Association between vitamin D hypovitaminosis and severe forms of COVID-19.

OBJECTIVE: Hypovitaminosis D may be associated with an increased susceptibility to infection, more severe COVID-19 forms, and a higher risk of death. The objective of this study was to investigate any possible connections between vitamin D status [as measured by serum 25-hydroxyvitamin D (25(OH)D) levels] and COVID-19 severity. PATIENTS AND METHODS: In 2021, a cross-sectional study of consecutive adult COVID-19 patients was conducted. Anthropometric data, comorbidities, hospital setting, length of stay, respiratory support, outcome data, and vitamin D status were all evaluated. RESULTS: The length of hospitalization among participants (n = 74; mean age 57.64 ± 17.83 years, 55.4% male) was 18.58 ± 10 days, the majority of the hospital setting was a medical ward (67.6%), and the respiratory support in the form of mechanical ventilation was represented by 12.2%. Hypertension (54.1%), obesity (64.9%), and overweight (64.9%) were the most common cardiometabolic risk factors. In the study group, 44.6% of participants had severe vitamin D deficiency (< 30 nmol/l), while 8.1% had vitamin D insufficiency (50 - 74.9 nmol/l). Furthermore, patients with severe COVID-19 (semi-intensive care unit, intensive care unit) had significantly lower serum 25(OH)D levels (32.9 vs. 20.5 nmol/l; p = 0.007). Participants with severe vitamin D deficiency were older and had more prevalent hypertension, requiring mechanical ventilation; 24.2% experienced a fatal outcome. CONCLUSIONS: Severe vitamin D deficiency may contribute significantly to the influence of other cardiometabolic risk factors in COVID-19.

Impact of treatment modalities on quality of life and depression in type 2 diabetes.

OBJECTIVE: Type 2 diabetes mellitus (T2DM) is a chronic disease with numerous complications that increase mortality and reduce the quality of life (QoL). The current study compares QoL in T2DM patients treated with insulin to those treated with oral antihyperglycemics (OAHs), as well as the frequency and severity of depression in patients. SUBJECTS AND METHODS: This prospective cross-sectional study included 200 patients with insulin or OAHs. Triglycerides, total cholesterol, low-density lipoprotein, and high-density lipoprotein cholesterol levels were measured. The Beck Depression Inventory and the SF-36 Quality of Life Questionnaire were used to assess depression symptoms and QoL in response to different treatment modalities. RESULTS: Insulin-treated patients have a longer duration of illness, higher preprandial glycemic levels, lower scores in three of four dimensions of the SF-36 physical component, and a lower score in the SF-36 psychological component's emotional role dimension. Patients on insulin have milder depression symptoms than those with OAHs. Depression symptoms, according to the findings, worsen QoL and glycemic control in insulin-treated patients. CONCLUSIONS: According to these findings, any treatment modality's success in T2DM patients primarily depends on psychological support and preventive measures that promote and maintain mental health.

A non-invasive method for estimating the severity of liver steatosis and the risk of fibrosis in non-obese type 2 diabetes patients with NAFLD.

Context: Prognostic considerations include assessing the risk of liver fibrosis in people with non-alcoholic fatty liver disease (NAFLD). Objectives: This study evaluates the use of hematologic and metabolic parameters regarding liver steatosis and fibrosis scores (FLI and Fib-4) in non-obese type 2 diabetes mellitus (t2DM) patients with NAFLD. Methods: Subjects underwent abdominal ultrasound examinations, and FLI and Fib-4 scores were calculated to evaluate liver steatosis and the risk of liver fibrosis non-invasively: 61 non-obese NAFLD subjects with t2DM were included in the cohort study and were divided into 2 groups depending on the t2DM treatment regimen. Results: Fib-4 and WBC count demonstrated a significant inverse correlation (OR = 0.509, p = 0.007). WBC count had an R2 of 0.237, indicating that this marker could account for up to 23.7% of a variation in Fib-4. Fib-4 and FFA had positive correlation which did not achieve statistically significant prediction (OR=7.122, p=0.062). Additionally, a significant prediction of HbA1c (OR=1.536, p=0.016) and haemoglobin (OR=1.071, p=0.020) for FLI was revealed. Conclusion: HbA1c and other haematological and metabolic parameters, such as haemoglobin and WBC, may be another non-invasive tool for determining whether non-obese NAFLD patients with t2DM are at risk of developing liver steatosis and fibrosis.

Hypothesis related to the regulation of inducible nitric oxide synthase during carotid endarterectomy.

Sudden occlusion of an artery caused by a thrombus or emboli is the most frequent cause of acute brain ischemia (ABI). Carotid endarterectomy (CEA) represents the gold standard for preventing strokes of carotid origin. However, neuronal damage caused by ischemia and/or reperfusion may contribute to a poor clinical outcome after CEA. In response to shear stress caused by hypoxic-ischemic conditions in patients undergoing CEA, stimulation of the hypothalamic-pituitaryadrenal axis leads to biological responses known as hypermetabolic stress, characterized by hemodynamic, metabolic, inflammatory and immunological changes. These changes maintain homeostasis and assist recovery, but an unregulated inflammatory response could lead to further tissue damage and death of neurons. Nitric oxide (NO) is an important signaling molecule involved in several physiological and pathological processes, including ABI. However, an excess of NO could have detrimental effects. We hypothesized that the hypoxic-ischemic state induced by carotid clamping leads to overexpression of inducible NO synthase and that uncontrolled production of NO could adversely affect outcome after CEA.

17ß-estradiol inhibits hepatic iNOS via the activation of the estrogen receptor ER-α and inhibition of erk1/2-mir-221 axis.

17ß-Estradiol (E2) is known to negatively regulate inducible nitric oxide (NO) synthase (iNOS) expression via estrogen receptor alpha (ER-α) activation in aortic vascular smooth muscle cells.Therefore, we sought to determine whether E2 can inhibit iNOS in vivo in hepatic tissue via the activation of ER-α and whether extracellular signal-regulated kinases 1/2 (ERK1/2)-miR-221 axis is involved in this process. Male Wistar rats were treated with a bolus injection of E2 intraperitoneally (40 µg/kg), and 24 hours after treatment the animals were sacrificed and the livers excised. The protein levels of iNOS, p50 and p65 subunits of nuclear factor κB (NFκB), ERα, ERK1/2 and protein kinase B (Akt), as well as the association of ERα/Src in liver lysates were assessed by Western blot. The expression of hepatic miR-221 was analyzed by qRT-PCR. Results show that E2 reduced hepatic iNOS protein expression (p less than 0.01), the protein level of ERα (p less than 0.05), ERK1/2 (p less than 0.05), Akt phosphorylation (p less than 0.001) and miR-221 expression (p less than 0.05). In contrast, hepatic ERα/Src kinase association level (p less than 0.05) increased after E2 treatment. Our results indicate that E2 inhibits hepatic iNOS via molecular mechanisms involving the activation of the ER-α and inhibition of ERK1/2-miR-221 axis.

Use of acellular collagen matrix for the closure of the open oral wound in bone regeneration.

Mucograft is collagen matrix was designed for use in open healing situations due to its compact outer layer. The technique presented in this article is an attempt to avail this attribute for covering open oral wound in guided bone regeneration (GBR) procedure. The essential idea of this technique is to avoid scoring of periosteum, submucosa and muscle layer for buccal flap advancement. Therefore, we used mucograft to cover bone substitute and barrier membrane in GBR surgical procedure. Thus, we avoided periostal-releasing incisions (PRI) and gained reposition of the flap to original level without impairing the attached keratinized gingiva. Buccal flap advancement in situations of shallow vestibulum, shortly attached gingiva and strong muscle pull may reduce or eliminate attached gingiva with an adverse effect on extended survival of placed implants. This technique promises to be beneficial for the preservation of the soft tissue around dental implants after GBR procedure.

Effects of Intracerebroventricularly (ICV) Injected Ghrelin on Cardiac Inducible Nitric Oxide Synthase Activity/Expression in Obese Rats.

The aim of this study was to examine the effects of ghrelin on regulation of cardiac inducible nitric oxide synthase (iNOS) activity/expression in high fat (HF), obese rats.For this study, male Wistar rats fed with HF diet (30% fat) for 4 weeks were injected every 24 h for 5 days intracerebroventricularly (ICV) with ghrelin (0.3 nmol/5 µl) or with an equal volume of phosphate buffered saline (PBS). Control rats were ICV injected with an equal volume of PBS. Glucose, insulin and nitric oxide (NO) concentrations were measured in serum, while arginase activity and citrulline concentrations were measured in heart lysate. Protein iNOS and regulatory subunit of nuclear factor-κB (NFκB-p65), phosphorylation of enzymes protein kinase B (Akt) at Ser(473), and extracellular signal-regulated kinases 1/2 (ERK1/2) at Tyr(202)/Tyr(204) were determined in heart lysate by Western blot. For gene expression of iNOS qRT-PCR was used.Results show significantly (p<0.01) higher serum NO production in ghrelin treated HF rats compared with HF rats. Ghrelin significantly reduced citrulline concentration (p<0.05) and arginase activity (p<0.01) in HF rats. In ghrelin treated HF rats, gene and protein expression of iNOS and NFκB-p65 levels were significantly (p<0.05) increased compared with HF rats. Increased phosphorylation of Akt (p<0.01) and decreased (p<0.05) ERK1/2 phosphorylation were detected in HF ghrelin treated rats compared with HF rats hearts.Results from this study indicate that exogenous ghrelin induces expression and activity of cardiac iNOS via Akt phosphorylation followed by NFκB activation in HF rats.

Involvement of the ADAM 12 in thrombin-induced rat's VSMCs proliferation.

Cardiovascular disease is the largest single cause of mortality and its major underlying pathology is atherosclerosis. The proliferation of vascular smooth muscle cells (VSMCs) is a key event in the pathogenesis of the various vascular diseases, including atherosclerosis and hypertension. Thrombin (Thr) is involved in the abnormal proliferation of VSMCs associated with atherosclerosis and hypertension. ADAMs (A Disintegrin And Metalloproteinase) are transmembrane metalloproteinases, belonging to the adamalysins group, that are distinct from matrix metalloproteinases (MMPs) in a way as they have an extracellular disintegrin domain and cytoplasmic domain that can associate with intracellular proteins. There is limited knowledge about the presence of ADAM metalloproteinase activity in Thr-induced VSMCs proliferation. Therefore, this review examines recent findings in signaling mechanisms employed by Thr in modulating the regulation of proliferation of VSMCs with particular emphasis on involvement of ADAM 12 which has been identified as an important mediator of VSMCs hypertrophy and vascular diseases. These findings are critical for understanding the role of Thr in vascular biology and vascular diseases.

Interaction between insulin and estradiol in regulation of cardiac glucose and free fatty acid transporters.

The estrogen binding to specific extranuclear receptors (ER) activates several intracellular pathways that are activated by insulin as well. Moreover, insulin and estradiol (E2) influence cardiac energy substrates, blood glucose and free fatty acids (FFAs), and both hormones exert cardio-beneficial effects. In view of these facts, we suggest that cross-talk between their signaling pathways might have an important role in regulation of cardiac energy substrate transport. Ovariectomized rats were treated with insulin, estradiol (E2), or their combination 20, 30, or 40 min before analysis of blood glucose and FFA level, as well as cardiac plasma membranes (PM) and low density microsomes (LDM) content of glucose (GLUT4 and GLUT1) and FFA (CD36) transporters. Insulin, given alone, or in combination with E2, decreased plasma glucose level at all time points, but did not influence FFA level, while E2 treatment itself did not change glucose and FFA concentration. Insulin increased PM GLUT4 and GLUT1 content 30 and 40 min after treatment and the increases were partially accompanied by decrease in transporter LDM content. E2 increased PM content and decreased LDM content only of GLUT4 at 30 min. Insulin generally, and E2 at 20 min increased CD36 content in PM fraction. Both hormones decreased CD36 LDM content 20 min after administration. Effect of combined treatment mostly did not differ from single hormone treatment, but occasionally, particularly in distribution of GLUT4, combined treatment emphasized single hormone effect, suggesting that insulin and E2 act synergistically in regulation of energy substrate transporters in cardiac tissue.

Insulin regulation of proliferation involves activation of AKT and ERK 1/2 signaling pathways in vascular smooth muscle cells.

This investigation used primary cultured rat vascular smooth muscle cells (VSMCs) to examine the effect of insulin (INS) on proliferation of VSMCs. In this study, we investigated the role of protein kinase B (Akt) and p42/44 mitogen-activated protein kinase (ERK 1/2) signaling pathways in mediating the mitogenic action of INS in VSMCs. Incubation of rat VSMCs with INS (100 nM) for 10 min resulted in an increase of Akt phosphorylation by 6-fold (p<0.001) and ERK 1/2 phosphorylation by 3-fold (p<0.001). Pretreatment for 15 min with 10 muM of PI3K/Akt inhibitor LY294002 or with 20 muM PD98059, inhibitor of ERK 1/2, significantly reduced INS-stimulated Akt and ERK 1/2 phosphorylation by 76 and 75%, respectively. Prolonged treatment of VSMCs with INS for 24 h did not have an effect on either Akt or ERK1/2 phosphorylation. Incubation of rat VSMCs with INS resulted in an increase of VSMCs proliferation by 87% (p<0.001.) The effect of INS on VSMCs proliferation was significantly reduced by 68% by pretreatment with LY294002 (p>0.01) and by 71% (p>0.01) by pretreatment with PD98059. These results indicate that INS acts through Akt and ERK 1/2 signaling pathways to up-regulate proliferation of VSMC's.

Sodium nitroprusside regulates the relaxation of the longitudinal muscle in the gut.

Nitric oxide (NO) has been shown to mediate nonadrenergic-noncholinergic relaxation in gastrointestinal (GI) smooth muscle cells. As GI smooth muscles relaxations are partly dependent on NO, we decided to investigate the effect of sodium nitroprusside (SNP) on the longitudinal muscle contraction of the isolated guinea pig ileum. Increasing concentrations of SNP (10(-10)M, 10(-9)M, 10(-8)M, 10(-7)M, 10(-6)M and 10(-5)M) reduced ileum contractions stimulated by electrical stimulation (ES) (8-76%; p < 0.05) and by acetylcholine (Ach) (23-62%; p < 0.05) significantly and in a concentration-dependent manner. Furthermore, treatment with an inhibitor of the soluble guanylate cyclase, methylene blue (10 mM), antagonized significantly the relaxing effect of SNP (0-39%; p < 0.05, p < 0.01, p < 0.001 for ES- and 4-27%; p < 0.05 for Ach-induced contractions). The results show that treatment with 1 microM manganese-containing superoxide dismutase (MnSOD) and 10 microM L-arginine (L-arg) caused a significant decrease in SNP induced relaxations (6-55%; p < 0.05, p < 0.001 and 2-46%; p < 0.05, p < 0.01 for ES- and 15-28%; p < 0.05, p < 0.01, p < 0.001 and 12-32%; p < 0.05, p < 0.01 for Ach-induced contractions, respectively). In conclusion, our data suggest that SNP, which releases NO, is able to depress longitudinal muscle contraction of the isolated guinea pig ileum, suggesting that exogenous application of NO inhibits intestinal contractions of smooth muscle cells and that cGMP mediates the response to NO. In addition, MnSOD and L-arg decreased the relaxing effect of SNP on the isolated ileum of the guinea pig.

Insulin signaling in the liver and uterus of ovariectomized rats treated with estradiol.

We used rat hepatic and uterine tissues to examine the impact of estradiol (E2) on insulin (INS) signaling. Ovariectomized (OVX) female Wistar rats were treated with E2 (20 microg/kg b.wt., i.p.) and used for the experiment 6h after E2 administration. To highlight E2 effects on tyrosine phosphorylation of INS receptor (IR) and INS receptor substrates (IRSs) and IRSs association with p85 subunit of phosphatidylinositol 3-kinase (PI3-K) in the context of INS signaling, E2-treated OVX rats were also injected with INS (20 IU, i.p.), 30 min before the experiment. Treatment with E2 did not change the levels of plasma INS and glucose (Glu). However, it significantly decreased the free fatty acid (FFA) level and increased uterine weight. Furthermore, the results show that E2 had no effect on the content of hepatic IR protein, but significantly increased IR protein content in the uterus and decreased IR tyrosine phosphorylation in both the liver and uterus. Compared to the control, hepatic IRS-1 and IRS-2 were significantly decreased and increased, respectively, after E2 treatment. Protein content of both molecules, IRS-1 and IRS-2, was increased in uterine tissue after E2 administration. Protein content of the p85 subunit of PI3-K and that of protein kinase B (Akt) were increased in the uterus, with no changes in the liver. The results suggest that E2 treatment induces tissue-specific changes in INS signaling. The consequences of E2 treatment on INS signaling molecules are more apparent in the uterus, but their physiological relevance for INS action is probably greater in the liver.

Comparative analysis of tryptophan oxygenase activity and glucocorticoid receptor under the influence of insulin.

This investigation addresses the interaction of insulin (INS) and glucocorticoid (GC) signaling in the hepatic regulation of tryptophan oxygenase (TO) enzyme activity in the rat. Male Wistar rats (200-250 g b.w) received an injection of the different doses of INS (10, 25, 50, 70 and 100 microg/200 g b.w., i.p.) and were used for experiments 3 h and 18 h after INS administration. This study shows that maximum of TO activity was found at dose of 50 microg of INS with peak increases observed at 3 h and 18 h after injection of INS, while INS had no effect on TO activity in adrenalectomized rats. The analysis of INS effects on glucocorticoid receptor-complex (GC/GR complex) stability shows that complexes from INS-treated rats are less stable than those from control animals. In addition, INS-stimulated stability of glucocorticoid receptor (GR) protein was significantly increased from the controls. Furthermore, the results show that GC/GR complexes from INS-treated rats could be activated and accumulated at higher rate in cell nuclei of control animals. These data support the involvement of INS in modulation of GC signaling pathway which mediates, in part, the activity of TO.

Effects of gamma-radiation on cell growth, cycle arrest, death, and superoxide dismutase expression by DU 145 human prostate cancer cells.

Gamma-irradiation (gamma-IR) is extensively used in the treatment of hormone-resistant prostate carcinoma. The objective of the present study was to investigate the effects of 60Co gamma-IR on the growth, cell cycle arrest and cell death of the human prostate cancer cell line DU 145. The viability of DU 145 cells was measured by the Trypan blue exclusion assay and the 3(4,5-dimethylthiazol-2-yl)-2,5,diphenyltetrazolium bromide test. Bromodeoxyuridine incorporation was used for the determination of cell proliferation. Cell cycle arrest and cell death were analyzed by flow cytometry. Superoxide dismutase (SOD), specifically CuZnSOD and MnSOD protein expression, after 10 Gy gamma-IR, was determined by Western immunoblotting analysis. Gamma-IR treatment had a significant (P < 0.001) antiproliferative and cytotoxic effect on DU 145 cells. Both effects were time and dose dependent. Also, the dose of gamma-IR which inhibited DNA synthesis and cell proliferation by 50% was 9.7 Gy. Furthermore, gamma-IR induced cell cycle arrest in the G2/M phase and the percentage of cells in the G2/M phase was increased from 15% (control) to 49% (IR cells), with a nonsignificant induction of apoptosis. Treatment with 10 Gy gamma-IR for 24, 48, and 72 h stimulated CuZnSOD and MnSOD protein expression in a time-dependent manner, approximately by 3- to 3.5-fold. These data suggest that CuZnSOD and MnSOD enzymes may play an important role in the gamma-IR-induced changes in DU 145 cell growth, cell cycle arrest and cell death.

Effects of gamma-radiation on cell growth, cycle arrest, death, and superoxide dismutase expression by DU 145 human prostate cancer cells

Gamma-irradiation (gamma-IR) is extensively used in the treatment of hormone-resistant prostate carcinoma. The objective of the present study was to investigate the effects of 60Co gamma-IR on the growth, cell cycle arrest and cell death of the human prostate cancer cell line DU 145. The viability of DU 145 cells was measured by the Trypan blue exclusion assay and the 3(4,5-dimethylthiazol-2-yl)-2,5,diphenyltetrazolium bromide test. Bromodeoxyuridine incorporation was used for the determination of cell proliferation. Cell cycle arrest and cell death were analyzed by flow cytometry. Superoxide dismutase (SOD), specifically CuZnSOD and MnSOD protein expression, after 10 Gy gamma-IR, was determined by Western immunoblotting analysis. gamma-IR treatment had a significant (P < 0.001) antiproliferative and cytotoxic effect on DU 145 cells. Both effects were time and dose dependent. Also, the dose of gamma-IR which inhibited DNA synthesis and cell proliferation by 50 percent was 9.7 Gy. Furthermore, gamma-IR induced cell cycle arrest in the G2/M phase and the percentage of cells in the G2/M phase was increased from 15 percent (control) to 49 percent (IR cells), with a nonsignificant induction of apoptosis. Treatment with 10 Gy gamma-IR for 24, 48, and 72 h stimulated CuZnSOD and MnSOD protein expression in a time-dependent manner, approximately by 3- to 3.5-fold. These data suggest that CuZnSOD and MnSOD enzymes may play an important role in the gamma-IR-induced changes in DU 145 cell growth, cell cycle arrest and cell death.