Dysregulated miRNAs network in the critical COVID-19: An important clue for uncontrolled immunothrombosis/thromboinflammation.

Known as a pivotal immunohemostatic response, immunothrombosis is activated to restrict the diffusion of pathogens. This beneficial intravascular defensive mechanism represents the close interaction between the immune and coagulation systems. However, its uncontrolled form can be life-threatening to patients with the critical coronavirus disease 2019 (COVID-19). Hyperinflammation and ensuing cytokine storm underlie the activation of the coagulation system, something which results in the provocation of more immune-inflammatory responses by the thrombotic mediators. This vicious cycle causes grave clinical complications and higher risks of mortality. Classified as an evolutionarily conserved family of the small non-coding RNAs, microRNAs (miRNAs) serve as the fine-tuners of genes expression and play a key role in balancing the pro/anticoagulant and pro-/anti-inflammatory factors maintaining homeostasis. Therefore, any deviation from their optimal expression levels or efficient functions can lead to severe complications. Despite their extensive effects on the molecules and processes involved in uncontrolled immunothrombosis, some genetic agents and uncontrolled immunothrombosis-induced interfering factors (e.g., miRNA-single nucleotide polymorphysms (miR-SNPs), the complement system components, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, and reactive oxygen species (ROS)) have apparently disrupted their expressions/functions. This review study aims to give an overview of the role of miRNAs in the context of uncontrolled immunothrombosis/thromboinflammation accompanied by some presumptive interfering factors affecting their expressions/functions in the critical COVID-19. Detecting, monitoring, and resolving these interfering agents mafy facilitate the design and development of the novel miRNAs-based therapeutic approaches to the reduction of complications incidence and mortality in patients with the critical COVID-19.

Cytokine storm in the pathophysiology of COVID-19: Possible functional disturbances of miRNAs.

SARS-CoV-2, as the causative agent of COVID-19, is an enveloped positives-sense single-stranded RNA virus that belongs to the Beta-CoVs sub-family. A sophisticated hyper-inflammatory reaction named cytokine storm is occurred in patients with severe/critical COVID-19, following an imbalance in immune-inflammatory processes and inhibition of antiviral responses by SARS-CoV-2, which leads to pulmonary failure, ARDS, and death. The miRNAs are small non-coding RNAs with an average length of 22 nucleotides which play various roles as one of the main modulators of genes expression and maintenance of immune system homeostasis. Recent evidence has shown that Homo sapiens (hsa)-miRNAs have the potential to work in three pivotal areas including targeting the virus genome, regulating the inflammatory signaling pathways, and reinforcing the production/signaling of IFNs-I. However, it seems that several SARS-CoV-2-induced interfering agents such as viral (v)-miRNAs, cytokine content, competing endogenous RNAs (ceRNAs), etc. preclude efficient function of hsa-miRNAs in severe/critical COVID-19. This subsequently leads to increased virus replication, intense inflammatory processes, and secondary complications development. In this review article, we provide an overview of hsa-miRNAs roles in viral genome targeting, inflammatory pathways modulation, and IFNs responses amplification in severe/critical COVID-19 accompanied by probable interventional factors and their function. Identification and monitoring of these interventional elements can help us in designing the miRNAs-based therapy for the reduction of complications/mortality rate in patients with severe/critical forms of the disease.

Efficient roles of miR-146a in cellular and molecular mechanisms of neuroinflammatory disorders: An effectual review in neuroimmunology.

Known as one of the most sophisticated systems of the human body, the nervous system consists of neural cells and controls all parts of the body. It is closely related to the immune system. The effects of inflammation and immune reactions have been observed in the pathogenesis of some neurological disorders. Defined as the gene expression regulators, miRNAs participate in cellular processes. miR-146a is a mediator in the neuroimmune system, leaving substantial effects on the homeostasis of immune and brain cells, neuronal identities acquisition, and immune responses regulation in the nervous system. Its positive efficiency has been proven in modulating inflammatory reactions, hemorrhagic complications, and pain. Moreover, the miR-146a targets play a key role in the pathogenesis of these illnesses. Based on the performance of its targets, miR-146a can have various effects on the disease progress. The abnormal expression/function of miR-146a has been reported in neuroinflammatory disorders. There is research evidence that this molecule qualifies as a desirable biomarker for some disorders and can even be a therapeutic target. This study aims to provide a meticulous review regarding the roles of miR-146a in the pathogenesis and progression of several neuroinflammatory disorders such as multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, temporal lobe epilepsy, ischemic stroke, etc. The study also considers its eligibility for use as an ideal biomarker and therapeutic target in these diseases. The awareness of these mechanisms can facilitate the disease management/treatment, lead to patients' amelioration, improve the quality of life, and mitigate the risk of death.

A comprehensive review on miR-146a molecular mechanisms in a wide spectrum of immune and non-immune inflammatory diseases.

MicroRNAs (miRNAs) are single-strand endogenous and non-coding RNA molecules with a length of about 22 nucleotides, which regulate genes expression, through modulating the translation and stability of their target mRNAs. miR-146a is one of the most studied miRNAs, due to its central role in immune system homeostasis and control of the innate and acquired immune responses. Accordingly, abnormal expression or function of miR-146a results in the incidence and progression of immune and non-immune inflammatory diseases. Its deregulated expression pattern and inefficient function have been reported in a wide spectrum of these illnesses. Based on the existing evidence, this miRNA qualifies as an ideal biomarker for diagnosis, prognosis, and activity evaluation of immune and non-immune inflammatory disorders. Moreover, much attention has recently been paid to therapeutic potential of miR-146a and several researchers have assessed the effects of different drugs on expression and function of this miRNA at diverse experimental, animal, besides human levels, reporting motivating results in the treatment of the diseases. Here, in this comprehensive review, we provide an overview of miR-146a role in the pathogenesis and progression of several immune and non-immune inflammatory diseases such as Rheumatoid arthritis, Systemic lupus erythematosus, Inflammatory bowel disease, Multiple sclerosis, Psoriasis, Graves' disease, Atherosclerosis, Hepatitis, Chronic obstructive pulmonary disease, etc., discuss about its eligibility for being a desirable biomarker for these disorders, and also highlight its therapeutic potential. Understanding these mechanisms underlies the selecting and designing the proper therapeutic targets and medications, which eventually facilitate the treatment process.

The role of ß-d-mannuronic acid, as a new non-steroidal anti-inflammatory drug on expression of miR-146a, IRAK1, TRAF6, NF-κB and pro-inflammatory cytokines following a clinical trial in rheumatoid arthritis patients.

Context: miR-146a, its targets (IRAK1, TRAF6) and NF-κB transcription factor play a fundamental role in rheumatoid arthritis (RA). Positive effects of drug ß-d-mannuronic acid (M2000) were proven on their expression in the HEK-Blue hTLR2 cell line, and results of its phase III clinical trial on RA patients were encouraging.Objective: This research aimed to investigate the effects of M2000 on expression of these genes and serum levels of IL-6 and TNF-α as pro-inflammatory cytokines in RA patients.Material and methods: In this study (Trial Registration Number: IRCT2017100213739N10), 12 RA patients (according to the American College of Rheumatology criteria) and 12 healthy subjects (as control group) were selected. The gene expression of miR-146a, IRAK1, TRAF6, and NF-κB were measured at the baseline and after 12 weeks M2000 therapy, using quantitative real-time PCR method. Moreover, the serum levels of IL-6 and TNF-α were evaluated at the similar times by ELISA method.Results: Our findings showed that the gene expression of miR-146a, IRAK1, TRAF6, and NF-κB significantly decreased after 12 weeks M2000 therapy in RA patients (0.81-, 0.68-, 0.79-, 0.82-fold, with p < .05, p < .01, p < .01, p < .05, respectively). Furthermore, the serum levels of IL-6 and TNF-α significantly reduced in these patients after 12 weeks M2000 therapy (both with p < .05).Conclusions: The present research results determined the part of molecular mechanisms of drug M2000 in RA treatment, based on the expression and function modification of miR-146a, IRAK1, TRAF6, NF-κB, IL-6 and TNF-α.

Immunopharmacological effect of ß-d-mannuronic acid (M2000), as a new immunosuppressive drug, on gene expression of miR-155 and its target molecules (SOCS1, SHIP1) in a clinical trial on rheumatoid arthritis patients.

The positive impacts of ß-d-mannuronic acid (M2000) on the gene expression of miR-155, its target molecules (SOCS1 and SHIP1), and NF-κB transcription factor were demonstrated in a study using the HEK293-TLR2 cell line. This new drug has been approved as a safe and effective medication by a randomized, multinational, phase III clinical trial on RA patients. The present study aimed to evaluate the oral administration effect of M2000 on the expression levels of the mentioned genes in RA patients. This research was conducted on 12 RA patients and 12 healthy individuals. After extraction of total RNA from PBMCs of patients and synthesis of cDNA, the expression levels of miR-155, SOCS1, SHIP1, and NF-κB genes were measured through quantitative Real-time PCR at baseline and after 12 weeks of M2000 therapy. Our findings showed that the miR-155 gene expression level significantly decreased in the M2000-treated patients compared with the baseline (0.76-fold, with p < .05). The expression levels of SOCS1 and SHIP1 genes significantly increased in the patients treated with M2000 compared with the before treatment (1.46-, 1.54-fold, with p < .01, p < .05, respectively). In addition, it was found that the gene expression level of the NF-κB transcription factor significantly reduced in M2000-treated patients compared with the baseline (0.81-fold, with p < .05). This study showed that the oral administration of M2000 was able to reduce the expression of the miR-155, increase the expression of SOCS1 and SHIP1, and decrease the NF-κB gene expression (Trial Registration Number: IRCT2017100213739N10).

International multicenter randomized, placebo-controlled phase III clinical trial of ß-D-mannuronic acid in rheumatoid arthritis patients.

BACKGROUND: The oral administration of drug ß-D-mannuronic acid (M2000) showed a potent therapeutic effect in phase I/II study in rheumatoid arthritis (RA) patients. Here, our aim is to assess the efficacy and safety of this new drug in RA patients under a multinational, randomized placebo-controlled phase III clinical trial. METHOD: Patients (n = 288) with active disease at baseline and inadequate response to conventional drugs were randomly allocated to three groups; (1) receiving mannuronic acid at a dose of two capsules (500 mg) per day orally for 12 weeks, (2) placebo-controlled, and (3) conventional. The primary endpoints were the America College of Rheumatology 20 response (ACR20), 28-joint disease activity score (DAS28) and Modified Health Assessment Questionnaire-Disability Index (M-HAQ-DI). In addition, the participants were followed-up for safety assessment. RESULTS: In this phase III trial, after 12 weeks of treatment, there was a significant reduction in ACR20 between mannuronic-treated patients compared to placebo and conventional groups. Moreover, there was a similar significant improvement for DAS28 following mannuronic therapy. The statistical analysis showed a significant reduction in the swollen and tender joint count in mannuronic-treated patients compared with the placebo group. On the other side, mannuronic acid showed no-to-very low adverse events in comparison to placebo. CONCLUSION: The results of this multinational, phase III clinical trial provided a potent evidence base for the use of ß-D-mannuronic acid as a new highly safe and efficient drug in the treatment of RA.

Anti-diabetic effect of ß-D-mannuronic acid (M2000) as a novel NSAID with immunosuppressive property on insulin production, blood glucose, and inflammatory markers in the experimental diabetes model.

This research aimed to evaluate the anti-diabetic effects of ß-d-mannuronic acid (M2000) on blood glucose, insulin production, and inflammatory markers in streptozotocin-induced diabetic rats. Our data showed that the final fasting serum glucose level was significantly lower in the M2000-treated group compared to the diabetic control group (p < .05). In addition, the final fasting serum insulin level significantly increased in the M2000-treated group compared to the diabetic control group (p < .05). Our finding revealed that the serum level of hs-CRP and IL-6 decreased significantly in the M2000-treated group compared to the diabetic control group (p < .05). This study showed that M2000, as a new NSAID, was able to decrease serum glucose levels and increase serum insulin levels and this drug could significantly decrease the inflammatory markers in the M2000-treated group. Collectively, treatment with M2000 might be recommended reducing the severity of diabetes-induced inflammatory symptoms.

Effect of ß-D-Mannuronic Acid (M2000) on Oxidative Stress Enzymes' Gene Using Healthy Donor Peripheral Blood Mononuclear Cells for Evaluating the Anti-Aging Property.

OBJECTIVE: This research aimed to study the anti-aging and anti-inflammatory effects of low and high doses of the ß-D-mannuronic (M2000) on gene expression of enzymes involved in oxidative stress (including SOD2, GST, GPX1, CAT, iNOS, and MPO) in peripheral blood mononuclear cells (PBMCs) of healthy donors under in vitro conditions. METHODS: The PBMCs were separated and the RNAs were then extracted and the cDNAs synthesized, and expression levels of the mentioned genes were detected by qRT-PCR. RESULTS: Our results indicated that the high dose of this drug could significantly reduce the expression level of the SOD2 gene compared to the lipopolysaccharide (LPS) group (p < 0.0001). Moreover, it was found that the high dose of this drug could significantly decrease the expression level of the GST gene compared to the LPS group (p < 0.0001). However, no significant reductions were observed in expression levels of the CAT and GPX1 genes compared to the LPS group. Furthermore, our data revealed that the level of iNOS and MPO gene expression was significantly reduced, in both doses of M2000, respectively, compared to the LPS group (p < 0.0001). CONCLUSION: This research showed that M2000 as a novel NSAID with immunosuppressive properties could modify oxidative stress through lowering expression levels of the SOD2, GST, iNOS, and MPO genes compared to the healthy expression levels, with a probable reduction of the risk of developing inflammatory diseases related to age and aging.

Assessment of immunological profile in ankylosing spondylitis patients following a clinical trial with guluronic acid (G2013), as a new NSAID with immunomodulatory properties.

The present research aims to study the effects of guluronic acid (G2013) on gene expression levels of the T-bet, GATA3, RORγt, AHR, and FOXP3 transcription factors and on gene expression of their related cytokines following oral administration of this drug in ankylosing spondylitis (AS) patients. In this trial (clinical trial identifier: IRCT2016091813739N4), 14 AS patients and 12 age- and sex-matched healthy individuals were enrolled. The level of transcription factors' gene expression and expression of their related cytokines were measured by quantitative real-time PCR, before and 3 months after G2013 therapy. Our data indicated that the gene expression levels of the T-bet and IFN-γ were not significantly reduced during 12 weeks of treatment with G2013 (p > 0.05). The findings showed that the gene expression levels of the GATA3 and IL-4 increased significantly during 12 weeks of treatment with G2013 (p < 0.05). In addition, gene expression levels of the RORγt, IL-17, AHR, and IL-22 decreased significantly during the 12-week treatment with G2013 (p < 0.05). Moreover, the gene expression level of the FOXP3 increased significantly during 12 weeks of treatment with G2013, but the gene expression level of IL-10 did not increase significantly (p < 0.05, p > 0.05, respectively). The present study showed that oral intake of G2013 was able to modify the severity of articular and inflammatory symptoms of AS through reducing the gene expression levels of the RORγt, IL-17, AHR, and IL-22 and increasing the gene expression levels of the GATA3, IL-4, and FOXP3.

Cardioprotective effect of ß-d-mannuronic acid (M2000) as a novel NSAID on gene expression of oxLDL scavenger receptors in the experimental diabetic model.

CONTEXT: The investigations have shown that patients with diabetes have the elevated levels of glucose and oxLDL. These two play an important role in increased expression levels of oxLDL scavenger receptors on the surface of macrophages and endothelial cells that leads to deposition of oxLDL and macrophages in vascular walls. OBJECTIVE: The present study intends to show the effects of ß-d-mannuronic acid (M2000) on the expression profile of ox-LDL scavenger receptors (including SR-A, LOX-1, CD36, and CD68) in an experimental model of diabetes. MATERIALS AND METHODS: Eighteen Sprague-Dawley rats were randomly divided into three 6-member groups of the healthy control, diabetic control, and treated rats by M2000. Diabetes was induced in rats by intraperitoneal (IP) administration of 60 mg/kg streptozotocin. The treated rats were given daily intraperitoneal injections of M2000 with a dose of 25 mg/kg for 28 days and at the end of the 28th day, their aortas were removed. The qRT-PCR technique was then used to evaluate the expression levels of the proposed gene. RESULTS: The gene expression levels of the SR-A, LOX-1, CD36, and CD68 significantly declined in the diabetic group that received M2000 compared with untreated diabetic rats. CONCLUSIONS: The M2000, as a novel NSAID is able to modify by lowering the gene expression levels of SR-A, LOX-1, CD36, and CD68 in treated rats compared to the untreated diabetic group, which may play an important role in preventing the complications that could lead to a cardioprotective efficacy.

Modification of Sexual Hormones in Rheumatoid Arthritis Patients by M2000 (ß-D-mannuronic Acid) as a Novel NSAID with Immunosuppressive Property.

BACKGROUND: Based on in-vitro, in-vivo and human studies, the ß-D-mannuronic acid (M2000) has been introduced as a novel non-steroidal anti-inflammatory drug (NSAID) with immunosuppressive properties. OBJECTIVE: This study aimed to evaluate the efficacy of this drug on serum level of sex hormones (Estradiol, Progesterone, and DHEAS) in rheumatoid arthritis (RA) patients. METHODS: The present research was performed on 10 RA patients who had an inadequate response to conventional treatments (clinical trial identifier: IRCT2014011213739N2). During this trial, the patients were permitted to continue the conventional therapy along with adding M2000 orally at a dose of 500 mg twice daily for 12 weeks. Serum samples were collected in a normal group, patient group (at baseline) and treatment group (after 12 weeks). The samples were tested for evaluating the serum level of Estradiol, Progesterone, and DHEAS using chemiluminescent microparticle immunoassay. RESULTS: Data showed that the serum level of estradiol was reduced (both in men and women) during the treatment with M2000 (after 12 weeks), but there was no significant difference in the non-treated group with M2000 (p > 0.05). In addition, the serum level of progesterone and DHEAS significantly increased following the 12-week administration of M2000 in both male and female patients, compared to the non-treated group with M2000 (p < 0.001, p < 0.05, p < 0.05, p < 0.01, respectively). CONCLUSION: The present research showed that the sex hormones might be modified by M2000 therapy in RA patients by increasing the serum level of progesterone and DHEAS compared to healthy individuals.

A phase I/II randomized, controlled, clinical trial for assessment of the efficacy and safety of ß-D-mannuronic acid in rheumatoid arthritis patients.

BACKGROUND: Following the potent efficacy of ß-D-mannuronic acid (M2000) in phase I/II trial in ankylosing spondylitis patients, the present clinical trial was conducted to evaluate the efficacy, safety, and tolerability of this novel drug in rheumatoid arthritis (RA) patients who had inadequate response to conventional therapy. METHOD: The study was a 12-week randomized, controlled, phase I/II clinical trial with two treatment arms: M2000 and conventional treatment. Patients who had RA according to the modified American College of Rheumatology (ACR) criteria, with active disease at baseline also inadequate response to conventional therapy, were enrolled in this study. M2000 was administrated at a dose of two capsules (500 mg) per day orally during a period of 12 weeks. The primary endpoint was the proportion of patients fulfilling the ACR 20% improvement criteria after 12 weeks of M2000 therapy. Moreover, the patients were also followed up for safety. RESULTS: There were no statistically significant differences between treatment and conventional groups at baseline characteristics. The ACR20 response rate was significantly higher among M2000-treated patients than conventional-treated control, so that 74% of patients in treatment group showed an ACR20 response after 12 weeks of M2000 therapy (74 versus 16%; P = 0.011). 10% of M2000-treated patients and 57.1% of conventional-treated patient's adverse events occurred during this study. CONCLUSION: Treatment with M2000 in combination with conventional therapy showed a significantly superior efficacy along with a high safety profile compared to conventional-treated patients. Thereby, M2000 might be suggested as a suitable option in the treatment of RA.

Oral administration effects of ß-d-mannuronic acid (M2000) on Th17 and regulatory T cells in patients with ankylosing spondylitis.

BACKGROUND: To explore the effects of ß-d-mannuronic acid (M2000) on levels of Th17, regulatory T (Treg) cells and their related cytokines in patients with ankylosing spondylitis (AS). METHODS: 30 AS patients and 15 age and sex-matched healthy individuals were enrolled in this study. The frequencies of Th17 and Treg cells and serum levels of related cytokines were measured by flow cytometry analysis and ELISA respectively, before (baseline) and 3 months after M2000 therapy. RESULTS: Significantly higher baseline Th17 cells and serum IL-17, TNF-α and IL-6 were observed in AS patients than in normal controls, whereas baseline levels of Treg cells and serum IL-10 were not significantly different between AS patients and healthy controls. After M2000 therapy, frequencies of Th17 and serum levels of IL-17 and IL-6 significantly decreased in AS patients. The frequencies of Treg cells and serum level of IL-10 were not significantly changed, in comparison to before therapy. Moreover, the correlation analysis showed that frequencies of Th17 and levels of IL-17, TNF-α and IL-6 were positively correlated with Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and Bath Ankylosing Spondylitis Functional Index (BASFI) scores, whereas Treg cells were revealed to be negatively correlated with BASDAI and BASFI scores. CONCLUSIONS: It can be concluded that the oral administration of M2000 as a novel NSAID with the immunosuppressive property that down-regulates Th17 and Th17-related cytokines and facilitates the correction of the Th17/Treg imbalance can be effective in the process of AS treatment.

Effects of guluronic acid (G2013) on SHIP1, SOCS1 induction and related molecules in TLR4 signaling pathway.

OBJECTIVE: This research aimed to study the anti-inflammatory and immunomodulatory effects of guluronic acid (G2013) on gene expression of TLR4, MyD88, SHIP1, SOCS1, NF-κB, and assessment of the level of IL-1ß as a pro-inflammatory cytokine in HEK-Blue hTLR4 cell line. METHODS: The cytotoxicity of G2013 was assessed by the MTT assay. The mRNA expression levels of the mentioned genes were measured by qRT-PCR. IL-1ß concentration in culture media was determined using ELISA method. RESULTS: MTT assay demonstrated that G2013 (before the concentration of 125µg/ml) had no cytotoxic effect on HEK-Blue hTLR4 cells. Our results indicated that the low and high doses of this drug could significantly reduce the gene expression of TLR4 and MyD88, as compared to the control group (p<0.05). Moreover, it was found that the low dose of this drug could significantly increase the gene expression of SHIP1 and SOCS1, as compared to the control group (p<0.05). Furthermore, the study findings revealed that the level of NF-κB gene expression significantly reduced, in both doses of G2013 compared to the control group (p<0.05, p<0.01, respectively). Our data showed that the level of IL-1ß in culture media decreased by both doses of this drug in comparison to control group (p<0.05). CONCLUSION: This study indicates that G2013 is able to induce SHIP1, SOCS1 and reduce TLR4, MyD88, NF-κB at the level of gene expression and decrease IL-1ß as a pro-inflammatory cytokine which might be recommended for reduction of inflammatory reactions.

Anti-angiogenesis effect of ß-D-mannuronic acid (M2000) as a novel NSAID with immunosuppressive properties under experimental model.

Angiogenesis is a process through which new capillaries are formed from pre-existing ones, which contributes significantly to the pathogenesis of numerous diseases, such as cancer and chronic inflammatory disorders. The ß-D-mannuronic acid (M2000) is a novel non-steroidal anti-inflammatory drug (NSAID) with immunosuppressive effects and is a matrix metalloproteinase (MMP) inhibitor. This research aimed to study the anti-angiogenesis effects of M2000 under in vitro and in vivo models. The cytotoxic and anti-proliferative effects of M2000 were examined using the trypan blue method and the MTT assay, respectively. The 3D collagen-cytodex model and the chick chorioallantoic membrane (CAM) assay were then used to evaluate the anti-angiogenesis property of M2000. Cytotoxicity assay revealed that M2000 (at concentrations of less than 100 µg/mL) had no cytotoxic effect on human umbilical vein endothelial cells (HUVECs). It was also illustrated that M2000 had little or no anti-proliferative effect on HUVECs. In addition, the anti-angiogenesis effects of M2000 were shown to be marginal in the in vitro model and both significant and dose-dependent in the in vivo status. This study showed that M2000 could be considered as an anti-angiogenic molecule which more likely exerts its activity mainly via indirect effects on endothelial cells and its anti-inflammatory effects may partly be attributable to its anti-angiogenic activity. Therefore, it could be recommended as a candidate for prevention and treatment of cancer, chronic inflammatory diseases, and other angiogenesis-related disorders.

Introduction of ß-d-mannuronic acid (M2000) as a novel NSAID with immunosuppressive property based on COX-1/COX-2 activity and gene expression.

BACKGROUND: The NSAIDs which inhibit the cyclooxygenase (COX) enzymes are among medications widely used to treat pain and inflammation. These drugs cause digestive complications resulting in inhibition of the COX-1 enzyme, while the inhibition of the COX-2 enzyme has therapeutic effects. Therefore research focuses on the production of medications that specifically inhibit the COX-2 enzyme. This study aimed to study the effects of ß-d-mannuronic (M2000) acid on the gene expression and activity of COX-1/COX-2 enzymes in order to introduce a novel NSAID for treating inflammatory diseases. METHODS: The mRNA expression levels of COXs were analyzed with qRT-PCR. Prostaglandin E2 (PGE2) concentration in culture media was determined using ELISA method. RESULTS: Our results indicated that the M2000 at low and high dose could significantly reduce the gene expression level of COX-2 compared to the LPS group (p<0.0001), but no significant reduction was observed in the gene expression level of COX-1 compared to the LPS group. Moreover, it was noticed that this drug strongly and significantly reduced the activity of COX-1/COX-2 enzymes at the three concentrations of 5, 50 and 500 mMol/ml compared to the LPS and arachidonic acid groups (p<0.0001). CONCLUSIONS: This study showed that drug M2000 as a novel NSAID with immunosuppressive property is able strongly to inhibit the activity of COX-1/COX-2 enzymes, with suppressing the gene expression of COX-2 specifically. Therefore, based on gene expression findings this drug might be categorized and introduced as a novel NSAID with selective COX-2 inhibitory effect.

An in vitro evaluation of anti-aging effect of guluronic acid (G2013) based on enzymatic oxidative stress gene expression using healthy individuals PBMCs.

BACKGROUND: Aging is usually associated with increased levels of oxidants, and may result in damages caused by oxidative stress. There is a direct relationship between aging and increased incidence of inflammatory diseases. The present research intended to study the anti-aging and anti-inflammatory effects of the drug G2013 (guluronic acid) at low and high doses on the genes expression of a number of enzymes involved in oxidative stress (including SOD2, GPX1, CAT, GST, iNOS, and MPO) in peripheral blood mononuclear cells (PBMCs) of healthy individuals under in vitro conditions. METHODS: Venous blood samples were taken from 20 healthy individuals, the PBMCs were isolated and their RNAs extracted and their cDNAs were synthesized, and the genes expression levels were measured using the qRT-PCR technique. RESULTS: Our results indicated that this drug could, at both low and high doses, significantly reduce the expression of the genes for SOD2, GPX1, CAT, and GST compared to the LPS group (p<0.0001). Moreover, it was noticed that the drug is able to significantly reduce gene expression levels at the high dose and at both doses (low and high), for iNOS and MPO compared to the LPS group (p<0.0001), respectively. CONCLUSIONS: The present research showed that G2013, as a novel NSAID drug with immunomodulatory properties, could modulate the expression levels of the genes for SOD2, GPX1, CAT, GST, iNOS, and MPO, to the level of healthy gene expression, and possibly it might reduce the pathological process of aging and age-related inflammatory diseases.

Pharmacological effects of ß-d-mannuronic acid (M2000) on miR-146a, IRAK1, TRAF6 and NF-κB gene expression, as target molecules in inflammatory reactions.

BACKGROUND: Impaired expression and function of microRNAs (miRNAs) are involved in the pathogenesis of many autoimmune and inflammatory diseases. Moreover, there is a close relationship between TLRs and miRNAs and impairment in regulating their expression which can play a vital role in the immunopathogenesis of many inflammatory reactions. This research aimed to study the pharmaceutical effects of M2000 (ß-d-mannuronic acid) on the expression of miR-146a and its two target molecules (IRAK1 and TRAF6), and the transcription factor NF-κB in the HEK-Blue hTLR2 cell line. METHODS: The cytotoxicity of M2000 was assessed by the MTT assay, and the qRT-PCR technique was employed in the presence and absence of M2000 treatment to measure gene-expression levels of miR-146a, IRAK1, TRAF6, and NF-κB. RESULTS: MTT assay indicated that M2000 (before the concentration of 500µg/ml) had no cytotoxic effect on HEK-Blue hTLR2 cells. Our results showed that M2000 at low and high doses (5 and 25µg/well) could significantly reduce gene expression levels of miR-146a (p<0.01). Furthermore, it was found that this medication at two different doses could considerably decrease IRAK1 and TRAF6 gene expression (p<0.001). Moreover, this study revealed that expression level of NF-κB also significantly declined at these two doses (p<0.01). CONCLUSIONS: This study for the first time shows that M2000 as a novel NSAID with immunosuppressive properties is able to modify TLR signaling through suppressing the adaptor molecules IRAK1 and TRAF6, the transcription factor NF-κB and miR-146a as a new therapeutic approach.