Investigation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in vitro inflammation model at molecular level.

In our study, we aimed to create an inflammation model in endothelial and macrophage cell lines and to examine the changes in the expression of hyperpolarization activated cyclic nucleotide gated (HCN) channels at the molecular level. HUVEC and RAW cell lines were used in our study. 1 µg/mL LPS was applied to the cells. Cell media were taken 6 h later. TNF-α, IL-1, IL-2, IL-4, IL-10 concentrations were measured by ELISA method. Cell media were cross-applied to cells for 24 h after LPS. HCN1/HCN2 protein levels were determined by Western-Blot method. HCN-1/HCN-2 gene expressions were determined by qRT-PCR method. In the inflammation model, a significant increase in TNF-α, IL-1, and IL-2 levels was observed in RAW cell media compared to the control. While no significant difference was observed in IL-4 level, a significant decrease was observed in IL-10 level. While a significant increase in TNF-α level was observed in HUVEC cell medium, no difference was observed in other cytokines. In our inflammation model, an 8.44-fold increase in HCN1 gene expression was observed in HUVEC cells compared to the control group. No significant change was observed in HCN2 gene expression. 6.71-fold increase in HCN1 gene expression was observed in RAW cells compared to the control. The change in HCN2 expression was not statistically significant. In the Western-Blot analysis, a statistically significant increase in HCN1 level was observed in the LPS group in HUVEC cells compared to the control; no significant increase in HCN2 level was observed. While a statistically significant increase in HCN1 level was observed in the LPS group in RAW cells compared to the control; no significant increase in HCN2 level was observed. In immunofluorescence examination, it was observed that the level of HCN1 and HCN2 proteins in the cell membrane of HUVEC and RAW cells increased in the LPS group compared to the control group. While HCN1 gene/protein levels were increased in RAW and HUVEC cells in the inflammation model, no significant change was observed in HCN2 gene/protein levels. Our data suggest that the HCN1 subtype is dominant in endothelium and macrophages and may play a critical role in inflammation.

The Relationship Between COVID-19 Disease Severity and Zonulin Levels.

Introduction Zonulin is a protein that plays a role in the reversible regulation of epithelial permeability. As zonulin is released in large amounts into the intestinal lumen, it disrupts the integrity of the tight junctions and causes continuous migration of antigens to the submucosa. Consequently, it can trigger inflammatory processes and severe immune reactions. In severe cases, SARS-CoV-2 may have a major impact on the clinical manifestations of the disease by directly or indirectly affecting intestinal cells and triggering systemic inflammation. Therefore, our study aimed to investigate the role of one of the possible mediators, zonulin, in the severity of the COVID-19 infection. Methods  Thirty COVID-19 patients and 35 healthy controls were included in the study. Blood samples were taken from the patients on the 1st, 4th, and 8th days of hospitalization. Serum zonulin levels were determined by enzyme-linked immunosorbent assay (ELISA). Complete blood count (white blood cell [WBC], neutrophil, lymphocyte, and platelet), biochemical parameters (serum lactic acid dehydrogenase [LDH], erythrocyte sedimentation rate [ESR], C-reactive protein [CRP], D-dimer, ferritin, fibrinogen levels) were determined and chronic systemic disease states of the patients were assessed. Results  Serum zonulin levels were notably higher in the healthy control group compared to the patient group (p=0.003). Although there was an increase in the zonulin values by time in hospitalization, this rising was not significant. Conversely, ESR and CRP levels were significantly higher in the patient group (p<0.001). There was no significant difference between the two groups regarding gender, age, and WBC counts. Conclusion  The serum zonulin levels of COVID-19 patients with the mild clinical course were lower than the healthy control group. Moreover, serum zonulin levels were not correlated with ESR, CRP, and other inflammation markers. Our results suggest that low serum zonulin levels in COVID-19 patients might represent a mild disease course.

Effects of Suberoylanilide Hydroxamic Acid (SAHA) on the Inflammatory Response in Lipopolysaccharide-Induced N9 Microglial Cells.

INTRODUCTION: Epigenetics has shown promising results for understanding the different behaviors of microglia under the context of neuroinflammation. However, to our knowledge, the results of this complex mechanism with novel pharmacological agents such as histone deacetylase inhibitors (HDACis) are still missing. In this study, we aimed to investigate the effects of suberoylanilide hydroxamic acid (SAHA), a pan-HDACi, on the lipopolysaccharide (LPS)-induced neuroinflammation model in the N9 microglial cells. METHODS: Microglial cells were treated with SAHA (0.25, 0.5, 1.0, 1.25, 1.5 µM) and LPS (100 ng/mL) for 24 hours. Then, levels of the pro/anti-inflammatory cytokines interleukin-1 beta (IL-1ß), IL-6, tumor necrosis factor alpha (TNF-α), and IL-10 were determined by the enzyme-linked immunosorbent assay. The total cellular HDAC activity was determined by colorimetric analysis. Additionally, the expression levels of nuclear factor kappa-B (NF-κB) were quantified via western blotting. RESULTS: SAHA (1.0 and 1.25 µM) attenuated the LPS-induced inflammatory response of microglial cells via decreasing NF-κB expression and pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α) in the N9 microglial cells. Moreover, SAHA treatment improved IL-10 levels and prevented the LPS-induced increase in the HDAC activity in the microglial cells. CONCLUSION: Our results suggest SAHA attenuates the LPS-induced inflammatory response in the N9 microglial cells, and regulation of histone acetylation with HDACis might be a rational approach for the treatment of neuroinflammation.

The effects of agomelatine in cisplatin-induced toxicity on the kidney and liver tissues: In vivo study

Abstract Nephrotoxicity and hepatotoxicity are frequently seen adverse effects during cisplatin chemotherapy. In this study, we investigated the effects of agomelatine on cisplatin-induced toxicity in the kidney and liver. Animals were administered with a single dose of cisplatin (7 mg/kg, i.p.) and treated with agomelatine (20 and 40 mg/kg, p.o) for seven days. Renal and hepatic functions were evaluated by measuring concentrations of creatinine, BUN, AST and ALT in the serum. Oxidative stress and protein peroxidation were assessed by measuring SOD, CAT, GSH and AOPP levels in both tissues. Serum PON-1 levels were also evaluated. Histopathological analysis was performed to determined structural changes in the kidney and liver. Agomelatine (20 mg/kg) treatment approximately halved cisplatin-related increase in serum creatinine, BUN, AST and ALT levels. Agomelatine (20 mg/kg) significantly prevented the cisplatin-induced excessive decrease in SOD, CAT, GSH in both tissues and serum PON-1 levels. Agomelatine (20 and 40 mg/kg) protected the structural integrity of the kidney against cisplatin-insult. Although agomelatine (40 mg/kg) protected the kidney and showed parallel results with 20 mg/kg biochemically, it failed to show the same liver tissue effects in both analyses. Although agomelatine protected against cisplatin-induced toxicity in the kidney and liver, care should be taken with higher doses for possible hepatotoxicity.

Cannabinoid receptor-1 has an effect on CD200 under rotenone and alpha-synuclein induced stress.

Decades after identifying cannabinoids and their beneficial effects on Parkinson's disease (PD), many gaps are still missing. Although, CB2-dependent actions have been shown as underlying positive effects of cannabinoid treatment, in recent years, another receptor of cannabinoids, CB1, emerged as a valuable player in cannabinoid-induced neuroprotection. Remarkably, the effects of CB1 are mainly related to immune cells in the CNS, microglia, and astrocytes. However, oxidative stress, α-syn accumulation, and immune disbalance are essential aspects of both neurons and glial cells. Therefore, in this study, we investigated the effects of the CB1 on both α-syn and rotenone-treated SH-SY5Y and C8-D1A cells. ACEA and AM-251 were used as CB1 agonists and antagonists. Cell viability, IL-1ß, IL-6, TNF-α levels, and CD200 expressions were determined in culture mediums. Our results demonstrated that preformed fibril form (pFF) of α-syn did not cause any significant change in SH-SY5Y cells compared to C8-D1A cells. Rotenone significantly increased the expression of IL-1ß, IL-6, and TNF-α levels in both cells. pFF α-syn and rotenone treatment caused a decrease in CD200 expression. Surprisingly both ACEA and AM-251 alleviated rotenone-induced increase in cytokine levels in both cell lines. Although ACEA prevented pFF α-syn induced increase in cytokine levels and decrease in CD200 expression in C8-D1A cells, AM-251 failed to affect CD200 expression levels. Additionally, ACEA + AM-251 abolished the protective effects of both ACEA and AM-251 against rotenone and α-syn insults in both cell lines. The current study suggests that cannabinoid receptor agonism alleviates rotenone and α-syn-dependent inflammation in neurons and astrocytes.

Cannabinoid receptors modulate LPS-induced increase of class-II transactivator expression levels in a microglial cell line.

Microglial antigen generation (MAG) is an essential process in regulating disease states and homeostasis of the central nervous system. MAG is considered as responsible autoimmune mechanism in neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's diseases. Neuroprotective and regulator effects of cannabinoid receptors on these disease states and modulation with pharmacological agents are urgent subjects in recent decades. Although different aspects of microglial immune response have been investigated, specific effects of these receptor subtypes in the MAG are still unclear. Therefore, in the current study, we have investigated the effects of CB1 and CB2 receptors on antigen generation by investigating MHC-II and its master regulator CIITA by specific cannabinoid agents (ACEA, AM-251, CP 55,940, and SR144528) in the LPS-induced BV-2 cells. Additionally, the effects of drug treatments on inflammatory status were measured by determining IL-1ß, IL-6, and TNF-α levels. LPS-induced increase in MHC-II and CIITA expression was inhibited by specific CB1 agonist, ACEA, and nonselective cannabinoid agonist CP 55,940. A combination with specific CB1 antagonist AM-251 prevented these inhibitory effects of ACEA and CP 55,940 on both MHC-II and CIITA expression. Although specific CB2 antagonist, SR144528, also prevented the inhibitory effect of CP 55,940 on MHC-II, it did not affect CIITA expression. LPS-induced IL-1ß, IL-6, and TNF-α increase both attenuated with CP 55,940 and ACEA treatments. Although both selective cannabinoid antagonists inhibited this effect, preventive effects were more dominant on CB1 receptors. Our results demonstrated that CB1 receptors majorly mediates LPS-induced MHC-II and its regulator CIITA expression in microglial cells.

Agomelatine confers neuroprotection against cisplatin-induced hippocampal neurotoxicity.

Neurotoxicity caused by cisplatin is a major obstacle during chemotherapy. Oxidative stress and inflammation are considered the primary mechanism behind neuronal damage which affects the continuing chemotherapy regimen. Agomelatine was recently described as a neuroprotective compound against toxic insults in the nervous systems. It is an analog of the well-known antioxidant and anti-inflammatory compound melatonin and currently used for depression and sleep disturbances. In the current study, we investigated the possible neuroprotective role of agomelatine against cisplatin-induced oxidative, inflammatory, and behavioral alterations in male rats. Our results show that agomelatine prevented cisplatin-induced neurotoxicity in the HT-22 mouse hippocampal neuronal cell line. Additionally, agomelatine treatment inhibited cisplatin-induced behavioral deficits and neuronal integrity in vivo. For the evaluation of the effect of agomelatine on oxidative stress and inflammation, GSH, MDA, TNF, and IL-6 levels were analyzed in HT-22 cells and hippocampal tissues. Agomelatine significantly attenuated oxidative stress and inflammation due to the cisplatin insult in vitro and in vivo. Also, agomelatine treatment ameliorated the neuronal pathology in the hippocampus, which is strongly related to cognition and memory. Taken together, our results indicate that in males, the neuroprotective effect of agomelatine is mediated through its antioxidant and anti-inflammatory actions abrogating functional deficits.

Alterations in the matrix metalloproteinase-3 promoter methylation after common chemotherapeutics: in vitro study of paclitaxel, cisplatin and methotrexate in the MCF-7 and SH-SY5Y cell lines.

Cancer treatment is a complex process due to the several encountered obstacles during therapy, such as metastasis, angiogenesis, and drug resistance. The methylation status of elements that are thought to play crucial roles in these mechanisms is considered valuable targets. Matrix metalloproteinase-3 (MMP-3), one of the possible targets, is a well-known endopeptidase and secreted by several types of cancer cells. Paclitaxel, cisplatin, and methotrexate are frequently used for several malignancies, individually or in combination. Therefore, the aims of this study is that demonstration of possible effects of different doses of single or jointly application of these agents with maintaining their antiproliferative activity in clinically relevant cell lines, as well as revealing epigenetic results of this pharmacological alteration with exploring promoter methylation status of the MMP-3 gene. Cell viability was determined with Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Further methylation-specific PCR (MSP) experiments for determining the promoter methylation status of MMP-3 were performed according to the obtained IC50 values of the drug treatments. The MMP-3 promoter methylation status was analayzed with MSP and determined with agarose gel electrophoresis. As a result, methotrexate and paclitaxel treatment significantly methylated the MMP-3 promoter; however, cisplatin caused MMP-3 promoter unmethylation in MCF-7 and SH-SY5Y cells. Our study indicates that decreasing the dose of clinically prevalent chemotherapeutic agents while maintaining the same tumor-killing potency might be a rational strategy for treatment. In addition to avoiding adverse effects of these compounds, decreasing treatment doses will bring substantial benefits for patient life-quality.

Resveratrol inhibits Src tyrosine kinase, STAT3, and Wnt signaling pathway in collagen induced arthritis model.

Resveratrol, a phytochemical, acts several cellular signaling pathways and has anti-inflammatory potentials. The purpose of this study is to research the therapeutic effect of resveratrol in collagen-induced arthritis (CIA) model in rats and whether resveratrol affects the activities of signaling pathways those are potent pathogenic actors of rheumatoid arthritis. Arthritis was induced by intradermal injection of chicken type II collagen combined with incomplete Freund's adjuvant in Wistar albino rats. One day after the onset of arthritis (day 14), resveratrol (20 mg/kg/day) was given via oral gavage, until day 29. The paws of the rats were obtained for further analysis. Tissue Wnt5a, mitogen-activated protein kinase (MAPK), Src tyrosine kinase and signal transducer, and activator of transcription-3 (STAT3) mRNA expressions were determined by real-time polymerase chain reaction. Resveratrol ameliorated the clinical and histopathological (perisynovial inflammation and cartilage-bone destruction) findings of inflammatory arthritis. The tissue mRNA expressions of Wnt5a, MAPK3, Src kinase, and STAT3 were increased in the sham group compared to the control group. Resveratrol supplement decreased their expressions. The present study shows that Src kinase, STAT3, and Wnt signaling pathway are active in the CIA model. Resveratrol inhibits these signaling pathways and ameliorates inflammatory arthritis. © 2018 BioFactors, 45(1):69-74, 2019.

Paricalcitol inhibits the Wnt/beta-catenin signaling pathway and ameliorates experimentally induced arthritis

Background/aim: The Wnt/ß-catenin pathway has important biological activities, including the differentiation of cells and joint formations. The aim of our study was to determine the effect of paricalcitol on experimentally induced arthritis. Materials and methods: Type II collagen combined with Freund's adjuvant was applied to induce arthritis in Wistar albino female rats. Paricalcitol (0.3 µg/kg daily) was subcutaneously injected starting 1 day after collagen applications (prophylactic group) or 1 day after the onset of arthritis (therapeutic group), until day 29. Results: The 29th day arthritis scores were lower compared to the 13th day scores in the paricalcitol groups (P < 0.05), while they were higher in the arthritis group (P < 0.05). Marked cartilage-bone destruction and extensive perisynovial inflammation were detected in the arthritis group. Decreased cartilage-bone destruction and perisynovial inflammation in the paws were observed in the paricalcitol groups. The tissue mRNA levels of DKK1, Wnt5a, and axin-2 were higher in the arthritis group than in the control group. In the paricalcitol groups, mRNA expressions were lower than in the arthritis group. Conclusion: The present study shows that the Wnt/ß-catenin signaling pathway is active in arthritis. Moreover, paricalcitol ameliorates arthritis via inhibiting the Wnt/ß-catenin pathway. Paricalcitol and the Wnt/ß-catenin pathway are candidates for research in human rheumatoid arthritis.