therapy. Here, we report the results of L-NMMA plus taxane in a cohort of MpBC patients. METHODS We conducted a phase 1B/2 trial, with L-NMMA (starting dose of 7.5 mg/kg which was escalated to recommended phase 2 dose of 20 mg/kg) and docetaxel (100 mg/m2) every 3 weeks. Daily amlodipine was given to prevent hypertension from L-NMMA. Primary objective was to assess clinical benefit rate (CBR), as assessed by the Response Evaluation Criteria in solid tumors (RECIST). Secondary objectives were to study overall response rate (ORR), progression free survival (PFS), overall survival (OS), dose limiting toxicities (DLT), response correlation with type of MpBC, body mass index (BMI) and ethnicity. Exploratory analysis included immune correlates for clinical response; staining for iNOS, M2 macrophages (CD 68, CD 163), N2 neutrophils (CD 15, arginase), immune infiltration (PD-L1, CD 8) and fibrosis marker (α- SMA). Fisher’s exact test was used to find the association between different patient’s characteristics and the main outcome. A p-value of 0.05 was considered statistically significant and all analyses were conducted using Stata V16.1 (StataCorp, College Station, Texas 77845 USA) RESULTS Of the total 35 TNBC patients recruited, 15 patients had MpBC (Phase 1B, n= 4; Phase 2, n=11); 86.6% (13/15) patients had metastatic breast cancer (MBC), with a median of 2 prior lines of therapy (range 0-5) and 13.3% (2/15) had anthracycline-refractory locally advanced breast cancer (LABC). The CBR was 40% (6/15); the ORR was 20% (3/15) with 1 PR in MBC , 1 pathological CR and 1 PR in LABC. Grade 3 or more toxicity was seen in 13.3% (2/15) patients; however, none was attributed to L-NMMA. The mPFS and mOS for MBC patients were 4.5 months (range 3-7m) and 12.8 months, respectively. The response was more likely to be in women of Caucasian ethnicity, BMI&amp;gt; 25 and non-spindle pathological features such as squamous differentiation, keratinized and myxo-chondroid tumors; albeit these were not statistically significant (Table 1). CONCLUSIONS Inhibition of iNOS pathway in MpBC is a promising and novel therapeutic option in this very challenging breast cancer subtype. The small study size is an impediment in identifying clinical factors which can predict a response. This warrants further evaluation of treatment with L-NMMA in chemo-refractory MpBC patients in a larger clinical trial. Table 1.Association between ethnicity, BMI and pathological characteristics with responseTotalNo responderResponderp-valueN=13N=7N=6Ethnicity0.19Caucasian10 (76.92%)4 (57.14%)6 (100.00%)Other3 (23.08%)3 (42.86%)0 (0.00%)Obesity1.00&amp;lt;25 BMI3 (23.08%)2 (28.57%)1 (16.67%)&amp;gt;=25 BMI10 (76.92%)5 (71.43%)5 (83.33%)Pathological features0.27Spindle5 (38.46%)4 (57.14%)1 (16.67%)Other8 (61.54%)3 (42.86%)5 (83.33%)Data are presented as n (%). *P-value from Fisher’s exact test. The 2 patients with adverse events were excluded from the analysis. Citation Format: Akshjot Puri, Adriana Ordonez, Ann C. Anselme, Liliana Guzman, Polly Niravath, Jenny C. Chang. Phase 1B/2 clinical trial targeting nitric oxide in the treatment of chemo-refractory metaplastic triple-negative breast cancer patients [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-17-07.

Scutellaria brevibracteata Stapf and active principles with anti-inflammatory effects through regulation of NF-κB/COX-2/iNOS pathways

Inflammation is a critical defense mechanism for body which is crucial for wound healing. However, when it spirals out of control and becomes chronic, it possesses a risk for diseases such as metabolic disorders or cancer. Scutellaria L. species are known with their potent anti-inflammatory effects. We aim to investigate the potential anti-inflammatory effects of Scutellaria brevibracteata Stapf and its active principles with underlying mechanisms using both in vitro and in vivo methods. Aqueous extract of S. brevibracteata (SB) was chromatographed on several columns to get 6 main fractions and 2 pure compounds. The levels of IL-6, NO, and TNF-α were measured in LPS-induced RAW 264.7 cells. In vivo carrageenan-induced paw edema test and immunoblotting in these paws were performed. SB was significantly reduced the inflammation in LPS-induced RAW cells via IL-6 and TNF-α cytokines at 200 μg/mL (P < 0.001) and paw edema via COX-2 and iNOS (P < 0.001). 3-O-methyl kaempferol, isolated from SB, reduced the production of NO, IL-6 and TNF-α in LPS-stimulated macrophages at 50 μM (P < 0.001); it significantly diminished inflammation-induced edema and COX-2, iNOS, and NF-κB protein levels were reduced compared to control group (P < 0.001). It was noticed that the methoxylation of C3 position has no effect on the anti-inflammatory mechanism of action comparing with kaempferol. In vivo studies indicated 3-O-methyl kaempferol was as effective as indomethacin. Considering the side effects caused by indomethacin, this compound can be evaluated as a novel drug candidate in inflammatory diseases with fewer side effects.

Down-regulation of miR-18b-5p protects against splenic hemorrhagic shock by directly targeting HIF-1α/iNOS pathway

Splenic hemorrhagic shock is a typical emergency in surgery, seriously threatening human beings' life. Emerging evidence shows that microRNAs (miRNAs) are closely related to inflammation and immunity in the body. However, the detailed effects and underlying mechanisms of miRNAs on the immune function of splenic hemorrhagic shock have not been revealed yet. In the present study, we construct the rat hemorrhagic shock model, and the rats are further cured with splenic blood transport clipping recanalization (SBTCR). MiR-18b-5p was highly expressed in the spleen of hemorrhagic shock rats detected by the qRT-PCR assay. Functionally, down-regulation of miR-18b-5p notably inhibited the levels of SOD1, iNOS and IL-6 in macrophages isolated from splenic tissues detected by qRT-PCR and ELISA assays. In addition, inhibition of miR-18b-5p significantly decreased the M1/M2 ratio of macrophages. Besides, knockdown of miR-18b-5p obviously reduced the Th1/Th2 ratio of CD4+ T cells. Moreover, HIF-1α was predicted as a target gene of miR-18b-5p, which was further confirmed by dual-luciferase reporter assay, and HIF-1α was negatively associated with miR-18b-5p. Furthermore, overexpression of HIF-1α partially restored the effects of miR-18b-3p on inflammation and immunity in macrophages. Taken together, miR-18b-5p may be a novel therapeutic candidate target in splenic hemorrhagic shock treatment.

Protective Effects of Mirazid on Gentamicin-induced Nephrotoxicity in Rats through Antioxidant, Anti-inflammatory, JNK1/ iNOS, and Apoptotic Pathways; Novel Mechanistic Insights

Background: As the use of Gentamicin became more widespread, the drug's harmful effects, particularly nephrotoxicity, became increasingly well-known. Antibacterial and anti-inflammatory properties have long been associated with Mirazid. This study aimed to investigate the frameworks for the protection of Mirazid against nephrotoxicity triggered by Gentamicin. Methods: Male albino rats were divided into three groups; the normal group received only saline. Nephrotoxicity was induced by Gentamicin (100 mg/kg; i.p.) for 10 days in the second group. In the third group; Mirazid (10 mg/kg; p.o.) was administered for 10 days before receiving Gentamicin. This was done to investigate the kidney/body weight index, serum creatinine, urea, lactate dehydrogenase (LDH), malondialdehyde (MDA), and Glutathione (GSH) levels. Moreover, immunohistochemical staining was done to study Jun N- terminal kinase 1 (JNK1), inducible nitric oxide synthase (iNOS), and caspase3 expressions along with histopathological changes. Additionally, a molecular docking study was performed for the seventeen isolated and identified compounds from myrrh, which is an oleo-gum resin obtained from the Commiphora species of plants (Burseraceae) against JNK1. Results: The Gentamicin group showed an increase in kidney/body weight index, serum creatinine, urea, LDH, and MDA, while decreasing GSH levels. Furthermore, immunohistochemical staining revealed increased JNK1, iNOS, and caspase3 expressions along with histopathological changes. Mirazid showed a significant decrease in all of these parameters and restored oxidant/antioxidant hemostasis. In addition, it has significantly preserved the histopathological architecture of tissues. Concerning the docking study, the isolated compound (12) was found to be superior to the co-crystallized inhibitor (18) with a binding score of -7.19 kcal/mol compared to -6.95, respectively. Conclusion: Current data demonstrated that Mirazid represented a viable approach to suppress the nephrotoxicity produced by Gentamycin through inhibiting JNK1/ iNOS pathways, thus preserving kidney function. Mirazid prophylactic efficacy is assumed to be due to its antioxidant, anti-inflammatory, and anti-apoptotic qualities.

Hempseed (Cannabis sativa) Peptides WVSPLAGRT and IGFLIIWV Exert Anti-inflammatory Activity in the LPS-Stimulated Human Hepatic Cell Line.

WVSPLAGRT (H2) and IGFLIIWV (H3) are two transepithelial transported intestinal peptides obtained from the hydrolysis of hempseed protein with pepsin, which exert antioxidant activity in HepG2 cells. Notably, both peptides reduce the H2O2-induced reactive oxygen species, lipid peroxidation, and nitric oxide (NO) production levels in HepG2 cells via the modulation of the nuclear factor erythroid 2-related factor 2 and the inducible nitric oxide synthase (iNOS) pathways, respectively. Due to the close link between inflammation and oxidative stress and with the objective of fostering the multifunctional behavior of bioactive peptides, in this study, the molecular characterization of the anti-inflammatory and immunomodulatory properties of H2 and H3 was carried out in HepG2 cells. In fact, both peptides were shown to modulate the production of pro (IFN-γ: −33.0 ± 6.7% H2, p = 0.011; −13.1 ± 2.0% H3, p = <0.0001; TNF: −17.6 ± 1.7% H2, p = 0.0004; −20.3 ± 1.7% H3, p = <0.0001; and IL-6: −15.1 ± 6.5% H3, p = 0.010)- and anti (IL-10: +9.6 ± 3.1% H2, p = 0.010; +26.0 ± 2.3% H3, p = < 0.0001)-inflammatory cytokines and NO (−9.0 ± 0.7% H2, p = <0.0001; −7.2 ± 1.8% H3, p = <0.0001) through regulation of the NF-κB and iNOS pathways, respectively, in HepG2 cells stimulated by lipopolysaccharides.

Carfilzomib inhibits the proliferation and apoptosis of multiple myeloma cells by inhibiting STAT1/COX-2/iNOS signaling pathway.

BackgroundThe ubiquitin-proteasome pathway (UPP) plays a key role in the intracellular degradation of abnormal and misfolded proteins in eukaryotic cells. Multiple myeloma (MM) is a common hematologic malignancy caused by clonal expansion of malignant plasma cells. Proteasome-targeted drugs such as carfilzomib, which is a selective proteasome inhibitor (PI), could play an important role in the treatment of diseases such as MM.MethodsMM cells were treated with different concentrations of carfilzomib and apoptosis as well as the viability of MM cells were measured by flow cytometry analysis and MTT assay. We also measured the effect of carfilzomib on the proliferation of myeloma cells by DNA incorporation of the pyrimidine analog BrdU. The effect of carfilzomib on apoptosis was detected by immunofluorescence TUNEL staining and western blot. We also verified its effect on the STAT1/COX-2/iNOS pathway by western blot.ResultsCarfilzomib inhibited the growth of MM cells in a concentration-dependent manner, with the strongest inhibitory activity on RPMI-8226 cells. Carfilzomib also induced apoptosis of MM cells in a concentration-dependent manner, with the strongest effect on RPMI-8226. BrdU assay was then performed with RPMI-8226 cells, and the results showed that carfilzomib inhibited cell proliferation in a concentration-dependent manner. Immunofluorescence TUNEL staining and western blot assays showed that carfilzomib induced apoptosis in a dose-dependent manner, and promoted the expression of apoptosis-related proteins such as cleaved-caspase-3, cleaved-caspase-3, Bax and Bcl2. Western blot also verified that carfilzomib promoted STAT1 inhibition and subsequently inhibited COX-2 and iNOS.ConclusionsInhibition of the STAT1/COX-2/iNOS signaling pathway by carfilzomib not only inhibited MM cell proliferation, but was also an important mechanism of inducing MM cell apoptosis.

Mulberry polyphenol extracts attenuated senescence through inhibition of Ras/ERK via promoting Ras degradation in VSMC.

Ageing is one of the major risk factors of human diseases, including cancer, diabetes, and cardiovascular disease. Mulberry exhibits a wide range of functions, such as anti-oxidant, anti-inflammation, and anti-diabetes. In this study, we investigated the role of mulberry polyphenol extract (MPE) in K-Ras-induced senescence of smooth muscle cells. Forced expression of K-Ras enhanced senescence of smooth muscle A7r5 cells as shown by the elevation of β-galactosidase activity. Treatment with MPE significantly repressed the Ras, phosphorylated ERK, and β-galactosidase level. MPE triggered the association of cyclins with their corresponding cyclin-dependent protein kinases and hyperphosphorylated retinoblastoma (RB). MPE also down-regulated the levels of K-Ras-induced CDK inhibitors. MPE enhanced the phosphorylated AMP-dependent protein kinase (AMPK) and inducible nitric oxide synthase (iNOS) level in the presence of K-Ras. Pretreatment with either L-NAME or AMPK inhibitor reversed the effects of MPE. In addition, L-NAME and AMPK inhibitor repressed the MPE-induced total and phosphorylated 3-hydroxy-3-methylglutaryl coenzyme A (HMG-Co A) level. MPE repressed K-Ras-induced G0/G1 arrest, whereas L-NAME and AMPK inhibitor blocked the effects of MPE. Our results indicated that MPE recovered the K-Ras-induced senescence of vascular smooth muscle cells through iNOS and AMPK-dependent pathway. Our findings suggested that MPE may prevent ageing-induced atherosclerosis.

Investigation of the intestinal trans-epithelial transport and antioxidant activity of two hempseed peptides WVSPLAGRT (H2) and IGFLIIWV (H3)

A preceding paper has shown that a hempseed peptic hydrolysate displays a cholesterol-lowering activity with a statin-like mechanism of action in HepG2 cells and a potential hypoglycemic activity by the inhibition of dipeptidyl peptidase-IV in Caco-2 cells. In the framework of a research aimed at fostering the multifunctional behavior of hempseed peptides, we present here the identification and evaluation of some antioxidant peptides from the same hydrolysate. After evaluation of its diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, a trans-epithelial transport experiment was performed using differentiated Caco-2 cells that permitted the identification of five transported peptides that were synthesized and evaluated by measuring the oxygen radical absorbance capacity (ORAC), the ferric reducing antioxidant power (FRAP), and the 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic) acid (ABTS), and diphenyl-2-picrylhydrazyl radical DPPH assays. The most active peptides, i.e. WVSPLAGRT (H2) and IGFLIIWV (H3), were then tested in cell assays. Both peptides were able to reduce the H2O2-induced reactive oxygen species (ROS), lipid peroxidation, and nitric oxide (NO) production levels in HepG2 cells, via the modulation of Nrf-2 and iNOS pathways, respectively.

Anti-inflammatory properties of Amomum maximum Roxb and Amomum muricarpum Elmer in the North of Vietnam

Inflammation is the body's homeostatic defense mechanism in which the immune system reacts to remove foreign bodies. Chronic inflammation can increase the risk for additional damage like autoimmune diseases, arthritis, diabetes and can result in death. Amomum maximum Roxb and Amomum muricarpum Elmer distributed widely in Vietnam have been used in traditional medicine for treatment of some gastrointestinal diseases. This study aimed to investigate the anti-inflammatory effects of the methanol extracts of A. maximum (AMM) and A. muricarpum Elmer (AMC) in murine macrophage RAW 264.7 cell line. The total extracts showed that the extracts exhibited low cytotoxicity and potent anti-inflammatory activities by suppressing excessive nitric oxide (NO). The IC50 values of AMC and AMM were found to be 12.67 ± 1.7 µg/mL and 42.7 ± 2.5 µg/mL, respectively. To elucidate the underlying mechanism, the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were evaluated using Western blot analysis. Our data demonstrated that AMC reduced the inflammatory response in a lipopolysaccharide (LPS)-induced RAW264.7 cell model via inhibition of iNOS and COX-2 while AMM seemed to modulate the inflammatory effect through the iNOS pathway only. In conclusion, AMM and AMC root extracts might be potential candidates for a study of naturally alternative anti-inflammatory drugs.

Abstract CT175: Targeting inducible nitric oxide in a first-in-class phase 1/2 trial in triple-negative breast cancer patients diminishes M2 macrophage polarization and led to a robust clinical response

Abstract Targeting inducible nitric oxide in a first-in-class phase 1/2 trial in triple-negative breast cancer patients diminishes M2 macrophage polarization and led to a robust clinical response. Andrew W. Chung*, Kartik Anand*, Polly Niravath* (co-first authors), Nakul Gupta, Luz A Venta, Mary R. Schwartz, Wei Qian, Yitian Xu, Licheng Zhang, Qiuying Chen, Nabeel Attarwala, John Kuhn, Tejal Patel, Angel Rodriguez, Anna Belcheva, Jorge Darcourt, Joe Ensor, Eric Bernicker, Ping-Ying Pan, Virginia Kaklamani, Shu Hsia Chen, Jenny C ChangIntroduction:The inducible nitric oxide signaling (iNOS) pathway has been associated with poor prognosis in triple-negative breast cancer (TNBC). Inhibition of iNOS pathway by using the pan-NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) in patient-derived xenograft (PDX) models has shown reduced tumor growth and enhanced survival. Here, we report a first-in-class phase 1/2 trial of L-NMMA plus taxane for patients with chemorefractory locally advanced (LA) TNBC or metastatic TNBC and present findings on molecular immune correlates and metabolites of response/resistance. Methods:Women with chemorefractory LA or metastatic TNBC without uncontrollable hypertension or heart disease, age &amp;gt;18 years, and ECOG performance status ≤2 were eligible. A Bayesian model averaging continuous reassessment method was used to determine the recommended phase 2 L-NMMA dose (RP2D). In phase 1, two dose levels of docetaxel (75 and 100 mg/m2) and seven dose levels of L-NMMA (5,7.5, 10, 15,17.5, and 20 mg/kg) were studied. Patients in phase 2 received a maximum of six (q 3 weeks) cycles of L-NMMA given via IV on D1-D5. All patients also received amlodipine (6 days/cycle) and aspirin daily, as hypertensive and thromboembolic prophylaxis. Toxicity was measured as per CTCAE v4.03. Pharmacokinetics/Pharmacodynamics, 38 circulating cytokines, and 30 differential metabolites were also assayed. Tissue imaging mass cytometry (IMC) with 35 cell surface markers was performed on paired biopsies. Results: From July 2016 to September 2020, a total of 35 patients were recruited (phase 1, n=15; phase 2, n= 24, including 4 RP2D patients from phase 1). RP2D dose was 20 mg/kg for L-NMMA and 100 mg/m2 for docetaxel. In phase 2, 54.2% had metastatic TNBC (with 5 median prior lines of chemotherapy) and 45.8% had LABC (anthracycline-refractory). Overall response rate (ORR) was 54.2% - 81.8% for LABC and 30.8% for metastatic TNBC. Radiologic complete response (CR) rate was 27.2% for LABC and 7.7% for metastatic TNBC. Among the LABC patients, 2 patients had pathological CR at surgery (18.2%). Grade ≥3 toxicity was noted in 21% of patients, however, none of grade ≥3 toxicity was attributed to L-NMMA. Compared to responder patients, correlative data showed that non-responders had a significant higher expression of biomarkers associated with M2 macrophage polarization including IL-10, adenosine and metabolite profile suggestive of aerobic glycolysis. In alignment, IMC analysis showed significant increase in intra-tumoral M2 macrophages in non-responders end of therapy biopsies. Conclusion: L-NMMA combination with taxanes was well tolerated with a robust response rate. Further investigation is warranted to test this combination in larger studies along with biomarker evaluation. Citation Format: Andrew W. Chung, Kartik Anand, Polly Niravath, Nakul Gupta, Luz A. Venta, Mary R. Schwartz, Wei Qian, Yitian Xu, Licheng Zhang, Qiuying Chen, Nabeel Attarwala, John Kuhn, Tejal A. Patel, Angel Rodriguez, Anna Belcheva, Jorge Darcourt, Joe E. Ensor, Eric Bernicker, Ping-Ying Pan, Virginia Kaklamani, Shu-Hsia Chen, Jenny C. Chang. Targeting inducible nitric oxide in a first-in-class phase 1/2 trial in triple-negative breast cancer patients diminishes M2 macrophage polarization and led to a robust clinical response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT175.

Purification and Structural Characterization of Sulfated Polysaccharides Derived from Brown Algae, Sargassum binderi: Inhibitory Mechanism of iNOS and COX-2 Pathway Interaction.

Among the components derived from brown algae, anionic sulfated polysaccharides, which contain sulfated fucose as the major monosaccharide, exert significant biological activities. In this study, we purified and structurally characterized sulfated polysaccharides from brown algae, Sargassum binderi (S. binderi; SBPs), and evaluated their biological activity in vitro and in vivo. The SBPs were separated based on their charges and their biophysical properties were investigated according to their functional groups, structural features, and molecular weights using FTIR, NMR, and MALS. Among all the SBPs, Fraction 4 (SBP-F4), with an average molecular weight of 2.867 × 105 g/mol, had the highest polysaccharide and sulfate contents (75.15 ± 0.25% and 24.08 ± 0.18%, respectively). The biological activities of SBP-F4 were investigated further in vitro and in vivo. Our results showed that SBP-F4 significantly suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins in LPS-activated macrophages. Moreover, in the LPS-treated zebrafish model, a significant decrease in cell death and NO production was observed. Collectively, these results show that SBPs not only exert protective effects against LPS-induced cytotoxicity but also inhibit the activation and anti-inflammatory activity of macrophages. Therefore, polysaccharides derived from S. binderi are potential anti-inflammatory agents for use in clinical settings.