Beta mRNA Levels Research Articles

Reynosin protects neuronal cells from microglial neuroinflammation by suppressing NLRP3 inflammasome activation mediated by NADPH oxidase

Neuroinflammation, mediated by the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing-3 (NLRP3) inflammasome, is a significant contributor to the pathogenesis of neurodegenerative diseases (NDDs). Reynosin, a natural sesquiterpene lactone (SL), exhibits a broad spectrum of pharmacological effects, suggesting its potential therapeutic value. However, the effects and mechanism of reynosin on neuroinflammation remain elusive. The current study explores the effects and mechanisms of reynosin on neuroinflammation using mice and BV-2 microglial cells treated with lipopolysaccharide (LPS). Our findings reveal that reynosin effectively reduces microglial inflammation in vitro, as demonstrated by decreased CD11b expression and lowered interleukin-1 beta (IL-1β) and interleukin-18 (IL-18) mRNA and protein levels. Correspondingly, in vivo, results showed a reduction in the number of Iba-1 positive cells and alleviation of morphological alterations, alongside decreased expressions of IL-1β and IL-18. Further analysis indicates that reynosin inhibits NLRP3 inflammasome activation, evidenced by reduced transcription of NLRP3 and caspase-1, diminished NLRP3 protein expression, inhibited apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization, and decreased caspase-1 self-cleavage. Additionally, reynosin curtailed the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, demonstrated by reduced NADP+ and NADPH levels, downregulation of gp91phox mRNA, protein expression, suppression of p47phox expression and translocation to the membrane. Moreover, reynosin exhibited a neuroprotective effect against microglial inflammation in vivo and in vitro. These collective findings underscore reynosin’s capacity to mitigate microglial inflammation by inhibiting the NLRP3 inflammasome, thus highlighting its potential as a therapeutic agent for managing neuroinflammation.

Stemness and Cell Cycle Regulators and Their Modulation by Retinoic Acid in Ewing Sarcoma.

Retinoic acid (RA) regulates stemness and differentiation in human embryonic stem cells (ESCs). Ewing sarcoma (ES) is a pediatric tumor that may arise from the abnormal development of ESCs. Here we show that RA impairs the viability of SK-ES-1 ES cells and affects the cell cycle. Cells treated with RA showed increased levels of p21 and its encoding gene, CDKN1A. RA reduced mRNA and protein levels of SRY-box transcription factor 2 (SOX2) as well as mRNA levels of beta III Tubulin (TUBB3), whereas the levels of CD99 increased. Exposure to RA reduced the capability of SK-ES-1 to form tumorspheres with high expression of SOX2 and Nestin. Gene expression of CD99 and CDKN1A was reduced in ES tumors compared to non-tumoral tissue, whereas transcript levels of SOX2 were significantly higher in tumors. For NES and TUBB3, differences between tumors and control tissue did not reach statistical significance. Low expression of CD99 and NES, and high expression of SOX2, were significantly associated with a poorer patient prognosis indicated by shorter overall survival (OS). Our results indicate that RA may display rather complex modulatory effects on multiple target genes associated with the maintenance of stem cell's features versus their differentiation, cell cycle regulation, and patient prognosis in ES.

Effects of Dietary Sodium Propionate on Growth Performance, Fillet Texture, Hematologic and Plasma Biochemical Parameter, Immune Responses, and Intestine Histology of Juvenile Trachinotus ovatus

Seven graded levels of sodium propionate (SP) diets with 0 (SP1), 0.2% (SP2), 0.4% (SP3), 0.6% (SP4), 0.8% (SP5), 1.0% (SP6), and 1.2% (SP7) were prepared to feed Trachinotus ovatus (initial body weight: 8.64 ± 0.08 g) for 56 days. The results showed that increasing dietary SP levels quadratically increased significantly final body weight (FBW), weight gain rate (WGR), and specific growth rate (SGR) of T. ovatus but linearly and quadratically decreased significantly viscerosomatic index (VSI) and hepatosomatic index (HSI) of T. ovatus (P<0.05). In the SP4 treatment, FBW, WGR, and SGR presented the highest values. Both positive linear and quadratic trends were detected between crude lipid content of whole fish, adhesiveness of dorsal muscle, white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB), blood performance, high-density lipoprotein cholesterol (HDL-c), intestinal villus height, and dietary SP level, while negative linear and quadratic trends were found between firmness of dorsal muscle, triglyceride (TG), glucose (GLU), and dietary SP level (P<0.05). The increasing SP led to quadratic increases in lymphocyte (Lym), complement 3 (C3), chymotrypsin, villus number, and muscle layer thickness, and a quadratic decrease in hepatic malondialdehyde (MDAP<0.05). A significant negative linear trend was found between the content of glutamic-pyruvic transaminase (GPT) and dietary SP level, while significant positive linear trends were presented between C4, immunoglobulin M (IgM), α-amylase and dietary SP level (P<0.05). The increasing SP resulted in linear and quadratic increases in superoxide dismutase (SOD), total antioxidant capacity (T-AOC) of livers and C3, C4, IgM of head kidney (P<0.05). The expression levels of tumor necrosis factor alpha (TNF-α) and interleukin-8 (IL-8) were linearly and quadratically decreased, while the mRNA levels of growth factor beta (TGF-β) were linearly and quadratically increased with the increasing SP level (P<0.05). In conclusion, SP could be considered as a beneficial feed additive for enhancing growth and immunity of fish. And dietary SP level at 0.6% is optimal for the growth of Trachinotus ovatus based on a quadratic regression model of WGR.

Glucocorticoid Receptor (GR) Activation Is Associated with Increased cAMP/PKA Signaling in Castration-Resistant Prostate Cancer.

In castration-resistant prostate cancer (CRPC), increased glucocorticoid receptor (GR) expression and ensuing transcriptional activity have been proposed as an oncogenic "bypass" mechanism in response to androgen receptor (AR) signaling inhibition (ARSi). Here, we report that GR transcriptional activity acquired following ARSi is associated with the upregulation of cyclic adenosine monophosphate (cAMP)-associated gene expression pathways in both model systems and metastatic prostate cancer patient samples. In the context of ARSi, the expression of GR-mediated genes encoding cAMP signaling pathway-associated proteins can be inhibited by treatment with selective GR modulators (SGRMs). For example, in the context of ARSi, we found that GR activation resulted in upregulation of protein kinase inhibitor beta (PKIB) mRNA and protein levels, leading to nuclear accumulation of the cAMP-dependent protein kinase A catalytic subunit (PKA-c). Increased PKA-c, in turn, is associated with increased cAMP response element-binding protein phosphorylation and activity. Furthermore, enzalutamide and SGRM combination therapy in mice bearing CRPC xenografts delayed CRPC progression compared with enzalutamide therapy alone, and reduced tumor PKIB mRNA expression. Supporting the clinical importance of GR/PKA signaling activation in CRPC, we found a significant enrichment of both cAMP pathway signaling-associated gene expression and high NR3C1 (GR) activity in patient-derived xenograft models and metastatic human CRPC samples. These findings suggest a novel mechanism linking CRPC-induced GR transcriptional activity with increased cAMP signaling in AR-antagonized CRPC. Furthermore, our findings suggest that GR-specific modulation in addition to AR antagonism may delay GR+ CRPC time to recurrence, at least in part, by inhibiting tumor cAMP/PKA pathways.

Pre- and postnatal developmental exposure to the polychlorinated biphenyl mixture aroclor 1221 alters female rat pituitary gonadotropins and estrogen receptor alpha levels

Polychlorinated-biphenyls (PCBs) are industrial compounds, which were widely used in manufacturing of electrical parts and transformers. Despite being banned in 1979 due to human health concerns, they persist in the environment. In humans and experimental model systems, PCBs elicit toxicity in part by acting as endocrine-disrupting chemicals (EDCs). Aroclor 1221 (A1221) is a weakly estrogenic PCB mixture known to alter reproductive function in rodents. EDCs can impact hormone signaling at any level of the hypothalamic-pituitary-gonadal (HPG) axis, and we investigated the effects of A1221 exposure during the prenatal and postnatal developmental periods on pituitary hormone and steroid receptor expression in female rats. Examining offspring at 3 ages, postnatal day 8 (P8), P32 and P60, we found that prenatal exposure to A1221 increased P8 neonate pituitary luteinizing hormone beta (Lhb) mRNA and LHβ gonadotrope cell number while decreasing LH serum hormone concentration. No changes in pituitary hormone or hormone receptor gene expression were observed peri-puberty at P32. In reproductively mature rats at P60, we found pituitary follicle stimulating hormone beta (Fshb) mRNA levels increased by prenatal A1221 exposure with no corresponding alterations in FSH hormone or FSHβ expressing cell number. Estrogen receptor alpha (ERα) mRNA and protein levels were also increased at P60, but only following postnatal A1221 dosing. Together, these data illustrate that exposure to the PCB A1221, during critical developmental windows, alters pituitary gonadotropin hormone subunits and ERα levels in offspring at different phases of maturation, potentially impacting reproductive function in concert with other components of the HPG axis.

XANTHOHUMOL REDUCES THE EXPRESSION OF PRO-INFLAMMATORY AND OXIDATIVE STRESS MARKERS IN THE LUNG OF SENESCENCE-ACCELERATED PRONE MICE (SAMP8)

<h3>Objective</h3> Aging consists of a series of deleterious changes in cells and tissues along time, which lead to altered homeostasis. Nowadays we consider oxidative stress and inflammation the most relevant mechanisms of cell damage and dysfunction during aging. Both clinical and subclinical antigen exposure along life trigger a chronic inflammatory and oxidative response in the host. The lung, due to its position between the internal and the external milieu, is particularly exposed to pathogens and pollutants. An upregulation of oxidative stress and inflammation in lung tissue is expected. Xanthohumol, the major prenylated chalcone found in hops, has been shown to present anti-inflammatory and antioxidant properties in several organs, including liver, brain, and the reproductive system. Its effect on the aging lung has yet to be elucidated. For this reason, the aim of the present study was to evaluate the possible anti-inflammatory role of xanthohumol on a model of male senescence-accelerated prone mice (SAMP8). Design and method: Animals: young and old animals, aged 2 and 10 months respectively, were maintained under a 12:12 h light-dark cycle in an environmentally-controlled room and fed ad libitum. They were divided into 4 experimental groups: young non-treated, old non-treated, old treated with 1 mg/kg/day xanthohumol, and old treated with 5 mg/kg/day xanthohumol. Young and old non-treated male senescence-accelerated resistant mice (SAMR1) were used as controls. Treatment: xanthohumol was dissolved in ethanol and added to the drinking water. Only ethanol was administered to the water of non-treated animals. After 30 days of treatment, animals were sacrificed and lungs were collected and immediately frozen in liquid nitrogen. Inflammation and oxidative stress markers: both mRNA and protein levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and heme oxygenase 1 (HO-1) were analyzed. mRNA nuclear factor NF-kappa-B (NFκB) levels were also quantified. mRNA and protein expressions were measured by RT-PCR and Western blotting, respectively. The results were analyzed by using ANOVA followed by Fisher's post-hoc test. <h3>Results</h3> Compared to the young animals, old non-treated SAMP8 mice presented significantly elevated (p<0.05) mRNA and protein levels of TNF-α, IL-1β, and HO-1. mRNA NFκB2 levels were also significantly increased (p<0.05). SAMR1 mice showed significantly decreased values (p<0.05). <h3>Conclusions</h3> Treatment with xanthohumol was able to decrease significantly (p<0.05) the age-induced elevation at any of the doses administered. Supplementing the diet with foodstuffs containing xanthohumol may be used as a potential tool for counteracting inflammation and oxidative stress in the aging lung.

Abstract 15519: Site- and Cell-Selective Treatment of Atherosclerosis Using a Combination of RNA Therapeutic Approaches

Atherosclerotic plaque formation in the arteries underlies acute coronary syndromes (ACS); therapies to treat atherosclerosis that are both site- and cell-selective are not available. The CANTOS clinical trial demonstrated the benefits of targeting inflammation to reduce adverse cardiac events, but the systemic immune suppression increased incidence of sepsis or death caused by infections, highlighting the need for local inflammation inhibition. To assess the local chronic inflammatory state associated with atherosclerotic plaques, coronary arteries were isolated from non-transplantable human hearts ( Fig. A) from donors ranging in age from 6-months to 72 years old. Donors aged 51-72 had various conditions including hypertension, hypercholesterolemia, and atherosclerosis. Strikingly, qPCR analysis showed an increase in interleukin 1 beta (IL-1β) mRNA levels corresponding to increasing age of the donor and presence of CVD risk factors, with almost a 20-fold increase in the 72 compared to the 6-month-old donor ( Fig. B ).To specifically target the atherosclerotic plaques and the plaque forming cells without affecting the endothelium, we used a cell-penetrating peptide (p5RHH) ( Fig. C ) that self-assembles with synthetic mRNA to form nanoparticles that exclusively target denuded regions of endothelium and atherosclerotic plaques ( Fig. D ). We will deliver small interfering RNA specific to IL-1β and a microRNA switch that consists of synthetic mRNA encoding a cell-cycle inhibitor, p27, that includes a single target site for the endothelial cell specific miR-126, which we previously showed to selectively inhibit intimal hyperplasia and infiltrating immune cells while sparing the vascular endothelium ( Fig. E ). This innovative combination of multiple RNA therapeutics into a single nanoparticle will advance the field of site- and cell-selective anti-atherosclerotic therapy.

Active kisspeptin DNA vaccines oral immunization disrupt mRNA hormone receptors expression in ram lambs

To evaluate the efficacy of oral immunization with active kisspeptin DNA vaccine on the expression of hormone receptor mRNA. For this study, ten 56-day-old Hu breed ram lambs were randomly assigned to the treatment and control groups (n = 5). Treatment Experimental group received C500/pKS-asd and the control group received C500/pVAX-asd (aspartate-β semialdehyde dehydrogenase orally on days 0, 28, and 56, and blood samples were taken at each immunization interval (14-day) and tissues samples were collected at the end of the experimental period (day 98). The collected samples were stored in the refrigerator at −20 °C and liquid nitrogen, respectively, for laboratory examination. Total RNA was extracted from samples using TRIzol reagent and quantitative real-time polymerase chain reaction (QPCR) was used to quantify the levels of KISS1, G protein-coupled receptor-54 (Kiss1r), and gonadotrophin-releasing hormone (GnRH) mRNA in the hypothalamus. Levels of luteinizing hormone receptor (LHR) and luteinizing hormone beta (LHβ) mRNA, and follicle-stimulating hormone receptor (FSHR) and follicle-stimulating hormone beta (FSHβ) mRNA in the testes and pituitary were analyzed, respectively. Further, gonadotropin-releasing hormone receptor (GnRHR) mRNA expression level in the pituitary was measured. Moreover, the Kiss1r concentration level in the blood was measured using an indirect ELISA. The concentration of Kiss1r in the blood was lower in the treatment group than in the control group (p < 0.05). The levels of testicular FSHR and LHR mRNA were significantly lower in the treatment group (p < 0.05) when compared to the control group. Furthermore, the treatment group's levels of hypothalamic KISS1, Kiss1r, and GnRH mRNA were significantly lower (p < 0.05) than the controls. LH, FSH, and GnRHR mRNA expression in the pituitary were also significantly lower in the treatment group (p < 0.01 and p < 0.05, respectively). These findings imply that oral immunization with active kisspeptin DNA vaccine suppresses hormone receptor mRNA expression in the ram lambs.

DDX5/METTL3-METTL14/YTHDF2 Axis Regulates Replication of Influenza A Virus.

ABSTRACTDEAD-box helicase 5 (DDX5), a member of the DEAD/H-box helicases, is known to participate in all aspects of RNA metabolism. However, its regulatory effect in antiviral innate immunity during replication of influenza virus remains unclear. Herein, we found that human DDX5 promotes replication of influenza virus in A549 cells. Moreover, our results further revealed that DDX5 relies on its N terminus to interact with the nucleoprotein (NP) of influenza virus, which is independent of RNA. Of course, we also observed colocalization of DDX5 with NP in the context of transfection or infection. However, influenza virus infection had no significant effect on the protein expression and nucleocytoplasmic distribution of DDX5. Importantly, we found that DDX5 suppresses antiviral innate immunity induced by influenza virus infection. Mechanistically, DDX5 downregulated the mRNA levels of interferon beta (IFN-β), interleukin 6 (IL-6), and DHX58 via the METTL3-METTL14/YTHDF2 axis. We revealed that DDX5 bound antiviral transcripts and regulated immune responses through YTHDF2-dependent mRNA decay. Taken together, our data demonstrate that the DDX5/METTL3-METTL14/YTHDF2 axis regulates the replication of influenza A virus.IMPORTANCE The replication and transcription of influenza virus depends on the participation of many host factors in cells. Exploring the relationship between viruses and host factors will help us fully understand the characteristics and pathogenic mechanisms of influenza viruses. In this study, we showed that DDX5 interacted with the NP of influenza virus. We demonstrated that DDX5 downregulated the expression of IFN-β and IL-6 and the transcription of antiviral genes downstream from IFN-β in influenza virus-infected A549 cells. Additionally, DDX5 downregulated the mRNA levels of antiviral transcripts via the METTL3-METTL14/YTHDF2 axis. Our findings provide a novel perspective to understand the mechanism by which DDX5 regulates antiviral immunity.

Brain glycogen content is increased in the acute and interictal chronic stages of the mouse pilocarpine model of epilepsy.

Glucose is the main brain fuel in fed conditions, while astrocytic glycogen is used as supplemental fuel when the brain is stimulated. Brain glycogen levels are decreased shortly after induced seizures in rodents, but little is known about how glycogen levels are affected interictally in chronic models of epilepsy. Reduced glutamine synthetase activity has been suggested to lead to increased brain glycogen levels in humans with chronic epilepsy. Here, we used the mouse pilocarpine model of epilepsy to investigate whether brain glycogen levels are altered, both acutely and in the chronic stage of the model. One day after pilocarpine‐induced convulsive status epilepticus (CSE), glycogen levels were higher in the hippocampal formation, cerebral cortex, and cerebellum. Opposite to expected, this was accompanied by elevated glutamine synthetase activity in the hippocampus but not the cortex. Increased interictal glycogen amounts were seen in the hippocampal formation and cerebral cortex in the chronic stage of the model (21 days post‐CSE), suggesting long‐lasting alterations in glycogen metabolism. Glycogen solubility in the cerebral cortex was unaltered in this epilepsy mouse model. Glycogen synthase kinase 3 beta (Gsk3b) mRNA levels were reduced in the hippocampal formations of mice in the chronic stage, which may underlie the elevated brain glycogen content in this model. This is the first report of elevated interictal glycogen levels in a chronic epilepsy model. Increased glycogen amounts in the brain may influence seizure susceptibility in this model, and this warrants further investigation.

Interleukin-15 receptor subunit alpha regulates interleukin-15 localization and protein expression in skeletal muscle cells.

What is the central question of this study? How are the dynamics of interleukin (IL)-15 and its receptors altered during the differentiation of myoblasts into myotubes, and how is IL-15 regulated? What is the main finding and its importance? The mRNA levels of IL-15 and interleukin-2 receptor subunits beta and gamma increase during skeletal muscle differentiation, whereas interleukin-15 receptor subunit alpha (IL-15RA) exhibits different kinetics. IL-15RA regulates the localization and expression of IL-15 at the protein level. Interleukin-15 (IL-15) is a myokine in the interleukin-2 (IL-2) family that is generated in the skeletal muscle during exercise. The functional effect of IL-15 involves muscle regeneration and metabolic regulation in skeletal muscle. Reports have indicated that interleukin-15 receptor subunit alpha (IL-15RA) acts by regulating IL-15 localization in immune cells. However, the dynamics of IL-15 and its receptors, which regulate the IL-15 pathway in skeletal muscle differentiation, have not yet been clarified. In this study, we investigated the mechanism of IL-15 regulation using a mouse skeletal muscle cell line, C2C12 cells. We found that the mRNA expression of IL-15, interleukin-2 receptor subunit beta (IL-2RB; CD122) and interleukin-2 receptor subunit gamma (IL-2RG; CD132) increased, but that IL-15RA exhibited different kinetics as differentiation progressed. We also found that IL-15, mainly present in the cytosol, pre-assembled with IL-15RA in the cytosol and fused to the plasma membrane. Moreover, IL-15RA increased IL-15 protein levels. Our findings suggest that genes involved in the IL-15 signalling complex are enhanced with the differentiation of myotubes and that IL-15RA regulates the protein kinetics of IL-15 signalling in skeletal muscle.

CD163 as a Potential Biomarker of Monocyte Activation in Ischemic Stroke Patients.

In ischemic stroke patients, a higher monocyte count is associated with disease severity and worse prognosis. The complex correlation between subset phenotypes and functions underscores the importance of clarifying the role of monocyte subpopulations. We examined the subtype-specific distribution of the CD163+ and CD80+ circulating monocytes and evaluated their association with the inflammatory status in 26 ischemic stroke patients and 16 healthy controls. An increased percentage of CD163+/CD16+ and CD163+/CD14++ events occurred 24 and 48 h after a stroke compared to the controls. CD163+ expression was more pronounced in CD16+ non-classical and intermediate monocytes, as compared to CD14+ classical subtype, 24 h after stroke. Conversely, the percentage of CD80+/CD16+ events was unaffected in patients; meanwhile, the percentage of CD80+/CD14+ events significantly increased only 24 h after stroke. Interleukin (IL)-1beta, TNF-alpha, and IL-4 mRNA levels were higher, while IL-10 mRNA levels were reduced in total monocytes from patients versus controls, at either 24 h or 48 h after stroke. The percentage of CD163+/CD16+ events 24 h after stroke was positively associated with NIHSS score and mRS at admission, suggesting that stroke severity and disability are relevant triggers for CD163+ expression in circulating CD16+ monocytes.

Melatonin Alleviates Acute Gouty Inflammation In Vivo and In Vitro.

The aim of this study was to identify the effects of melatonin on acute gouty inflammation and to investigate the underlying mechanisms. We found significantly lower serum melatonin levels in gout patients in the acute phase than in those in the remission phase or in normal individuals. The mRNA expression of melatonin receptor 2 (MT2) was also lower in gout patients than in normal individuals. To verify the in-vivo role of melatonin, a gouty arthritis model was established by intraarticular injection of monosodium urate (MSU, 1 mg) crystals into the paws of C57BL/6 mice. Joint inflammation in the mouse model was evaluated by measuring the thickness of the right paw/left paw, and the inflammation index was determined by examining infiltrating neutrophils with haematoxylin and eosin (H&E) staining. Melatonin was found to reduce both paw thickness and the inflammation index in the mouse model, and melatonin also reduced the mRNA levels of interleukin-1 beta (IL-1β), IL-6 and NLR family pyrin domain containing 3 (NLRP3) inflammasome. To mimic gouty inflammation in vitro, mouse peritoneal macrophages were stimulated with lipopolysaccharides (LPS) plus MSU. Melatonin was revealed to reduce IL-1β secretion by stimulated macrophages. The mRNA expression levels of IL-1β and IL-6 were also inhibited by melatonin. Western blot analysis showed that the expression of NLRP3, caspase-1 and pro-IL-1β was also inhibited by melatonin. In conclusion, our study demonstrated that melatonin alleviated gouty inflammation in vivo and in vitro, and the underlying mechanism may involve inhibiting the assembly of the NLRP3 inflammasome.

Garlic extract attenuates retinal oxidative stress and IL-1 expression in STZ-induced diabetic rats

Retinopathy is one of the most common complications of diabetes. Hyperglycemia damages to the tissues through induction of oxidative stress and inflammation. In this study, the effects of garlic extract on the oxidative stress status and interleukin1 beta (IL-1β) mRNA and protein levels in the retinal tissue of diabetic rats were investigated. Twenty-four male Wistar rats were divided into four groups: control rats (C), diabetic rats (D), garlic treated diabetic rats (D+G), and garlic treated normal rats (G). At the end of the treatment period, for determination of oxidative stress, lipid peroxidation (LPO), thiol group (SH), total antioxidant capacity (TAC), and total oxidant status (TOS) were assayed. The expression of mRNA and protein levels of IL-1β was determined in retinal homogenates by real-time PCR and ELISA, respectively. In diabetic rats, TAC and thiol group decreased (p<0.05 and p<0.001, respectively), whereas TOS and LPO increased (p<0.001 and p<0.01, respectively) compared with control rats. In garlic treated diabetic rats TAC and thiol group increased (p<0.05 and p<0.001, respectively), whereas TOS and LPO decreased (p<0.001) compared with untreated diabetic rats. The mRNA expression and protein levels of IL-1β increased in diabetic rats in comparison with control group (p<0.001). These parameters decreased in garlic treated diabetic rats compared with untreated diabetic rats (p<0.05 and p<0.001, respectively). These results indicate that garlic extract has antioxidant property, can decrease IL-1β protein and mRNA level, and therefore, it can be a good candidate for reduction of diabetic retinopathy.

MiR-200b suppresses TNF-α-induced AMTN production in human gingival epithelial cells.

Amelotin (AMTN) is an enamel protein that is localized in junctional epithelium (JE) of gingiva and suggested to be involved in the attachment between JE and tooth enamel. MicroRNA is a small non-coding RNA that regulates gene expression at post-transcriptional level by binding to the 3'-untranslated region (3'-UTR) of target mRNAs. In this study, we have analyzed the effects of miR-200b on the expression of AMTN in human gingival epithelial (Ca9-22) cells. Total RNAs and proteins were extracted from Ca9-22 cells transfected with miR-200b expression plasmid or miR-200b inhibitor and stimulated by TNF-α (10ng/ml, 12h). AMTN and inhibitor of kappa-B kinase beta (IKKβ) mRNA and protein levels were measured by qPCR and Western blot. Human AMTN 3'-UTR that contains putative miR-200b target sites were cloned downstream of -353AMTN luciferase (LUC) plasmid. Ca9-22 cells were transfected with -353AMTN 3'-UTR LUC constructs and miR-200b expression plasmid, and LUC activities were measured with or without stimulation by TNF-α. TNF-α-induced AMTN mRNA levels were partially inhibited by miR-200b overexpression and enhanced by miR-200b inhibitor. TNF-α-induced IKKβ mRNA and protein levels were almost completely inhibited by miR-200b. Transcriptional activities of -353AMTN 3'-UTR LUC constructs were induced by TNF-α and partially inhibited by miR-200b. IKKβ inhibitor IMD0354 and NF-κB inhibitor triptolide decreased TNF-α-induced LUC activities. Furthermore, both inhibitors reduced AMTN mRNA levels in the presence or absence of TNF-α. These results suggest that miR-200b suppresses AMTN expression by targeting to AMTN and IKKβ mRNAs in the human gingival epithelial cells.

Di-(2-ethylhexyl)-phthalate’e Maruz Kalan Alburnus tarichi (Güldenstӓdt, 1814) Erken Larvalarında Östrojen Reseptör ve P450 Aromataz mRNA Seviyeleri

Objective: The aim of this study is to determine the effect of phthalate on the early larval development of Alburnus tarichi.Methods: The larva (2 days after hatching) of Alburnus tarichi were exposed to 0.1, 1, and 10 µg/L phthalate for 6 days (during yolk sac nutrition), and the levels of estrogen receptor alfa, beta-1 and beta-2, and aromatase B and A mRNA levels were measured.Results: No difference was found in the estrogen receptor alpha, beta-1, beta-2, and aromatase-B mRNA levels with all 3 concentrations of phthalate. However, the aromatase-A mRNA level was significantly increased with 0.1 µg/L of phthalate, while no changes were observed with 1 and 10 µg/L of phthalate.Conclusion: These results suggest that short-time expose to environmentally relative concentrations of phthalate do not significantly affect genes in steroid synthesis during the early larval development of Alburnus tarichi. However, long-term applied studies including external nutrition are need to determination the effect of phthalate on steroid gene expression.

Astragaloside IV attenuates IL-1β secretion by enhancing autophagy in H1N1 infection.

Excessive secretion of inflammatory factors (cytokine storm) plays a significant role in H1N1-induced acute pneumonia, and autophagy acts as a cell-intrinsic mechanism to regulate inflammation. Astragaloside IV (AS-IV), originating from the astragalus root, possesses multiple pharmacological activities, such as anti-inflammation. However, the influences of AS-IV on H1N1-induced autophagy and inflammation have remained elusive. It has been reported that H1N1 infection leads to the accumulation of autophagosomes but obstructs autophagosomes incorporating into lysosomes, whereas the present study showed that AS-IV enhanced autophagy activation in H1N1 infection. Furthermore, we found that AS-IV promoted H1N1-triggered formation of autophagosomes and autolysosomes. Additionally, it was noted that AS-IV did not affect viral replication, mRNA level of interleukin-1 beta (IL-1β) and pro-IL-1β protein level, but significantly decreased secretion of IL-1β, and chloroquine (CQ, as an inhibitor of autophagy) increased secretion of IL-1β in H1N1 infection. In conclusion, AS-IV stimulates the formation of autophagosomes and the fusion of autophagosomes and lysosomes in H1N1 infection and may lead to decreased IL-1β secretion.

Methotrexate+Medical Ozone combined therapy in Rheumatoid Arthritis. From experimental models to clinical trials [abstract

INTRODUCTION: The aim of this work has been to investigate the medical ozone effects on 2 experimental models: PG/PS induced RA and synovitis followed for a clinical trial of efficacy.&#x0D; &#x0D; METHODS: mRNA levels of TNF-alpha and IL-1 beta, injury and protective biomarker of oxidative stress, morphology studies, A1 adenosine receptor role, Anti-Cyclic Citrullinated (Anti-CCP) levels and clinical variables were determined.&#x0D; &#x0D; RESULTS: Medical ozone ameliorated the expression of proinflammatory cytoquines, reduced oxidative damage while participation of A1 adenosine receptor in the antiinflammatory effects was demonstrated.&#x0D; In RA patients (n=60) MTX + ozone reduced the activity of the disease while MTX merely showed a tendency to decrease it. MTX + ozone reduced Anti-CCP levels as well as increased antioxidant system, and decreased oxidative damage whereas MTX did not change.&#x0D; &#x0D; CONCLUSIONS: Medical ozone was effective in the reduction of inflammatory response in 2 experimental studies. MTX + ozone increased the MTX clinical efficacy in RA patients. These results suggest that ozone can increase the efficacy of MTX probably because both share common therapeutic targets.

Antiviral Activity Against Infectious Bronchitis Virus and Bioactive Components of Hypericum perforatum L.

Hypericum perforatum L., also known as Saint John’s Wort, has been well studied for its chemical composition and pharmacological activity. In this study, the antiviral activities of H. perforatum on infectious bronchitis virus (IBV) were evaluated in vitro and in vivo for the first time. The results of in vitro experiments confirmed that the antiviral component of H. perforatum was ethyl acetate extraction section (HPE), and results showed that treatment with HPE significantly reduced the relative messenger ribonucleic acid (mRNA) expression and virus titer of IBV, and reduced positive green immunofluorescence signal of IBV in chicken embryo kidney (CEK) cells. HPE treatment at doses of 480–120 mg/kg for 5 days, reduced IBV induced injury in the trachea and kidney, moreover, reduced the mRNA expression level of IBV in the trachea and kidney in vivo. The mRNA expression levels of IL-6, tumor necrosis factor alpha (TNF-α), and nuclear factor kappa beta (NF-κB) significantly decreased, but melanoma differentiation-associated protein 5 (MDA5), mitochondrial antiviral signaling gene, interferon alpha (IFN-α), and interferon beta (IFN-β) mRNA levels significantly increased in vitro and in vivo. Our findings demonstrated that HPE had significant anti-IBV effects in vitro and in vivo, respectively. In addition, it is possible owing to up-regulate mRNA expression of type I interferon through the MDA5 signaling pathway and down-regulate mRNA expression of IL-6 and TNF-α via the NF-κB signaling pathway. Moreover, the mainly active compositions of HPE analyzed by high-performance liquid chromatography/electrospray ionization–mass spectroscopy (ESI-MS) are hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin, and a combination of these compounds could mediate the antiviral activities. This might accelerate our understanding of the antiviral effect of H. perforatum and provide new insights into the development of effective therapeutic strategies.

Mice lacking adenosine 2A receptor reveal increased severity of MCD-induced NASH.

Adenosine 2A receptor (A2AR) exerts a protective role in obesity-related non-alcoholic fatty liver disease. Here, we examined whether A2AR protects against non-alcoholic steatohepatitis (NASH). In C57BL/6J mice, feeding a methionine- and choline-deficient diet (MCD) resulted in significant weight loss, overt hepatic steatosis, and massive aggregation of macrophages in the liver compared with mice fed a chow diet. MCD feeding also significantly increased the numbers of A2AR-positive macrophages/Kupffer cells in liver sections although decreasing A2AR amount in liver lysates compared with chow diet feeding. Next, MCD-induced NASH phenotype was examined in A2AR-disrupted mice and control mice. Upon MCD feeding, A2AR-disruptd mice and control mice displayed comparable decreases in body weight and fat mass. However, MCD-fed A2AR-disrupted mice revealed greater liver weight and increased severity of hepatic steatosis compared with MCD-fed control mice. Moreover, A2AR-disupted mice displayed increased severity of MCD-induced liver inflammation, indicated by massive aggregation of macrophages and increased phosphorylation states of Jun-N terminal kinase (JNK) p46 and nuclear factor kappa B (NFκB) p65 and mRNA levels of tumor necrosis factor alpha, interleukin-1 beta, and interleukin-6. In vitro, incubation with MCD-mimicking media increased lipopolysaccharide (LPS)-induced phosphorylation states of JNK p46 and/or NFκB p65 and cytokine mRNAs in control macrophages and RAW264.7 cells, but not primary hepatocytes. Additionally, MCD-mimicking media significantly increased lipopolysaccharide-induced phosphorylation states of p38 and NFκB p65 in A2AR-deficient macrophages, but insignificantly decreased lipopolysaccharide-induced phosphorylation states of JNK p46 and NFκB p65 in A2AR-deficient hepatocytes. Collectively, these results suggest that A2AR disruption exacerbates MCD-induced NASH, which is attributable to, in large part, increased inflammatory responses in macrophages.