Chitosan-enclosed SLN improved SV-induced hepatocellular cell carcinoma death by modulation of IQGAP gene expression, JNK, and HDAC activities.

BACKGROUND: Hepatocellular carcinoma (HCC) is a global life-threatening problem and therapeutic interventions are still encountered. IQGAP genes are involved in HCC oncogenesis. The modulatory effect of statins on the expression of IQGAP genes is still unclear. This study aims to study the effect of free SV and chitosan (CS) decorated simvastatin (SV) loaded solid lipid nanoparticles (C-SV-SLNs) on HCC mortality. METHODS AND RESULTS: Plain, SV-SLN, and C-SV- SLN were prepared and characterized in terms of particle size (PS), zeta potential (ZP), and polydispersity index (PDI). The biosafety of different SLN was investigated using fresh erythrocytes, moreover, cytotoxicity was investigated using HepG2 cell lines. The effect of SLNs on IQGAPs gene expression as well as JNK, HDAC6, and HDAC8 activity was investigated using PCR and MOE-docking. The current results displayed that SV-SLNs have nanosized, negative ZP and are homogenous, CS decoration shifts the ZP of SLN into cationic ZP. Furthermore, all SLNs exhibited desirable biosafety in terms of no deleterious effect on erythrocyte integrity. SV solution and SV-SLN significantly increase the mortality of HepG2 compared to undertreated cells, however, the effect of SV-SLN is more pronounced compared to free SV. Remarkably, C-SV-SLN elicits high HepG2 cell mortality compared to free SV and SV-SLN. The treatment of HepG2 cells with SV solution, SV-SLN, or C-SV-SLN significantly upregulates the IQGAP2 gene with repression of IQGAP1 and IQGAP3 genes. MOE-docking studies revealed both SV and tenivastatin exhibit interactions with the active sites of JNK, HDAC6, and HDAC8. Moreover, tenivastatin exhibited greater interactions with magnesium and zinc compared to SV. CONCLUSIONS: This research provides novel insights into the therapeutic potential of SV, SV-SLN and C-SV-SLNs in HCC treatment, modulating critical signaling cascades involving IQGAPs, JNK, and HDAC. The development of C-SV-SLNs presents a promising strategy for effective HCC therapy.

Highly efficient mica-incorporated graphene oxide-based membranes for water purification and desalination.

Graphene oxide (GO) has become the most attractive material for membrane technology owing to its potential application as a nanofiller in water treatment, purification, and desalination. In this study, we incorporated mica as a cross-linking reagent to increase the interlayer spacing and stability of GO sheets and fabricated a mica/GO (MGO) membrane for the first time. The MGO membrane (260 ± 10 nm) exhibits 100% rejection for biomolecules such as tannic acid (TA) and bovine serum albumin (BSA) and >99% rejection for multiple probe molecules, such as methylene blue, methyl orange, congo red, and rhodamine B. The high rejection of membranes can be attributed to the surface interaction of mica with GO nanosheets through covalent interaction, which enhances the stability and separation efficiency of the membranes for probe ions and molecules. This ultrathin MGO membrane also exhibits much better water permeability at 870 ± 5 L m-2 h-1 bar-1, which is 10-100 times greater than that reported for pure GO and GO-based composite membranes. Additionally, the membrane shows high rejection for salt ions (70%). Furthermore, the stability of the MGO membranes was evaluated under various conditions, and the membranes demonstrated remarkable stability for up to 60 days in a neutral environment.

Dulaglutide reduces oxidative DNA damage and hypermethylation in the somatic cells of mice fed a high-energy diet by restoring redox balance, inflammatory responses, and DNA repair gene expressions.

Obesity is an established risk factor for numerous malignancies, although it remains uncertain whether the disease itself or weight-loss drugs are responsible for a greater predisposition to cancer. The objective of the current study was to determine the impact of dulaglutide on genetic and epigenetic DNA damage caused by obesity, which is a crucial factor in the development of cancer. Mice were administered a low-fat or high-fat diet for 12 weeks, followed by a 5-week treatment with dulaglutide. Following that, modifications of the DNA bases were examined using the comet assay. To clarify the underlying molecular mechanisms, oxidized and methylated DNA bases, changes in the redox status, levels of inflammatory cytokines, and the expression levels of some DNA repair genes were evaluated. Animals fed a high-fat diet exhibited increased body weights, elevated DNA damage, oxidation of DNA bases, and DNA hypermethylation. In addition, obese mice showed altered inflammatory responses, redox imbalances, and repair gene expressions. The findings demonstrated that dulaglutide does not exhibit genotoxicity in the investigated conditions. Following dulaglutide administration, animals fed a high-fat diet demonstrated low DNA damage, less oxidation and methylation of DNA bases, restored redox balance, and improved inflammatory responses. In addition, dulaglutide treatment restored the upregulated DNMT1, Ogg1, and p53 gene expression. Overall, dulaglutide effectively maintains DNA integrity in obese animals. It reduces oxidative DNA damage and hypermethylation by restoring redox balance, modulating inflammatory responses, and recovering altered gene expressions. These findings demonstrate dulaglutide's expediency in treating obesity and its associated complications.

Rituximab alleviates increased disomic sperm in DBA/1J mouse models of rheumatoid arthritis via restoration of redox imbalance.

Compared to the general population, patients with arthritis have a higher risk of fertility abnormalities, which have deleterious effects on both reproductive function and pregnancy outcomes, especially in patients wishing to conceive. These may be due to the disease itself or those of drug therapies. Despite the increasing use of rituximab in arthritis, limited data are available on its potential to induce aneuploidy in germ cells. Therefore, the aim of the current investigation was to determine if repeated treatment with rituximab affects the incidence of aneuploidy and redox imbalance in arthritic mouse sperm. Mice were treated with 250 mg/kg rituximab once weakly for 3 weeks, and then sperm were sampled 22 days after the last dose of rituximab. Fluorescence in situ hybridization assay with chromosome-specific DNA probes was used to evaluate the disomic/diploid sperm. Our results showed that rituximab had no aneuploidogenic effect on the meiotic stage of spermatogenesis. Conversely, arthritis induced a significantly high frequency of disomy, and treatment of arthritic mice with rituximab reduced the increased levels of disomic sperm. The occurrence of total diploidy was not significantly different in all groups. Reduced glutathione and8-hydroxydeoxyguanosine, markers of oxidative stress were significantly altered in arthritic animals, while rituximab treatment restored these changes. Additionally, arthritis severity was reduced after rituximab treatment. We conclude that rituximab may efficiently alleviate the arthritis-induced effects on male meiosis and avert the higher risk of abnormal reproductive outcomes. Therefore, treating arthritic patients with rituximab may efficiently inhibit the transmission of genetic anomalies induced by arthritis to future generations.

Histamine H4 Receptor Agonist, 4-Methylhistamine, Aggravates Disease Progression and Promotes Pro-Inflammatory Signaling in B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model.

We sought to assess the impact of 4-Methylhistamine (4-MeH), a specific agonist targeting the Histamine H4 Receptor (H4R), on the progression of experimental autoimmune encephalomyelitis (EAE) and gain insight into the underlying mechanism. EAE is a chronic autoimmune, inflammatory, and neurodegenerative disease of the central nervous system (CNS) characterized by demyelination, axonal damage, and neurodegeneration. Over the past decade, pharmacological research into the H4R has gained significance in immune and inflammatory disorders. For this study, Swiss Jim Lambert EAE mice were treated with 4-MeH (30 mg/kg/day) via intraperitoneal administration from days 14 to 42, and the control group was treated with a vehicle. Subsequently, we evaluated the clinical scores. In addition, flow cytometry was employed to estimate the impact of 4-Methylhistamine (4-MeH) on NF-κB p65, GM-CSF, MCP-1, IL-6, and TNF-α within CD19+ and CXCR5+ spleen B cells. Additionally, we investigated the effect of 4-MeH on the mRNA expression levels of Nf-κB p65, Gmcsf, Mcp1, Il6, and Tnfα in the brain of mice using RT-PCR. Notably, the clinical scores of EAE mice treated with 4-MeH showed a significant increase compared with those treated with the vehicle. The percentage of cells expressing CD19+NF-κB p65+, CXCR5+NF-κB p65+, CD19+GM-CSF+, CXCR5+GM-CSF+, CD19+MCP-1+, CXCR5+MCP-1+, CD19+IL-6+, CXCR5+IL-6+, CD19+TNF-α+, and CXCR5+TNF-α+ exhibited was more pronounced in 4-MeH-treated EAE mice when compared to vehicle-treated EAE mice. Moreover, the administration of 4-MeH led to increased expression of NfκB p65, Gmcsf, Mcp1, Il6, and Tnfα mRNA in the brains of EAE mice. This means that the H4R agonist promotes pro-inflammatory mediators aggravating EAE symptoms. Our results indicate the harmful role of H4R agonists in the pathogenesis of MS in an EAE mouse model.

Upregulation of Inflammatory Mediators in Peripheral Blood CD40+ Cells in Children with Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a common and severe neurodevelopmental disorder in early childhood, defined as social and communication deficits and repetitive and stereotypic behaviours. The aetiology is unknown in most cases. However, several studies have identified immune dysregulation as potentially promoting ASD. Among the numerous immunological findings in ASD, reports of increased pro-inflammatory markers remain the most consistently observed. C-C chemokine receptor type 1 (CCR1) activation is pro-inflammatory in several neurological disorders. Previous evidence has implied that the expression of chemokine receptors, inflammatory mediators, and transcription factors play a pivotal role in several neuroinflammatory disorders. There have also been reports on the association between increased levels of proinflammatory cytokines and ASD. In this study, we aimed to investigate the possible involvement of CCR1, inflammatory mediators, and transcription factor expression in CD40+ cells in ASD compared to typically developing controls (TDC). Flow cytometry analysis was used to determine the levels of CCR1-, IFN-γ-, T-box transcription factor (T-bet-), IL-17A-, retinoid-related orphan receptor gamma t (RORγt-), IL-22- and TNF-α-expressing CD40 cells in PBMCs in children with ASD and the TDC group. We further examined the mRNA and protein expression levels of CCR1 using real-time PCR and western blot analysis. Our results revealed that children with ASD had significantly increased numbers of CD40+CCR1+, CD40+IFN-γ+, CD40+T-bet+, CD40+IL-17A+, CD40+RORγt+, CD4+IL-22+, and CD40+TNF-α+ cells compared with the TDC group. Furthermore, children with ASD had higher CCR1 mRNA and protein expression levels than those in the TDC group. These results indicate that CCR1, inflammatory mediators, and transcription factors expressed in CD40 cells play vital roles in disease progression.

Inhibition of ITK Signaling Causes Amelioration in Sepsis-Associated Neuroinflammation and Depression-like State in Mice.

Sepsis affects millions of people worldwide and is associated with multiorgan dysfunction that is a major cause of increased morbidity and mortality. Sepsis is associated with several morbidities, such as lung, liver, and central nervous system (CNS) dysfunction. Sepsis-associated CNS dysfunction usually leads to several mental problems including depression. IL-17A is one of the crucial cytokines that is expressed and secreted by Th17 cells. Th17 cells are reported to be involved in the pathogenesis of depression and anxiety in humans and animals. One of the protein tyrosine kinases that plays a key role in controlling the development/differentiation of Th17 cells is ITK. However, the role of ITK in sepsis-associated neuroinflammation and depression-like symptoms in mice has not been investigated earlier. Therefore, this study investigated the efficacy of the ITK inhibitor, BMS 509744, in sepsis-linked neuroinflammation (ITK, IL-17A, NFkB, iNOS, MPO, lipid peroxides, IL-6, MCP-1, IL-17A) and a battery of depression-like behavioral tests, such as sucrose preference, tail suspension, and the marble burying test. Further, the effect of the ITK inhibitor on anti-inflammatory signaling (Foxp3, IL-10, Nrf2, HO-1, SOD-2) was assessed in the CNS. Our data show that sepsis causes increased ITK protein expression, IL-17A signaling, and neuroinflammatory mediators in the CNS that are associated with a depression-like state in mice. ITK inhibitor-treated mice with sepsis show attenuated IL-17A signaling, which is associated with the upregulation of IL-10/Nrf2 signaling and the amelioration of depression-like symptoms in mice. Our data show, for the first time, that the ITK inhibition strategy may counteract sepsis-mediated depression through a reduction in IL-17A signaling in the CNS.

The Exposure to Lead (Pb) Exacerbates Immunological Abnormalities in BTBR T+ Itpr3tf/J Mice through the Regulation of Signaling Pathways Relevant to T Cells.

Autism spectrum disorder (ASD) is a common neurodevelopmental illness characterized by abnormal social interactions, communication difficulties, and repetitive and limited behaviors or interests. The BTBR T+ Itpr3tf/J (BTBR) mice have been used extensively to research the ASD-like phenotype. Lead (Pb) is a hazardous chemical linked to organ damage in the human body. It is regarded as one of the most common metal exposure sources and has been connected to the development of neurological abnormalities. We used flow cytometry to investigate the molecular mechanism behind the effect of Pb exposure on subsets of CD4+ T cells in the spleen expressing IFN-γ, T-bet, STAT1, STAT4, IL-9, IRF4, IL-22, AhR, IL-10, and Foxp3. Furthermore, using RT-PCR, we studied the effect of Pb on the expression of numerous genes in brain tissue, including IFN-γ, T-bet, STAT1, STAT4, IL-9, IRF4, IL-22, AhR, IL-10, and Foxp3. Pb exposure increased the population of CD4+IFN-γ+, CD4+T-bet+, CD4+STAT1+, CD4+STAT4+, CD4+IL-9+, CD4+IRF4+, CD4+IL-22+, and CD4+AhR+ cells in BTBR mice. In contrast, CD4+IL-10+ and CD4+Foxp3+ cells were downregulated in the spleen cells of Pb-exposed BTBR mice compared to those treated with vehicle. Furthermore, Pb exposure led to a significant increase in IFN-γ, T-bet, STAT1, STAT4, IL-9, IRF4, IL-22, and AhR mRNA expression in BTBR mice. In contrast, IL-10 and Foxp3 mRNA expression was significantly lower in those treated with the vehicle. Our data suggest that Pb exposure exacerbates immunological dysfunctions associated with ASD. These data imply that Pb exposure may increase the risk of ASD.

Cadmium Exposure Is Associated with Behavioral Deficits and Neuroimmune Dysfunction in BTBR T+ Itpr3tf/J Mice.

Autism spectrum disorders (ASD) are neurobehavioral disabilities characterized by impaired social interactions, poor communication skills, and restrictive/repetitive behaviors. Cadmium is a common heavy metal implicated in ASD. In this study, we investigated the effects of Cd exposure on BTBR T+ Itpr3tf/J (BTBR) mice, an ASD model. We looked for changes in repetitive behaviors and sociability through experiments. We also explored the molecular mechanisms underlying the effects of Cd exposure, focusing on proinflammatory cytokines and pathways. Flow cytometry measured IL-17A-, IL-17F-, IL-21-, TNF-α-, STAT3-, and RORγt-expressing CD4+ T cells from the spleens of experimental mice. We then used RT-PCR to analyze IL-17A, IL-17F, IL-21, TNF-α, STAT3, and RORγ mRNA expression in the brain. The results of behavioral experiments showed that Cd exposure significantly increased self-grooming and marble-burying in BTBR mice while decreasing social interactions. Cd exposure also significantly increased the number of CD4+IL-17A+, CD4+IL-17F+, CD4+IL-21+, CD4+TNF-α+, CD4+STAT3+, and CD4+RORγt+ cells, while upregulating the mRNA expression of the six molecules in the brain. Overall, our results suggest that oral exposure to Cd aggravates behavioral and immune abnormalities in an ASD animal model. These findings have important implications for ASD etiology and provide further evidence of heavy metals contributing to neurodevelopmental disorders through proinflammatory effects.

Histamine H4 Receptor Antagonist Ameliorates the Progression of Experimental Autoimmune Encephalomyelitis via Regulation of T-Cell Imbalance.

Multiple sclerosis (MS) is a degenerative condition characterized by immune-mediated attacks on the central nervous system (CNS), resulting in demyelination and recurring T-cell responses. The histamine H4 receptor (H4R) is mainly expressed in cellular populations and plays a vital role in inflammation and immunological responses. The role of H4R in neurons of the CNS has recently been revealed. However, the precise role of H4R in neuronal function remains inadequately understood. The objective of this work was to investigate the impact of JNJ 10191584 (JNJ), a highly effective and specific H4R antagonist, on the development of experimental autoimmune encephalomyelitis (EAE) and to gain insight into the underlying mechanism involved. In this study, we examined the potential impact of JNJ therapy on the course of EAE in SJL/J mice. EAE mice were administered an oral dose of JNJ at a concentration of 6 mg/kg once a day, starting from day 10 and continuing until day 42. Afterward, the mice's clinical scores were assessed. In this study, we conducted additional research to examine the impact of JNJ on several types of immune cells, specifically Th1 (IFN-γ and T-bet), Th9 (IL-9 and IRF4), Th17 (IL-17A and RORγt), and regulatory T (Tregs; Foxp3 and TGF-ß1) cells in the spleen. In this study, we further investigated the impact of JNJ on the mRNA expression levels of IFN-γ, T-bet, IL-9, IRF4, IL-17A, RORγt, Foxp3, and TGF-ß1 in the brain. Daily treatment of JNJ effectively reduced the development of EAE in mice. The percentages of CD4+IFN-γ+, CD4+T-bet+, CD4+IL-9+, CD4+IRF4+, CD4+IL-17A+, and CD4+RORγt+ cells were shown to decrease, whereas the percentages of CD4+TGF-ß1+ and CD4+Foxp3+ cells were observed to increase in EAE mice treated with JNJ. Therefore, the HR4 antagonist positively affected the course of EAE by modulating the signaling of transcription factors. The identified results include possible ramifications in the context of MS treatment.

Structural Characteristics of PON1 with Leu55Met and Gln192Arg Variants Influencing Oxidative-Stress-Related Diseases: An Integrated Molecular Modeling and Dynamics Study.

Background and Objectives: PON1 is a multi-functional antioxidant protein that hydrolyzes a variety of endogenous and exogenous substrates in the human system. Growing evidence suggests that the Leu55Met and Gln192Arg substitutions alter PON1 activity and are linked with a variety of oxidative-stress-related diseases. Materials and Methods: We implemented structural modeling and molecular dynamics (MD) simulation along with essential dynamics of PON1 and molecular docking with their endogenous (n = 4) and exogenous (n = 6) substrates to gain insights into conformational changes and binding affinity in order to characterize the specific functional ramifications of PON1 variants. Results: The Leu55Met variation had a higher root mean square deviation (0.249 nm) than the wild type (0.216 nm) and Gln192Arg (0.202 nm), implying increased protein flexibility. Furthermore, the essential dynamics analysis confirms the structural change in PON1 with Leu55Met vs. Gln192Arg and wild type. Additionally, PON1 with Leu55Met causes local conformational alterations at the substrate binding site, leading to changes in binding affinity with their substrates. Conclusions: Our findings highlight the structural consequences of the variants, which would increase understanding of the role of PON1 in the pathogenesis of oxidative-stress-related diseases, as well as the management of endogenous and exogenous chemicals in the treatment of diseases.

Sinapic acid alleviates 5-fluorouracil-induced nephrotoxicity in rats via Nrf2/HO-1 signalling.

Fluoropyrimidine 5-fluorouracil (5-FU) is a DNA analogue broadly used in chemotherapy, though treatment-associated nephrotoxicity limits its widespread clinical use. Sinapic acid (SA) has potent antioxidant, anti-inflammatory, and anti-apoptotic effects, we investigated its protective effects against 5-FU-induced nephrotoxicity in a rat model. We designated four treatment groups each Group I (control) received five intraperitoneal saline injections (once daily) from days 17 to 21; Group II received five intraperitoneal injections of 5-FU (50 mg/kg/day) from days 17 to 21; Group III received an oral administration of SA (40 mg/kg) for 21 days and five intraperitoneal injections of 5-FU (50 mg/kg/day) from days 17 to 21; and Group IV received an oral administration of SA (40 mg/kg) for 21 days (n-six rats in each group). blood samples were collected on day 22 from each group. Animals were sacrificed and their kidneys removed, and instantly frozen. 5-FU caused oxidative stress, inflammation, and activation of the apoptotic pathway by upregulating Bax and Caspase-3 and downregulating Bcl-2. However, SA exposure reduced serum toxicity indicators, boosted antioxidant defences, and reduced kidney apoptosis, which was confirmed by histopathological analysis. Therefore, prophylactic administration of SA could inhibit 5-FU-induced renal injuries in rats via suppression of renal inflammation and oxidative stress, primarily through regulation of NF-κB and proinflammatory cytokines, inhibition of renal apoptosis, and restoration of tubular epithelial antioxidant activities and cytoprotective defences.

Crosstalk of TNF-α, IFN-γ, NF-kB, STAT1 and redox signaling in lipopolysaccharide/d-galactosamine/dimethylsulfoxide-induced fulminant hepatic failure in mice.

Purpose: The clinical study of fulminant hepatic failure is challenging due to its high mortality and relative rarity, necessitating reliance on pre-clinical models to gain insight into its pathophysiology and develop potential therapies. Methods and Results: In our study, the combination of the commonly used solvent dimethyl sulfoxide to the current-day model of lipopolysaccharide/d-galactosamine-caused fulminant hepatic failure was found to cause significantly greater hepatic damage, as indicated by alanine aminotransferase level. The effect was dose-dependent, with the maximum increase in alanine aminotransferase observed following 200 µl/kg dimethyl sulfoxide co-administration. Co-administration of 200 µl/kg dimethyl sulfoxide also remarkably increased histopathological changes induced by lipopolysaccharide/d-galactosamine. Importantly, alanine aminotransferase levels and survival rate in the 200 µl/kg dimethyl sulfoxide co-administration groups were both greater than those in the classical lipopolysaccharide/d-galactosamine model. We found that dimethyl sulfoxide co-administration aggravated lipopolysaccharide/d-galactosamine-caused liver damage by stimulating inflammatory signaling, as indicated by tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) levels. Further, nuclear factor kappa B (NF-kB) and transcription factor activator 1 (STAT1) were upregulated, as was neutrophil recruitment, indicated by myeloperoxidase activity. Hepatocyte apoptosis was also increased, and greater nitro-oxidative stress was noted, as determined based on nitric oxide, malondialdehyde, and glutathione levels. Conclusion: Co-treatment with low doses of dimethyl sulfoxide enhanced the lipopolysaccharide/d-galactosamine-caused hepatic failure in animals, with higher toxicity and greater survival rates. The current findings also highlight the potential danger of using dimethyl sulfoxide as a solvent in experiments involving the hepatic immune system, suggesting that the new lipopolysaccharide/d-galactosamine/dimethyl sulfoxide model described herein could be used for pharmacological screening with the goal to better understand hepatic failure and evaluate treatment approaches.

CC chemokine receptor 5 antagonist alleviates inflammation by regulating IFN-γ/IL-10 and STAT4/Smad3 signaling in a mouse model of autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an immunopathological disease that causes demyelination and recurrent episodes of T cell-mediated immune attack in the central nervous system. Experimental autoimmune encephalomyelitis (EAE) is a well-established mouse model of MS. The roles of T cells in MS/EAE have been well investigated, but little is known about the role of CCR5+ cells. In the present study, we investigated whether treatment with DAPTA, a selective CCR5 antagonist, could modulate the progression of EAE in the SJL/J mice. EAE mice were treated with DAPTA (0.01 mg/kg) intraperitoneally daily from day 14 to day 42, and the clinical scores were evaluated. We further investigated the effects of DAPTA on IFN-γ-, TGF-ß-, IL-10-, IL-17A-, IL-22-, T-bet, STAT4-, RORγT-, AhR-, Smad3-, and Foxp3-expressing CCR5+ spleen cells using flow cytometry analysis. We further explored the effects of DAPTA on mRNA/protein expression of IFN-γ, IL-10, IL-17A, IL-22, TGF-ß, T-bet, STAT4, RORγT, AhR, Foxp3, and NF-H in the brain tissue. The severity of clinical scores decreased in DAPTA-treated EAE mice as compared to that in the EAE control mice. Moreover, the percentage of CCR5+IFN-γ+, CCR5+T-bet+, CCR5+STAT4+, CCR5+IL-17A+, CCR5+RORγt+, CCR5+IL-22+, and CCR5+AhR+ cells decreased while CCR5+TGF-ß+, CCR5+IL-10+, CCR5+Smad3+, and CCR5+Foxp3+ increased in DAPTA-treated EAE mice. Furthermore, DAPTA treatment significantly mitigated the EAE-induced expression of T-bet, STAT4, IL-17A, RORγT, IL-22, and AhR but upregulated Foxp3, IL-10, and NF-H expression in the brain tissue. Taken together, our data demonstrated that DAPTA could ameliorate EAE progression through the downregulation of the inflammation-related cytokines and transcription factors signaling, which may be useful for the clinical therapy of MS.

Novel sulphonamide-bearing methoxyquinazolinone derivatives as anticancer and apoptosis inducers: synthesis, biological evaluation and in silico studies.

We synthesised a new series of sulphonamide-bearing quinazolinone derivatives 5-18 and evaluated their in vitro cytotoxicity in various cancer cell lines (A549, HepG-2, LoVo and MCF-7) and in normal human cells (HUVEC). Compounds 6 and 10 exhibited the higher activity against all the cancer cell lines compared with 5-flourourcil as positive control. The ability of the most promising compounds 6 and 10 to induce cell cycle arrest and apoptosis in breast cancer (MCF-7) cells was evaluated by flow cytometry. Reverse transcriptase-polymerase chain reaction and western blotting were used to evaluate the expression of apoptosis-related markers. We found that the 2-tolylthioacetamide derivative 6 and the 3-ethyl phenyl thioacetamide derivative 10 exhibited cytotoxic activity comparable to that of 5-fluorouracil as reference drug in MCF-7 and LoVo colon cancer cells. Cell cycle analysis showed a concentration-dependent accumulation of cells in the sub-G1 phase upon treatment with both compounds. The Annexin V-fluorescein isothiocyanate/propidium iodide assay showed that the compounds 6 and 10 increased the early and late apoptosis cell death modes in a dose-dependent manner. These compounds downregulated the expression of B-cell lymphoma-2 (Bcl-2), while increasing that of p53, Bcl-2-like protein 4, and caspase-7, at the mRNA and protein levels. Molecular docking of compounds 6 and 10 with Bcl-2 predicted them to show moderate - high binding affinity (6: -7.5 kcal/mol, 10: -7.9 kcal/mol) and interactions with key central substrate cavity residues. Overall, compounds 6 and 10 were found to be promising anticancer and apoptosis-inducing agents.

Cytotoxicity of Newly Synthesized Quinazoline-Sulfonamide Derivatives in Human Leukemia Cell Lines and Their Effect on Hematopoiesis in Zebrafish Embryos.

Many quinazoline derivatives with pharmacological properties, such as anticancer activity, have been synthesized. Fourteen quinazoline derivatives bearing a substituted sulfonamide moiety (4a-n) were previously synthesized and fully characterized. These compounds exerted antiproliferative activity against cell lines derived from solid tumors. Herein, the antileukemic activities of these compounds (4a-n) against two different leukemia cell lines (Jurkat acute T cell and THP-1 acute monocytic) were investigated. Our investigation included examining their activity in vivo in a zebrafish embryo model. Remarkably, compounds 4a and 4d were the most potent in suppressing cell proliferation, with an IC50 value range of 4-6.5 µM. Flow cytometry analysis indicated that both compounds halted cell progression at the G2/M phase and induced apoptosis in a dose-dependent manner. RT-PCR and Western blot analyses also showed that both compounds effectively induced apoptosis by upregulating the expression of proapoptotic factors while downregulating that of antiapoptotic factors. In vivo animal toxicity assays performed in zebrafish embryos indicated that compound 4d was more toxic than compound 4a, with compound 4d inducing multiple levels of teratogenic phenotypes in zebrafish embryos at a sublethal concentration. Moreover, both compounds perturbed the hematopoiesis process in developing zebrafish embryos. Collectively, our data suggest that compounds 4a and 4d have the potential to be used as antileukemic agents.

The Antiproliferative and Apoptotic Effects of a Novel Quinazoline Carrying Substituted-Sulfonamides: In Vitro and Molecular Docking Study.

In order to investigate for a new effective and safe anticancer drug, we synthesized a novel series of quinazoline containing biologically active substituted-sulfonamide moiety at 3- position 4a-n. The structure of the newly prepared compounds was proved by microanalysis, IR, 1H-NMR, 13C-NMR and mass spectral data. All the synthesized compounds were evaluated for their in vitro cytotoxic activity in numerous cancer cell lines including A549, HepG-2, LoVo and MCF-7 and normal HUVEC cell line. The two most active compounds 4d and 4f were then tested for their apoptosis induction using DNA content and Annexin V-FITC/PI staining. Moreover, apoptosis initiation was also confirmed using RT-PCR and Western blot. To further understand the binding preferences of quinazoline sulfonamides, docking simulations were used. Among the fourteen new synthesized compounds, we found that compounds 4d and 4f exerted the strongest cytotoxicity against MCF-7 cells with an IC50 value of 2.5 and 5 µM, respectively. Flow cytometry data revealed the ability of compounds 4d and 4f to mediate apoptosis and arrest cell cycle growth at G1 phase. Furthermore, RT-PCR and Western blot results suggested that both 4d and 4f activates apoptotic cell death pathway in MCF-7 cells. Molecular docking assessments indicated that compounds 4d and 4f fit perfectly into Bcl2's active site. Based on the biological properties, we conclude that both compounds 4d and 4f could be used as a new type of anticancer agent, which provides a scientific basis for further research into the treatment of cancer.

Chemokine Receptor 5 Antagonism Causes Reduction in Joint Inflammation in a Collagen-Induced Arthritis Mouse Model.

Rheumatoid arthritis (RA) is a chronic inflammatory disease mainly affecting the synovial joints. A highly potent antagonist of C-C chemokine receptor 5 (CCR5), maraviroc (MVC), plays an essential role in treating several infectious diseases but has not yet been evaluated for its potential effects on RA development. This study focused on evaluating the therapeutic potential of MVC on collagen-induced arthritis (CIA) in DBA/1J mice. Following CIA induction, animals were treated intraperitoneally with MVC (50 mg/kg) daily from day 21 until day 35 and evaluated for clinical score and histopathological changes in arthritic inflammation. We further investigated the effect of MVC on Th9 (IL-9, IRF-4, and GATA3) and Th17 (IL-21R, IL-17A, and RORγT) cells, TNF-α, and RANTES in CD8+ T cells in the spleen using flow cytometry. We also assessed the effect of MVC on mRNA and protein levels of IL-9, IL-17A, RORγT, and GATA3 in knee tissues using RT-PCR and western blot analysis. MVC treatment in CIA mice attenuated the clinical and histological severity of inflammatory arthritis, and it substantially decreased IL-9, IRF4, IL-21R, IL-17A, RORγT, TNF-α, and RANTES production but increased GATA3 production in CD8+ T cells. We further observed that MVC treatment decreased IL-9, IL-17A, and RORγt mRNA and protein levels and increased those of GATA3. This study elucidates the capacity of MVC to ameliorate the clinical and histological signs of CIA by reducing pro-inflammatory responses, suggesting that MVC may have novel therapeutic uses in the treatment of RA.

Development and Validation of an HPLC-UV Detection Assay for the Determination of Clonidine in Mouse Plasma and Its Application to a Pharmacokinetic Study.

An accurate and simple HPLC-UV method has been developed for the determination of clonidine in mouse plasma. A reversed phase C18 Nova Pack® column (125 mm × 4.6 mm i.d., × 3 µm particle size) was used as stationary phase. The mobile phase composition was a mixture of 0.1% diethylamine/acetonitrile (70:30, v/v) at pH 8 in an isocratic mode at flow rate was 1.0 mL/min. Detection was set at 210 nm. Tizanidine was used as an internal standard. The clonidine and tizanidine were extracted from plasma matrix using the deproteinization technique. The developed method exhibited a linear calibration range 100.0-2000 ng/mL and the lower limit of detection (LOD) and quantification (LOQ) were 31.0 and 91.9 ng/mL, respectively. The intra-day and inter-day accuracy and precision of the method were within 8.0% and 3.0%, respectively, relative to the nominal concentration. The developed method was validated with respect to linearity, accuracy, precision, and selectivity according to the US Food and drug guideline. Minimal degradation was demonstrated during the determination of clonidine under different stability conditions. The suggested method has been successfully applied during a pharmacokinetic study of clonidine in mouse plasma.

Inhibition of Bruton's tyrosine kinase and IL-2 inducible T-cell kinase suppresses both neutrophilic and eosinophilic airway inflammation in a cockroach allergen extract-induced mixed granulocytic mouse model of asthma using preventative and therapeutic strategy.

Asthma is a complex airways disease with a wide spectrum which ranges from eosinophilic (Th2 driven) to mixed granulocytic (Th2/Th17 driven) phenotypes. Mixed granulocytic asthma is a cause of concern as corticosteroids often fail to control this phenotype. Different kinases such as Brutons's tyrosine kinase (BTK) and IL-2 inducible T cell kinase (ITK) play a pivotal role in shaping allergic airway inflammation. Ibrutinib is primarily a BTK inhibitor, however it is reported to be an ITK inhibitor as well. In this study, we sought to determine the effect of Ibrutinib on Th1, Th17 and Th2 immune responses in a cockroach allergen extract (CE)-induced mixed granulocytic (eosinophilic and neutrophilic) mouse model in preventative mode. Ibrutinib attenuated neutrophilic inflammation at a much lower doses (25-75 µg/mouse) in CE-induced mixed granulocytic asthma whereas Th2/Th17 immune responses remained unaffected at these doses. However, at a much higher dose, i.e. 250 µg/mouse, Ibrutinib remarkably suppressed both Th17/Th2 and lymphocytic/neutrophilic/eosinophilic airway inflammation. At molecular level, Ibrutinib suppressed phosphorylation of BTK in neutrophils at lower doses and ITK in CD4 + T cells at higher doses in CE-treated mice. Further, effects of Ibrutinib were compared with dexamethasone on CE-induced mixed granulocytic asthma in therapeutic mode. Ibrutinib was able to control granulocytic inflammation along with Th2/Th17 immune response in therapeutic mode whereas dexamethasone limited only Th2/eosinophilic inflammation. Thus, Ibrutinib has the potential to suppress both Th17/Th2 and neutrophilic/eosinophilic inflammation during mixed granulocytic asthma and therefore may be pursued as alternative therapeutic option in difficult-to-treat asthma which is resistant to corticosteroids.