Vitamin K2 (MK-7) Intercepts Keap-1/Nrf-2/HO-1 Pathway and Hinders Inflammatory/Apoptotic Signaling and Liver Aging in Naturally Aging Rat.

Aging is a naturally occurring physiological process with a deleterious impact on various body organs and humans' well-being. The aging population is increasing worldwide, which imposes the need for the exploration of nutritional options that can intercept the impact of the aging processed on various body organs. Vitamin K2 (VK2) is a fat-soluble vitamin with emerging evidence on its therapeutic merits. In the current study, natural aging induced a significant liver deterioration with a disrupted Keap-1/Nrf-2/HO-1 axis and increased COX-2, iNOS and TNF-α expression and apoptotic and fibrotic changes. VK2 administration, on the other hand, improved the biochemical indices of liver function (total protein, albumin, ALT and AST); the suppressed hepatic expression of Keap-1 and increased the hepatic expression of Nrf-2 with a parallel increase in the hepatic activity of HO-1. Subsequently, the liver content and hepatic expression of TNF-α, COX-2 and iNOS were significantly retracted. In context, the liver content and hepatic expression of the fibrotic biomarkers TGFß and TIMP significantly retracted as well. Moreover, the TUNEL assay confirmed the retraction of liver apoptotic changes. Of notice, electron transmission microscope examination confirmed the preservation of mitochondrial functions and preservation of the ultra-microscopical structures. In conclusion, the VK2-mediated interception of aging-induced Keap-1/Nrf-2/HO-1 signaling suppressed the hepatic contents of inflammatory and fibrotic biomarkers, as well as apoptotic changes with preservation of the hepatic architectural and functional status. VK2 can be presumed to be an effective nutritional supplement to the aging population to spare the liver, amongst other body organs, against aging-induced deleterious injury.

Beta-Sitosterol Facilitates GLUT4 Vesicle Fusion on the Plasma Membrane via the Activation of Rab/IRAP/Munc 18 Signaling Pathways in Diabetic Gastrocnemius Muscle of Adult Male Rats.

Nutritional overload in the form of high-fat and nonglycolysis sugar intake contributes towards the accelerated creation of reactive oxygen species (ROS), hyperglycemia, and dyslipidemia. Glucose absorption and its subsequent oxidation processes in fat and muscle tissues alter as a consequence of these modifications. Insulin resistance (IR) caused glucose transporter 4 (GLUT4) translocation to encounter a challenge that manifested itself as changes in glycolytic pathways and insulin signaling. We previously found that beta (ß)-sitosterol reduces IR in fat tissue via IRS-1/PI3K/Akt facilitated signaling due to its hypolipidemic and hypoglycemic activity. The intention of this research was to see whether the phytosterol ß-sitosterol can aid in the translocation of GLUT4 in rats fed on high-fat diet (HFD) and sucrose by promoting Rab/IRAP/Munc 18 signaling molecules. The rats were labeled into four groups, namely control rats, HFD and sucrose-induced diabetic control rats, HFD and sucrose-induced diabetic rats given oral dose of 20 mg/kg body wt./day of ß-sitosterol treatment for 30 days, and HFD and sucrose-induced diabetic animals given oral administration of 50 mg/kg body wt./day metformin for 30 days. Diabetic rats administered with ß-sitosterol and normalized the titers of blood glucose, serum insulin, serum testosterone, and the status of insulin tolerance and oral glucose tolerance. In comparison with the control group, ß-sitosterol effectively regulated both glycolytic and gluconeogenesis enzymes. Furthermore, qRT-PCR analysis of the mRNA levels of key regulatory genes such as SNAP23, VAMP-2, syntaxin-4, IRAP, vimentin, and SPARC revealed that ß-sitosterol significantly regulated the mRNA levels of the above genes in diabetic gastrocnemius muscle. Protein expression analysis of Rab10, IRAP, vimentin, and GLUT4 demonstrated that ß-sitosterol had a positive effect on these proteins, resulting in effective GLUT4 translocation in skeletal muscle. According to the findings, ß-sitosterol reduced HFD and sucrose-induced IR and augmented GLUT4 translocation in gastrocnemius muscle through insulin signaling modulation via Rab/IRAP/Munc 18 and glucose metabolic enzymes. The present work is the first of its kind to show that ß-sitosterol facilitates GLUT4 vesicle fusion on the plasma membrane via Rab/IRAP/Munc 18 signaling molecules in gastrocnemius muscle.

Raisins Preserve Thyroid Gland Function and Structure in an Animal Model of Hypercholesterolemia.

Background: Statins are among the first line of pharmacological treatment of lipid disorders and lowering serum cholesterol, but they have many side effects. Aim: The study aim was to evaluate the role of raisins in protecting the thyroid function and structure in a rat model of hypercholesterolemia, through biochemical and histopathological investigation. Materials and Methods: Thirty male rats were randomly divided into three groups (n = 10 each) of albino rats included the control, high cholesterol diet (HCD)-fed for 13 weeks and HCD plus Raisins were included in this study. Blood levels of glucose, insulin, cholesterol, lipids, thyroid-stimulating hormone (TSH), T3, T4, oxidants/anti-oxidants were assessed. Thyroid gland was processed and examined histopathologically using light and electron microscopy. Results: Feeding HCD resulted in hypercholesterolemia in rats after 13 weeks as evidence by lipid profile. Ingestion of raisins along with HCD resulted in a significant (P < 0.001) decrease in the levels of insulin, blood glucose, thyroxine (T4) and malondialdehyde (MDA), while the levels of TSH, T3 and total anti-oxidant capacity significantly (P < 0.001) elevated. Raisins histologically alleviated the HCD-induced structural changes in the thyroid glands that included degenerated mitochondria and increased lipid droplets in the cytoplasm. Conclusions: Simultaneous administration of raisins along with HCD, administrated for a short time, could modulate the negative effect on thyroid gland structure and function.

Hair Follicle-Related MicroRNA-34a Serum Expression and rs2666433A/G Variant in Patients with Alopecia: A Cross-Sectional Analysis.

Alopecia areata (AA) is a type of immune-mediated alopecia. Recent studies have suggested microRNAs' (miRNAs) implication in several cellular processes, including epidermal and hair follicle biology. Single nucleotide polymorphisms (SNPs) can modify gene expression levels, which may induce an autoimmune response. This case−control study included 480 participants (240 for each case/control group). MicroRNA-34a gene (MIR-34A) rs2666433A/G variant was genotyped using real-time allelic discrimination polymerase chain reaction (PCR). Additionally, circulatory miR-34a levels were quantified by quantitative reverse transcription PCR (qRT-PCR). On comparing between alopecia and non-alopecia cohorts, a higher frequency of A variant was noted among patients when compared to controls­A allele: 28 versus 18% (p < 0.001); A/A genotype: 9 versus 2%; A/G genotype: 39 versus 32% (p < 0.001). A/A and A/G carriers were more likely to develop alopecia under heterozygote comparison (OR = 1.83, 95% CI = 1.14−2.93), homozygote comparison (OR = 4.19, 95% CI = 1.33−13.1), dominant (OR = 2.0, 95% CI = 1.27−3.15), recessive (OR = 3.36, 95% CI = 1.08−10.48), over-dominant (OR = 1.65, 95% CI = 1.04−32.63), and log additive (OR = 1.91, 95% CI = 1.3−2.82) models. Serum miR-34a expression levels were upregulated in alopecia patients with a median and quartile fold change of 27.3 (1.42−2430). Significantly higher levels were more pronounced in A/A genotype patients (p < 0.01). Patients carrying the heterozygote genotype (rs2666433 * A/G) were two times more likely to develop more severe disease grades. Stratified analysis by sex revealed the same results. A high expression level was associated with concomitant autoimmune comorbidities (p = 0.001), in particular SLE (p = 0.007) and vitiligo (p = 0.049). In conclusion, the MIR34A rs2666433 (A/G) variant is associated with AA risk and severity in the studied population. Furthermore, high miR-34a circulatory levels could play a role in disease pathogenesis.

MYD88, NFKB1, and IL6 transcripts overexpression are associated with poor outcomes and short survival in neonatal sepsis.

Toll-like receptor (TLR) family signature has been implicated in sepsis etiopathology. We aimed to evaluate the genetic profile of TLR pathway-related key genes; the myeloid differentiation protein 88 (MYD88), IL1 receptor-associated kinase 1 (IRAK1), the nuclear factor kappa-B1 (NFKB1), and interleukin 6 (IL6) in the blood of neonates with sepsis at the time of admission and post-treatment for the available paired-samples. This case-control study included 124 infants with sepsis admitted to the neonatal intensive care unit and 17 controls. The relative gene expressions were quantified by TaqMan Real-Time qPCR and correlated to the clinic-laboratory data. MYD88, NFKB1, and IL6 relative expressions were significantly higher in sepsis cases than controls. Higher levels of MYD88 and IL6 were found in male neonates and contributed to the sex-based separation of the cases by the principal component analysis. ROC analysis revealed MYD88 and NFKB1 transcripts to be good biomarkers for sepsis. Furthermore, patients with high circulatory MYD88 levels were associated with poor survival, as revealed by Kaplan-Meier curves analysis. MYD88, NFKB1, and IL6 transcripts showed association with different poor-outcome manifestations. Clustering analysis split the patient cohort into three distinct groups according to their transcriptomic signature and CRP levels. In conclusion, the study TLR pathway-related transcripts have a gender-specific signature, diagnostic, and prognostic clinical utility in neonatal sepsis.

Chronic valproic acid administration enhances oxidative stress, upregulates IL6 and downregulates Nrf2, Glut1 and Glut4 in rat's liver and brain.

Valproic acid (VPA) is a powerful antiepileptic drug that was associated with several neurological and hepatic problems especially with increasing its dose and duration. These problems may be metabolic in origin and related to glucose homeostasis. So, the present study investigated the effect of different doses and durations of VPA on the expression of glucose transporters (Glut1 and Glut4), oxidative stress and inflammatory cytokine (IL-6) in the liver and specific brain regions. Seventy-two male Sprague-Dawley rats were randomly allocated into three equal groups: (1) saline group, (2) 200 mg VPA group and (3) 400 mg VPA group. By the end of experiments, the expressions of Glut1, Glut4 nuclear factor erythroid-like 2 related factor (Nrf2), IL-6 and oxidative stress markers [malondialdehyde (MDA) and glutathione (GSH)] in the liver, corpus striatum, prefrontal cortex (PFC) and cerebellum were assessed. We found that administration of VPA (200 mg and 400 mg) caused a significant decrease in the Glut1 and Glut4 expression in different tissues in a dose- and time-dependent manner (P < 0.01). Also, VPA (200 and 400 mg) caused a significant increase in MDA with a decrease in GSH in tissues at different times. Moreover, VPA (200 and 400 mg) caused significant upregulation in IL-6 expression and downregulation in Nrf2 expression (P < 0.01). The results suggest that increasing the dose and time of VPA therapy downregulates Glut1 and Glut4 in the liver and brain which may impair glucose uptake in these tissues. This effect was associated with enhanced oxidative stress, downregulation of nrf2 and upregulation of IL-6 in liver and brain tissues.

Thymoquinone reverses nonalcoholic fatty liver disease (NAFLD) associated with experimental hypothyroidism.

OBJECTIVES: To assess the efficacy of thymoquinone (TQ), the most active constituent in Nigella sativa, which is a medicinal plant from the Ranunculaceae family, in restoring the normal liver structure after 6-propyl-2-thiouracil (PTU)-induced hypothyroidism and explore the mechanism behind this. MATERIALS AND METHODS: Hypothyroidism was induced in rats by injection of PTU [6 mg∕kg body weight (b.w.)] for six weeks. Twenty-four adult male Wistar rats were divided into four groups; the control, TQ-treated at the dose 400 mg∕kg b.w., untreated hypothyroidism and TQ-treated hypothyroid groups. Serum levels of thyroid hormones and antioxidant profile were measured. Real-time polymerase chain reaction was used to assess gene expression of catalase (CAT). Liver was histopathologically examined using routine and immunohistochemical techniques. RESULTS: Livers of rats with hypothyroidism displayed nonalcoholic fatty liver disease (NAFLD) in the form of steatosis as well as nonalcoholic steatohepatitis (NASH). Moreover, there was an intralobular inflammatory reaction associated with significant (p<0.05) increases in the density of resident hepatic macrophages [cluster of differentiation 68 (CD68)+ cells], as well as in activated hepatic stellate cells, alpha-smooth muscle actin (α-SMA) index in livers with hypothyroidism. Resolution of hypothyroid NAFLD was observed in livers after treatment with TQ. The significantly increased (p<0.05) steatosis, lobular inflammation, NAFLD activity scores, α-SMA index as well as CD68+ cells induced by hypothyroidism were corrected after TQ administration. Up-regulation of the CAT gene in livers with hypothyroidism after treatment with TQ supported our hypothesis of its antioxidant mechanistic hepatoprotective action. CONCLUSIONS: TQ efficiently restores the normal liver histology in hypothyroid rats with up-regulation of the antioxidant CAT gene.

Effects of GLP-1 Receptor Activation on a Pentylenetetrazole-Kindling Rat Model.

Objectives: To study the possible anti-seizure and neuroprotective effect of glucagon like peptide 1 (GLP1) analogue (liraglutide) in a pentylenetetrazole (PTZ) induced kindled rat model and its underlying mechanisms. Methods: Thirty Sprague Dawley rats were allocated into 3 equal groups; i) Normal group: normal rats received normal saline, ii) PTZ (kindling) group: received PTZ (50 mg/Kg intraperitoneally (i.p.)) every other day for 2 weeks and iii) PTZ + GLP1 group: same as the PTZ group but rats received liraglutide (75 µg/kg i.p. daily) for 2 weeks before PTZ injection. Seizure severity score, seizure latency and duration were assessed. Also, the expression of caspase-3 (apoptotic marker) and ß-catenin (Wnt pathway) by western blotting, markers of oxidative stress (GSH, CAT and MDA) by biochemical assay and the expression of LC3 (marker of autophagy) and heat shock protein 70 (Hsp70) by immunostaining were assessed in hippocampal regions of brain tissues. Results: PTZ caused a significant increase in Racine score and seizure duration with a significant decrease in seizure latency. These effects were associated with a significant increase in MDA, ß-catenin, caspase-3, Hsp70 and LC3 in brain tissues (p < 0.05). Meanwhile, liraglutide treatment caused significant attenuation in PTZ-induced seizures, which were associated with significant improvement in markers of oxidative stress, reduction in LC3, caspase-3 and ß-catenin and marked increase in Hsp70 in hippocampal regions (p < 0.05). Conclusion: Activation of GLP1R might have anticonvulsant and neuroprotective effects against PTZ-induced epilepsy. These effects could be due to suppression of oxidative stress, apoptosis and autophagy and upregulation of Hsp70.

Effects of heme oxygenase-1 upregulation on isoproterenol-induced myocardial infarction.

The present study was designed to examine the effect of heme oxygenase-1 (HO-1) induction by cobalt protoporphyrin (CoPP) on the cardiac functions and morphology, electrocardiogram (ECG) changes, myocardial antioxidants (superoxide dismutase [SOD] and glutathione [GSH]), and expression of heat shock protein (Hsp) 70 and connexin 43 (Cx-43) in myocardial muscles in isoproterenol (ISO) induced myocardial infarction (MI). Thirty two adult male Sprague Dawely rats were divided into 4 groups (each 8 rats): normal control (NC) group, ISO group: received ISO at dose of 150 mg/kg body weight intraperitoneally (i.p.) for 2 successive days; ISO + Trizma group: received (ISO) and Trizma (solvent of CoPP) at dose of 5 mg/kg i.p. injection 2 days before injection of ISO, with ISO at day 0 and at day 2 after ISO injections; and ISO + CoPP group: received ISO and CoPP at a dose of 5 mg/kg dissolved in Trizma i.p. injection as Trizma. We found that, administration of ISO caused significant increase in heart rate, corrected QT interval, ST segment, cardiac enzymes (lactate dehydrogenase, creatine kinase-muscle/brain), cardiac HO-1, Hsp70 with significant attenuation in myocardial GSH, SOD, and Cx-43. On the other hand, administration of CoPP caused significant improvement in ECG parameters, cardiac enzymes, cardiac morphology; antioxidants induced by ISO with significant increase in HO-1, Cx-43, and Hsp70 expression in myocardium. In conclusions, we concluded that induction of HO-1 by CoPP ameliorates ISO-induced myocardial injury, which might be due to up-regulation of Hsp70 and gap junction protein (Cx-43).

Overexpression of heat shock protein HSP90AA1 and translocase of the outer mitochondrial membrane TOM34 in HCV-induced hepatocellular carcinoma: A pilot study.

OBJECTIVE: Identification of new molecular markers to enhance early diagnosis, prognosis and/or treatment of hepatocellular carcinoma (HCC) is a need. TOM34 (34 kDa-translocase of the outer mitochondrial membrane) protein expression deregulation has demonstrated to be involved in the growth of many cancers. Here, we aimed at evaluating serum TOM34 and some heat shock proteins (HSPA4, HSPA1B, and HSP90AA1) expressions in hepatitis C virus (HCV)-related cirrhosis and HCV-induced HCC relative to controls and correlating these expressions to the clinicopathological data. METHODS: Serum specimens were collected from 90 patients with HCV associated complications (30 cirrhotic, 30 early HCC and 30 late HCC) and 60 controls. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed for relative quantification of the four target genes using the Livak method. In silico network analysis was also executed to explore the contribution of the genes in liver cancer. RESULTS: The serum TOM34 and HSP90AA1 transcripts were significantly upregulated in HCC patients compared to cirrhotic ones with more up-regulation in late HCC patients. Receiver operating characteristic analysis showed the optimum cutoff value of 0.625 corresponding to 71.7% sensitivity and 56.7% specificity, and an area under the curve (AUC) of 0.705 to discriminate HCC from cirrhotic groups (P = .002). In multivariate analysis, ordination plot showed obvious demarcation between the study groups caused by the higher levels of TOM34 among other variables. CONCLUSIONS: TOM34 and its partner HSP90AA1 might be used as a potential biomarker for monitoring HCV-induced HCC progression in the Egyptian population. Future large-scale validation studies are warranted.

Longevity-Related Gene Transcriptomic Signature in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) (grade IV astrocytoma) has been assumed to be the most fatal type of glioma with low survival and high recurrence rates, even after prompt surgical removal and aggressive courses of treatment. Transcriptional reprogramming to stem cell-like state could explain some of the deregulated molecular signatures in GBM disease. The present study aimed to quantify the expression profiling of longevity-related transcriptional factors SOX2, OCT3/4, and NANOG to evaluate their diagnostic and performance values in high-grade gliomas. Forty-four specimens were obtained from glioblastoma patients (10 females and 34 males). Quantitative real-time polymerase chain reaction was applied for relative gene expression quantification. In silico network analysis was executed. NANOG and OCT3/4 mRNA expression levels were significantly downregulated while that of SOX2 was upregulated in cancer compared to noncancer tissues. Receiver operating characteristic curve analysis showed high diagnostic performance of NANOG and OCT3/4 than SOX2. However, the aberrant expressions of the genes studied were not associated with the prognostic variables in the current population. In conclusion, the current study highlighted the aberrant expression of certain longevity-associated transcription factors in glioblastoma multiforme which may direct the attention towards new strategies in the treatment of such lethal disease.

Structure and functional impact of seed region variant in MIR-499 gene family in bronchial asthma.

BACKGROUND: Small non-coding RNAs (microRNAs) have been evolved to master numerous cellular processes. Genetic variants within microRNA seed region might influence microRNA biogenesis and function. The study aimed at determining the role of microRNA-499 (MIR-499) gene family polymorphism as a marker for susceptibility and progression of bronchial asthma and to analyze the structural and functional impact of rs3746444 within the seed region. METHODS: Genotyping for 192 participants (96 patients and 96 controls) in the discovery phase and 319 subjects (115 patients and 204 controls) in the replication phase was performed via Real Time-Polymerase Chain Reaction technology. Patients underwent the methacholine challenge test and biochemical analysis. Gene structural and functional analysis, target prediction, annotation clustering, and pathway enrichment analysis were executed. Predicted functional effect of rs37464443 SNP was analyzed. RESULTS: miR-499 gene family is highly implicated in inflammation-related signaling pathways. Rs374644 (A > G) in MIR499A and MIR499B within the seed region could disrupt target genes and create new genes. The G variant was associated with high risk of developing asthma under all genetic association models (G versus A: OR = 3.27, 95% CI = 2.53-4.22; GG versus AA: OR = 9.52, 95% CI = 5.61-16.5; AG versus AA: OR = 2.13, 95% CI = 1.24-3.46; GG + AG versus AA: OR = 4.43, 95% CI = 2.88-6.82). GG genotype was associated with poor pre-bronchodilator FEV1 (p = 0.047) and the worst bronchodilator response after Salbutamol inhalation, represented in low peaked expiratory flow rate (p = 0.035). CONCLUSIONS: miR-499 rs3746444 (A > G) polymorphism was associated with asthma susceptibility and bronchodilator response in Egyptian children and adolescents. Further functional analysis is warranted to develop more specific theranostic agents for selecting targeted therapy.