Mitochondrial active and relaxed state respiration after heat shock mRNA response in the heart.

Induction of Heat Shock Proteins results in cytoprotection. Beneficial effect results from transcription and translational cellular components' involvement that defends metabolism and thus induce ischemic protection of the tissue. Mitochondrial respiration is also involved in stress- induced conditions. It is not a uniform process. Cytochrome c Oxidase (CytOx) representing complex IV of the Electron Transfer Chain (ETC) has a regulatory role for mitochondrial respiratory activity, which is tested in our study after hsp induction. Moreover, protein translation for mitochondrial components was probed by the detection of MT-CO1 for Subunit 1 of CytOx neosynthesis. Wistar rats were subjected to whole-body hyperthermia at 42.0-42.5 °C for 15 min followed by a normothermic recovery period. Heat shock response was monitored time dependent from LV biopsies of all control and heat treated animals with PCR-analysis for hsp 32, 60, 70.1, 70.2, 90 and MT-CO1 expression at 15, 30, 45, 60, 120 and 360 min recovery (n = 5 in each group), respectively. Enzymatic activity of CytOx were evaluated polarographically. High energy phosphates were detected by chromatographic analysis. The mRNA expression of MT-CO1 peaked at 60 min and was accompanied by hsp 32 (r = 0.457; p = 0.037) and hsp 70.2 (r = 0.615; p = 0.003) upregulation. With hsp induction, mitochondrial respiration was increased initially. Enzymatic activity reconciled from active into relaxed status wherein CytOx activity was completely inhibited by ATP. Myocardial ATP content increased from stress induced point i.e. < 1 µmol g-1 protein w/w to finally 1.5 ±â€¯0.53 µmol g-1 protein w/w at 120 min recovery interval. Hyperthermic, myocardial hsp- induction goes along with increased CytOx activity representing an increased "active" mitochondrial respiration. In parallel, de -novo holoenzyme assembly of CytOx begins as shown by MT-CO1 upregulation at 60 min recovery time crossing with a final return to the physiological "relaxed" state and ATP -inhibited respiration.

Induction of haem oxygenase-1 increases infection of dog macrophages by L. infantum.

We aimed to induce and inhibit HO-1, ascertaining its effect on infection rate, parasite load and the levels of superoxide, reactive oxygen species (ROS), nitric oxide (NO), TNF-alpha and IL-10 in cultured macrophages from healthy dogs infected by Leishmania infantum. Macrophages obtained from 15 healthy dogs were cultured alone or infected with L. infantum, with or without association of HO-1 inducer and inhibitor. The infection rate and the parasite load were determined by the number of infected macrophages and number of promastigotes per macrophage, respectively. HO-1 levels and gene expression, as well as IL-10 and TNF-alpha levels were also measured in these cultures. Superoxide, ROS and NO levels in macrophages were measured through flow cytometry. Induction of HO-1 increased the infection rate and parasite load, while its inhibition decreased the infection rate and IL-10 production. There was a positive correlation between HO-1 and infection rate or parasite load. Increased infection rate was associated with decreased superoxide, ROS and NO levels. Induction of HO-1 metabolism in dogs infected by L. infantum is possibly one of the mechanisms responsible for increasing the infection of macrophages, mainly through reduction in the oxidative and nitrosative metabolisms of these cells.

Expression of a stress-inducible heme oxygenase-1 in NK cells is maintained in the process of human aging.

Introduction: Heme oxygenase-1 (HO-1) is a stress-inducible heat shock protein (HSP32) that exerts cytoprotective effects against oxidative stress and inflammation, and is involved in the maintenance of cellular homeostasis. This study aimed to evaluate the expression of HO-1 in natural killer (NK) cells from individuals of different age groups after stimulation with various factors, and to analyze the relationships between the concentration of this cytoprotective protein and parameters corresponding to oxidative stress and inflammation, that is, NOD-like receptor protein 3 (NLRP3), glutathione (GSH), GSH disulfide (GSSG), and interleukin 6 (IL-6). Methods: The study population comprised three age groups: young adults (age range, 19-23 years), older adults aged under 85 years (age range, 73-84 years), and older adults aged over 85 years (age range, 85-92 years). NLRP3, GSH, and GSSG concentrations were measured in serum, whereas the HO-1 concentration and IL-6 expression were studied in NK cells cultivated for 48 h and stimulated with IL-2, lipopolysaccharide (LPS), or phorbol 12-myristate 13-acetate (PMA) with ionomycin. Results: The analysis of serum NLRP3, GSH, and GSSG concentrations revealed no statistically significant differences among the studied age groups. However, some typical trends of aging were observed, such as a decrease in GSH concentration and an increase in both GSSG level, and GSSG/GSH ratio. The highest basal expression of IL-6 and lowest basal content of HO-1 were found in NK cells of adults over 85 years of age. The NK cells in this age group also showed the highest sensitivity to stimulation with the applied factors. Moreover, statistically significant negative correlations were observed between HO-1 and IL-6 expression levels in the studied NK cells. Conclusions: These results showed that NK cells can express HO-1 at a basal level, which was significantly increased in activated cells, even in the oldest group of adults. The reciprocal relationship between HO-1 and IL-6 expression suggests a negative feedback loop between these parameters.

Sex differences in hearing impairment due to diet-induced obesity in CBA/Ca mice.

BACKGROUND: Obesity is an independent risk factor for hearing loss. Although attention has focused on major obesity comorbidities such as cardiovascular disease, stroke, and type 2 diabetes, the impact of obesity on sensorineural organs, including the auditory system, is unclear. Using a high-fat diet (HFD)-induced obese mouse model, we investigated the impact of diet-induced obesity on sexual dimorphism in metabolic alterations and hearing sensitivity. METHODS: Male and female CBA/Ca mice were randomly assigned to three diet groups and fed, from weaning (at 28 days) to 14 weeks of age, a sucrose-matched control diet (10 kcal% fat content diet), or one of two HFDs (45 or 60 kcal% fat content diets). Auditory sensitivity was evaluated based on the auditory brainstem response (ABR), distortion product otoacoustic emission (DPOAE), and ABR wave 1 amplitude at 14 weeks of age, followed by biochemical analyses. RESULTS: We found significant sexual dimorphism in HFD-induced metabolic alterations and obesity-related hearing loss. Male mice exhibited greater weight gain, hyperglycemia, increased ABR thresholds at low frequencies, elevated DPOAE, and lower ABR wave 1 amplitude compared to female mice. The hair cell (HC) ribbon synapse (CtBP2) puncta showed significant sex differences. The serum concentration of adiponectin, an otoprotective adipokine, was significantly higher in female than in male mice; cochlear adiponectin levels were elevated by HFD in female but not male mice. Adiponectin receptor 1 (AdipoR1) was widely expressed in the inner ear, and cochlear AdipoR1 protein levels were increased by HFD, in female but not male mice. Stress granules (G3BP1) were significantly induced by the HFD in both sexes; conversely, inflammatory (IL-1ß) responses were observed only in the male liver and cochlea, consistent with phenotype HFD-induced obesity. CONCLUSIONS: Female mice are more resistant to the negative effects of an HFD on body weight, metabolism, and hearing. Females showed increased peripheral and intra-cochlear adiponectin and AdipoR1 levels, and HC ribbon synapses. These changes may mediate resistance to HFD-induced hearing loss seen in female mice.

Subcutaneous transplantation of engineered islet/adipose-derived mesenchymal stem cell sheets in diabetic pigs with total pancreatectomy.

INTRODUCTION: Intraportal islet transplantation is a promising therapeutic approach for patients with type 1 diabetes mellitus (T1DM). However, despite being minimally invasive, the method has some limitations, such as short-term graft loss, portal venous thrombosis, and difficulty in collecting adequate amounts of islets. Subcutaneous islet transplantation on adipose-derived mesenchymal stem cell (ADSC) sheets has been suggested to overcome these limitations, and in this study, we have examined its feasibility in T1DM pigs. METHODS: Inguinal subcutaneous fat was harvested from young pigs and then isolated and cultured adequate ADSCs to prepare sheets. Islets were isolated from the pancreases of mature pigs and seeded on the ADSC sheets. T1DM pigs were generated by total pancreatectomy, and ADSC sheets with transplanted islets were administered subcutaneously to the waist (n = 2). The effects of the islets on the ADSC sheets and on blood glucose levels were evaluated. Insulin secretion was measured by insulin stimulation index. RESULTS: Islet viability was higher on ADSCs compared to islets alone (91.8 ± 4.3 vs. 81.7 ± 4.1%). The insulin stimulation index revealed higher glucose sensitivity of islets on ADSC sheets compared to islets alone (2.8 ± 2.0 vs. 0.8 ± 0.3). After transplantation, the blood glucose levels of two pigs were within the normal range, and sensitive insulin secretion was confirmed by intravenous glucose tolerance tests. After graftectomy, decreased insulin secretion and hyperglycemia were observed. CONCLUSIONS: Subcutaneous islet transplantation using ADSC sheets can regulate the blood glucose levels of T1DM pigs.

HO-1 dependent antioxidant effects of ethyl acetate fraction from Physalis alkekengi fruit ameliorates scopolamine-induced cognitive impairments.

Physalis alkekengi var. francheti is an indigenous herb well known for its anti-inflammatory, sedative, antipyretic, and expectorant properties. However, the information regarding the impacts of P. alkekengi fruits (PAF) in modulation of oxidative stress and learning memory are still unknown. This study therefore evaluated the antioxidant properties of ethyl acetate (EA) fraction of PAF and its impacts on learning and memory. The antioxidant activities of PAF were evaluated in LPS-induced BV2 microglial cells. The potent EA fraction then investigated and confirmed for its involvement of HO-1 pathway using hemin (HO-1 inducer) and ZnPP (HO-1 inhibitor) through Western blotting, DCFH-DA, and/or Griess assay. The involvements of PI3K/Akt, MEK, and p38 MAPK also investigated. Furthermore, we applied EA fraction to the animals at 100 and 200 mg/kg doses to check if the extract could improve scopolamine-induced memory deficits in passive avoidance and elevated plus maze tests. Our results demonstrated that the fractions from PAF significantly inhibited the generation of intracellular reactive oxygen species (ROS) induced by LPS in concentration-dependent manners. In comparison to other fractions, the EA fraction exhibited potent effect in suppressing intracellular ROS generation. Besides, EA fraction also induced the expression of HO-1 in time- and concentration-dependent manners. ZnPP significantly reversed the suppressive effect of EA fraction on LPS-induced ROS generation and NO production, which confirm the involvement of HO-1 signaling in EA-fraction-mediated antioxidant activities. Consistently, blocking of PI3K/Akt, MEK, and p38 MAPK pathways by PAF-EA suppressed the production of intracellular ROS, indicating their potential participation. In addition, one of the major constituents of EA fraction, luteolin-7-O-ß-D-glucoside, also demonstrated HO-1-dependent antioxidant effects in BV2 cells. Further, the EA fraction significantly (p < 0.05) improves scopolamine-induced memory deficits in mice. Taken together, our findings highlight the antioxidant effects of EA fraction of PAF which may be beneficial in treatment of different neurodegenerative diseases associated with free radicals.

The effects of increased heme oxygenase-1 on the lymphoproliferative response in dogs with visceral leishmaniasis.

Canine visceral leishmaniasis (CVL) is known to affect the cellular immunity of infected dogs, through impairing lymphoproliferation and microbicidal mechanisms. This study examined heme oxygenase-1 (HO-1) and its metabolites, oxidative stress and IL-10 levels in CVL and investigated correlations between these parameters. Additionally, the effects of HO-1 inhibition on the lymphoproliferative response and cytokine production in lymph node cells (LNCs) from infected dogs were evaluated. Forty-four dogs, 24 controls and 20 dogs with CVL were selected. Plasma and splenic levels of HO-1, haptoglobin, soluble CD163 receptor, ferritin and IL-10 were determined using capture ELISA. The HO-1 levels and relative gene expression in peripheral blood and bone marrow mononuclear cells were also determined. LNCs proliferation was evaluated with an HO-1 activator and with an HO-1 inhibitor, in the presence of the Leishmania infantum soluble antigen (SAgL), using flow cytometry. HO-1, IL-2, IFN-gamma and IL-10 were also determined in these cultures using capture ELISA. Infected dogs presented oxidative stress and increased HO-1 levels and relative gene expression, with correlation between oxidative stress and HO-1. The substances from heme metabolism and IL-10 were also elevated in the plasma and spleens of infected dogs. IL-10 and HO-1 levels were positively correlated with one another. Inhibition of HO-1 increased LNCs proliferation and decreased IL-10 and IL-2 production in the presence of SAgL. The increased HO-1 metabolism observed in CVL is probably associated with oxidative stress and increased IL-10, which could be one of the mechanisms responsible for inhibition of the lymphoproliferative response in sick dogs.

Nucleolin protects the heart from ischaemia-reperfusion injury by up-regulating heat shock protein 32.

AIMS: Nucleolin plays important roles in a variety of cellular processes. In this study, we aimed to investigate the role of nucleolin in cardiac ischaemia-reperfusion (I-R) injury. METHODS AND RESULTS: We investigated the expression pattern of nucleolin in hearts subjected to I-R, or neonatal rat cardiomyocytes subjected to hypoxia-re-oxygenation. We found that nucleolin expression was significantly down-regulated and the cleaved protein was present, both in vivo and in vitro. Gene transfection and RNA interference approaches were employed in cardiomyocytes to investigate the function of nucleolin. Over-expression of nucleolin was cytoprotective, whereas nucleolin ablation enhanced both hypoxia- and H2O2-induced cardiomyocyte death. Furthermore, transgenic mice with cardiac-specific over-expression of nucleolin were resistant to I-R injury as indicated by decreased cellular necrosis and decreased infarct size. The cardio-protective roles of nucleolin in cardiomyocytes, are attributable to the interaction of nucleolin with the mRNA of heat shock protein 32 (Hsp32), resulting in an increase of Hsp32 mRNA stability, and subsequent up-regulation of Hsp32 expression. The selective Hsp32 inhibitor, zinc protoporphyrin-IX, abrograted the cardiac protection mediated by nucleolin. CONCLUSION: This study has demonstrated that nucleolin is involved in the regulation of I-R-induced cardiac injury and dysfunction via the regulation of Hsp32, and may be a novel therapeutic target for ischaemic heart diseases.