Zahra Aghazadeh

Pharmacological effects of β-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 (β-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.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 (β-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 500 μg/ml) had no cytotoxic effect on HEK-Blue hTLR2 cells. Our results showed that M2000 at low and high doses (5 and 25 μg/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.

Anti-aging effects of M2000 (β-D-mannuronic acid) as a novel immunosuppressive drug on the enzymatic and non-enzymatic oxidative stress parameters in an experimental model

Abstract Background: The anti-aging property of β-D-mannuronic acid (M2000) as a novel non-steroidal anti-inflammatory and immunosuppressive agent was investigated on several determinants relative to the oxidative stress in an animal model. Methods: Sprague-Dawley rats were used for evaluating the safety and efficacy properties of M2000 on some oxidative stress enzymes, including the following: mitochondrial superoxide dismutase (SOD2), catalase (CAT), glutathione peroxidase (GPX1), glutathione S-transferase (GST), myeloperoxidase (MPO), and inducible nitric oxide synthase (iNOS) gene expression by real-time PCR. Malondialdehyde (MDA), carbonyl protein (PCO) (the lipid and protein oxidation marker, respectively), and total antioxidant capacity (TAC) were tested in serum by biochemical analysis. In addition, cortisol as a steroid hormone was surveyed by chemiluminescence immunoassay after 12 weeks of M2000 consumption. The rats were sacrificed 3 months after daily oral administration of M2000. Results: Our findings revealed the favorable effects of M2000 on several antioxidant enzyme and gene expression, including SOD2, CAT, GPX1, and GST; however, our results were not statistically significant. Moreover, there was no significant difference in MDA and PCO as lipid and protein oxidation markers, TAC, and cortisol compared with the control group following M2000 consumption. A slight weight increase in the M2000-treated group was also observed. Conclusions: Our data showed the anti-aging property of M2000 as a novel designed non-steroidal anti-inflammatory drug (NSAID) with immunosuppressive property on various oxidative stress determinants.

Effects of β-D-mannuronic acid, as a novel non-steroidal anti-inflammatory medication within immunosuppressive properties, on IL17 , RORγt , IL4 and GATA3 gene expressions in rheumatoid arthritis patients

Rheumatoid arthritis (RA) is the most common form of chronic inflammatory arthritis characterized by pain, swelling and destruction of joints, with a resultant disability. Disease-modifying anti-rheumatic drugs (DMARDs) and biological drugs can interfere with the disease process. In this study, the effect of β-d-mannuronic acid (M2000) as a novel non-steroidal anti-inflammatory drug (NSAID) with immunosuppressive and anti-inflammatory effects together with antioxidant effects was evaluated on IL17, RORγt, IL4 and GATA3 gene expression in 12 RA patients. Previously, M2000 driven from sodium alginate (natural product; patented, DEU: 102016113018.4) has shown a notable efficacy in experimental models of multiple sclerosis, RA and nephrotic syndrome. This study was performed on 12 patients with RA who had an inadequate response to conventional treatments. During this trial, patients were permitted to continue the conventional therapy excluding NSAIDs. M2000 was administered orally at a dose of 500 mg twice daily for 12 weeks. The peripheral blood mononuclear cells (PBMCs) were collected before and after treatment to evaluate the expression levels of IL4, GATA3, IL17 and RORγt. The gene expression results showed that M2000 has a potent efficacy, so that it could not only significantly decrease IL17 and RORγt levels but also increase IL4 and GATA3 levels after 12 weeks of treatment. Moreover, the gene expression results were in accordance with the clinical and preclinical assessments. In conclusion, M2000 as a natural novel agent has therapeutic and immunosuppressive properties on RA patients (identifier: IRCT2014011213739N2).

Evaluation of the efficacy and safety of β-d-mannuronic acid in patients with ankylosing spondylitis: A 12-week randomized, placebo-controlled, phase I/II clinical trial

Objective To evaluate the efficacy, safety and tolerability of &bgr;‐d‐mannuronic acid (M2000) in the treatment of ankylosing spondylitis (AS). Methods The study was a 12‐week randomized, double‐blind, placebo‐controlled, phase I/II clinical trial with 3 treatment arms: placebo, &bgr;‐d‐mannuronic acid and naproxen. Patients who had AS according to the modified New York criteria, with active disease at baseline were eligible for study. Primary outcome measure was the Assessment of SpondyloArthritis international Society (ASAS) 20 response rate at week 12. Results Of the 85 randomized patients, 27 were allocated to receive placebo, 28 naproxen, and 30 &bgr;‐d‐mannuronic acid. There were no statistically significant differences between treatment groups at baseline. Of the patients receiving &bgr;‐d‐mannuronic acid, 57.7% achieved an ASAS20 response at week 12, compared with 59% of the patients in the naproxen group (P > 0.05) and 19% of the patients in the placebo group (P = 0.007). In comparison with patients receiving placebo over the 12‐week treatment period, those receiving &bgr;‐d‐mannuronic acid and naproxen demonstrated statistically significantly greater improvement in all secondary endpoints. Interestingly, &bgr;‐d‐mannuronic acid reduced some parameters associated with inflammation more effectively than naproxen and placebo. The incidence of gastrointestinal and other adverse events were higher on naproxen than on &bgr;‐d‐mannuronic acid and placebo. Conclusion The present study demonstrated similar efficacy, but with a more favorable safety profile for &bgr;‐d‐mannuronic acid than naproxen and, therefore, suggest that &bgr;‐d‐mannuronic acid is suitable for the management of AS. Trial registration Iranian registry of clinical trials; www.irct.ir; IRCT2013062213739N1. Highlights&bgr;‐d‐Mannuronic acid has an acceptable safety profile in patients with AS.&bgr;‐d‐Mannuronic acid treatment led to an acceptable reduction in the signs and symptoms of AS.&bgr;‐d‐Mannuronic acid is suitable for the management of AS.

Immunomodulatory Effect of G2013 (α-L-Guluronic Acid) on the TLR2 and TLR4 in Human Mononuclear Cells

BACKGROUND Inhibition of Toll-like receptors (TLRs) signaling has been established as a new method for the development of anti-inflammatory drugs instead of NSAIDs (non-steroid anti-inflammatory drugs). Since the immunomodulatory role of G2013 (α-L-Guluronic acid) was reported in some recent experiments, we decided to assess the effects of G2013 on the protein expression of TLR2 and TLR4, their downstream signaling cascade, and the secretion of pro-inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs). METHODS After blood sampling from 16 healthy donors, PBMCs were isolated and treated with/without lipopolysaccharide (LPS), lipopolyteichoic acid (LTA), and G2013. Flow cytometry was done for detecting the protein expression of TLR2 and TLR4. MyD88, IκB, Tollip, and NF-κB mRNA expression were assessed by realtime PCR. ELISA was performed for assessing the concentration of IL-1β and IL-6. RESULTS G2013 at a concentration of 25 µg/mL (high dose) significantly downregulated NF-κB, IκB and MyD88 mRNA expression and suppressed the secretion of IL-1β by PBMCs. The findings indicate that G2013 may exert its regulatory effect under normal condition via Tollip in a dose dependence pathway. Our results demonstrated that G2013 had no profound impact on the protein expression of TLR2 and TLR4. CONCLUSION In conclusion, our findings point to the immunomodulatory effect of G2013 on the TLR2 and TLR4 signaling cascade and cytokine production by PBMCs. These findings could lead to an establishment of new safe anti-inflammatory drugs in the future.

Therapeutic effects of D-aspartate in a mouse model of multiple sclerosis

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis. EAE is mainly mediated by adaptive and innate immune responses that leads to an inflammatory demyelization and axonal damage. The aim of the present research was to examine the therapeutic efficacy of D-aspartic acid (D-Asp) on a mouse EAE model. EAE induction was performed in female C57BL/6 mice by myelin 40 oligodendrocyte glycoprotein (35-55) in a complete Freunds adjuvant emulsion, and D-Asp was used to test its efficiency in the reduction of EAE. During the course of study, clinical evaluation was assessed, and on Day 21, post-immunization blood samples were taken from the heart of mice for the evaluation of interleukin 6 and other chemical molecules. The mice were sacrificed, and their brain and cerebellum were removed for histological analysis. Our findings indicated that D-Asp had beneficial effects on EAE by attenuation in the severity and delay in the onset of the disease. Histological analysis showed that treatment with D-Asp can reduce inflammation. Moreover, in D-Asp-treated mice, the serum level of interleukin 6 was significantly lower than that in control animals, whereas the total antioxidant capacity was significantly higher. The data indicates that D-Asp possess neuroprotective property to prevent the onset of the multiple sclerosis.

Effect of β-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 β-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.

Therapeutic effects of pegylated-interferon-α2a in a mouse model of multiple sclerosis

Introduction Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS). EAE is mainly mediated by adaptive and innate immune responses that lead to an inflammatory demyelination and axonal damage. The aim of the present research was to examine the therapeutic efficacy of Peg interferon alpha 2a (Peg-IFN α-2a) as a serine protease inhibitor on EAE model. Material and methods EAE induction was performed in female C57BL/6 mice by myelin oligodendrocyte glycoprotein (35-55) (MOG35-55) in Complete Freund’s Adjuvant (CFA) emulsion, and Peg-IFN α-2a was used for the treatment of EAE. During the course of the study, clinical evaluation was assessed, and on day 21 post-immunisation blood samples were taken from the heart of mice for evaluation of IL-6, and enzymatic and non-enzymatic antioxidants. The mice were sacrificed and the brains and cerebellums were removed for histological analysis. Results Our findings indicated that Peg-IFN α-2a had beneficial effects on EAE by attenuation of the severity and a delay in the onset of disease. Histological analysis showed that treatment with Peg-IFN α-2a can reduce inflammation criteria. Moreover, in Peg-IFN α-2a-treated mice the serum level of IL-6 was significantly less than in controls, and total antioxidant capacity was significantly more than in the control animals. Conclusions These data indicate that Peg-IFN α-2a as an anti-serine protease with immunomodulatory properties may be useful for the treatment of MS.