Mitochondrial Common Deletion Level in Adipose Tissue Is Not Associated with Obesity but Is Associated with a Structural Change in Triglycerides as Revealed by FTIR Spectroscopy.

OBJECTIVE: Several studies have shown that mitochondrial metabolism may be disrupted if the rate of the specific 4,977 bp deletion of mitochondrial DNA (mtDNA) reaches a threshold. This study aimed to investigate the possible associations between the mtDNA4977 deletion load and obesity-related metabolic abnormalities in the adipose tissue. METHODS: The study included thirty obese individuals, who underwent bariatric surgery, and twelve control subjects. mtDNA4977 deletion, adenine nucleotides, and lactate levels, which show the bioenergetic status were evaluated in visceral adipose tissues. Fourier transform infrared (FTIR) spectroscopy was used to investigate the structural variations and composition of adipose tissues in the context of deletion load. RESULTS: There were no differences between the two groups in terms of mtDNA4977 deletion, adenine nucleotides, and lactate levels. The FTIR spectra indicated a few obesity-related alterations in adipose tissues that were not related to the mtDNA deletion load. Also, statistical analysis showed a correlation between the deletion load and a band shift of 1,744 cm-1, which assigns C = O stretching of the carbonyl group of the ester group in triglycerides and other esterified fatty acids, although it is not associated with obesity. CONCLUSIONS: Our data suggest that the mtDNA4977 deletion in visceral adipose tissues of obese individuals do not have a significant impact on the bioenergetic status. However, the increased accumulation of deletion may be associated with a specific change in the ester bond, indicating structural differences in the lipids. These findings shed light on our understanding of the tissue-specific distribution of mtDNA deletions and obesity-related adipose tissue pathogeneses.

Evaluation of the in vivo wound healing potential of the lipid fraction from activated platelet-rich plasma.

Previous in vitro studies suggest a direct relevance for the peptide-free lipid fraction (LF) of platelet-rich plasma (PRP) in biological mechanisms related to wound healing. However, there are no scientific reports to date on the wound healing activities of this lipid component in vivo. Thus, the present study provides a scientific evaluation for the wound healing potential of the lipid portion of the activated PRP. For the wound healing activity assessment, in vivo full-thickness excisional wounds were created on the dorsal skin of Sprague-Dawley rats. Lipid extract from pooled PRP was applied topically to the wounds on 0, 3, and 7 days after injury. Histological assessment of epidermal and dermal regeneration, granulation tissue thickness and angiogenesis by Sirius red and Masson's trichrome staining, in addition to immunohistochemical staining for transforming growth factor beta-1 (TGF-ß1), collagen type I (COL I), and collagen type III (COL III) were performed on skin biopsies at 3, 7 and 14 days. The total histological scores of the LF group were significantly higher than the 25% dimethylsulfoxide-control group. According to the immunohistochemical staining, the observed expression changes for TGF-ß1, COL I and III at 3, 7, and 14 days after wounding were significantly better in the study group than the control group. Furthermore, COL I/III ratio in the lipid extract-treated group at day 14 was much higher than that of the control group. Meanwhile, analysis of the data also indicated that the LF has less positive effects on all evaluated parameters than PRP. From the present data, it could be concluded that the peptide-free LF of PRP has potent wound healing capacity in vivo for cutaneous wounds, although not as much as that of PRP. Strengthening our understanding of the wound healing potential of lipid components of PRP and platelet-derived lipid factors may provide new avenues for improving the healing process of a wound with elevated protease activity.

Anesthesia may alter mRNA expression of certain wound healing-associated genes in dermal wound environment of the rats.

Some anesthetics including ketamine/xylazine and thiopental have been shown to alter the expression of genes related with inflammatory cytokines and chemokines in previous studies unassociated with wound healing, arising the question of whether commonly used anesthetics in wound healing models could interfere with the transcriptional responses of the genes associated with skin wound healing. The gene expression profile in wound biopsies of rats who received widely used anesthetics doses of intraperitoneal ketamine/xylazine (50 mg/kg and 10 mg/kg) or thiopental (50 mg/kg) in comparison with control rats was analyzed by monitoring the expression of genes effective on various phases of wound healing. The expression levels of 84 genes were determined on 3rd, 7th and 14th days of post-wounding using a qPCR array system. Of the genes either up or downregulated fivefolds or more, three (Egf, Col5a1 and Cxcl3) and two (Tgfa and Il2) genes were found to be the most responsive ones to ketamine/xylazine or thiopental anesthesia respectively in a period of 14 days after correction for multiple testing. However, up to 22 and 24 genes for ketamine/xylazine and thiopental were found to be differentially expressed in the same period without correction for multiple-comparisons testing (p < 0.05). In conclusion, our data suggest that ketamine/xylazine and thiopental may alter the transcriptional responses of some genes associated with wound healing in rats. We strongly suggest to consider the possible alteration effect of these anesthetics on gene expression in animal models of dermal wound healing.

Evidence that Extreme Dilutions of Paclitaxel and Docetaxel Alter Gene Expression of In Vitro Breast Cancer Cells.

BACKGROUND: Gene expression analysis of cells treated with extreme dilutions or micro amounts of drugs has been used to provide useful suggestions about biological responses. However, most of the previous studies were performed on medicines being prepared from a variety of herbal and metal sources. This study investigated the effects of ultramolecular dilution of the taxane anti-cancer drugs, which are not commonly used in homeopathic medicines, on mRNA expression profiles of five key genes (p53, p21, COX-2, TUBB2A and TUBB3) in the breast cancer cell line MCF-7. METHOD: MCF-7 cells were exposed to paclitaxel (Taxol) or docetaxel (Taxotere) preparations (6X, 5C and 15C dilutions prepared from pharmacological concentration of 25 nmol/L) for 72 hours. The cell culture groups were evaluated with the trypan blue dye exclusion method for the proliferation/cytotoxicity rates, immuno-staining ß-tubulin for microtubule organization, and reverse transcription polymerase chain reaction for gene expression levels.Fold-change in gene expression was determined by the ΔΔCt method. RESULTS: The administration of diluted preparations had little or no cytotoxic effect on MCF-7 cells, but altered the expression of genes analyzed with a complex effect. According to the ΔΔCt method with a five-fold expression difference (p < 0.05) as a cut-off level, ultra-high dilutions of paclitaxel and docetaxel showed differential effects on the studied genes with a concentration-independent activity. Furthermore, the dilutions disrupted the microtubule structure of MCF-7 cells, suggesting that they retain their biological activity. CONCLUSION: Despite some limitations, our findings demonstrate that gene expression alterations also occur with ultra-high dilutions of taxane drugs.

Investigation of preconditioning and the protective effects of nicotinamide against cerebral ischemia-reperfusion injury in rats.

This study investigated the antioxidant and neuroprotective effects of nicotinamide combined with ischemic preconditioning against cerebral ischemia reperfusion (CIR) injury. Thirty-five Wistar albino male rats were randomly divided into five groups: sham, preconditioned ischemia/reperfusion (IP+IR), ischemia/reperfusion (IR), preconditioned ischemia/reperfusion + nicotinamide (IP+IR+N), and ischemia/reperfusion + nicotinamide (IR+N). CIR was achieved with bilateral common carotid artery occlusion. IP+IR and IP+IR+N groups 30 min before ischemia; Three cycles of 10 sec ischemia/30 sec reperfusion followed by 20 min IR were applied. The IP+IR+N and IR+N groups received 500 mg/kg nicotinamide intraperitoneally. After 24 h of reperfusion, a neurological evaluation was performed and vertical pole test. Biochemically, malondialdehyde (MDA), glutathione (GSH) levels and catalase (CAT) activity were measured in blood and brain tissue samples. Rates of red neurons, sateliosis and spongiosis were determined histopathologically in the prefrontal cortex areas. After CIR, MDA levels increased significantly in serum and brain tissue in the IR group compared to the sham group, while GSH and CAT activity decreased in the brain tissue (p < 0.05). MDA levels in the tissues were found significantly decreased in the IR+N group compared to the IR group (p < 0.05). Administration of nicotinamide together with IP significantly decreased MDA levels in brain tissue and increased GSH and CAT activity (p < 0.05). Compared to the IR group, the morphological and neurological damage in the prefrontal cortex areas decreased in the IP+IR, IP+IR+N, and IR+N groups (p < 0.05). In addition, red neuron, sateliosis and spongiosis rates increased significantly in the IR group compared to the Sham, IP+IR+N, IR+N groups (p < 0.001 for all). In neurological evaluation, while the neurological score increased and the time on the vertical pole decreased significantly in the IR group, preconditioning, and nicotinamide groups reversed (p < 0.05). The study's results show that nicotinamide administration with ischemic preconditioning alleviates cerebral ischemia/reperfusion injury.

Are elevated mitochondrial DNA fragments in prostatic inflammation a potential biomarker for prostate cancer?

BACKGROUND: We sought to determine whether two soluble forms with different size of mtDNA are linked to prostatic inflammation, and whether they discriminate prostate cancer (PCa) from inflammatory prostatic conditions. METHODS: Histopathologically diagnosed prostatitis, PCa and benign prostatic hyperplasia patients (n = 93) were enrolled in this study and they were categorized as with and without prostate inflammation. Quantitative RT-PCR was used to analyze the levels of 79-bp and 230-bp fragments in urine and blood samples collected following prostate massage. RESULTS: The urine mtDNA-79 and mtDNA-230 were significantly increased in patients with prostate inflammation compared with those in without inflammation. Here, 79-bp fragment of apoptotic origin was significantly higher level than 230-bp fragment of necrotic origin. Although mtDNA-79 copy number in serum samples was also increased in patients with prostate inflammation, mtDNA-230 was similar in the two groups. Furthermore, mtDNA-79 and mtDNA-230 copy numbers in postprostate massage urine were higher (about 16-fold and 22-fold, respectively) than those from serum samples. ROC analysis showed that, although post-prostate massage urine have relatively higher performance than blood, ability to discriminate cases of both fragments was not better than that of serum total PSA. CONCLUSIONS: Our results demonstrate that shorter cf-mtDNA fragment size in particular, increase in the presence of prostate inflammation in post-prostatic massage urine but both fragments could never improve serum total PSA performance.

Erythroid microRNA and oxidant status alterations in l-thyroxine-induced hyperthyroid rats: effects of selenium supplementation.

BACKGROUND: Hypermetabolic state in hyperthyroidism causes oxidative stress. Erythrocytes are the cells that are involved in oxidant equilibrium in an organism and contain microRNA (miRNA). Selenium, which is an essential element for an organism, has antioxidant properties. The present study was aimed at investigating the effects of selenium supplementation in hyperthyroidism, on pro- and antioxidant enzymes, and miRNA (miR-144 and miR-451) expressions in the erythrocytes. METHODS: Forty-eight Sprague-Dawley male rats were divided into 6 groups; control group, group fed with 0.5 mg/kg sodium selenite; group fed with 1 mg/kg sodium selenite; hyperthyroid group; hyperthyroid group fed with 0.5 mg/kg sodium selenite; and hyperthyroid group fed with 1 mg/kg sodium selenite. Malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), miR-144, and miR-451 expression levels were studied in erythrocyte hemolysates. RESULTS: MDA levels were increased in the hyperthyroid group compared to the control group, and the group fed with 0.5 mg/kg sodium selenite (P<0.001 and P<0.01, respectively). GSH levels were increased in the hyperthyroid group and the hyperthyroid group fed with 0.5 mg/kg sodium selenite compared to the control group (P<0.001, and P<0.05, respectively). GSH levels of the hyperthyroid group fed with 1 mg/kg sodium selenite were decreased when compared with the hyperthyroid group (P<0.05). SOD levels of the hyperthyroid group were increased when compared with the control group (P<0.05, and P<0.001, respectively). Similarly, SOD levels of the hyperthyroid group fed with 1 mg/kg sodium selenite were lower than the hyperthyroid group (P<0.01). miR-144 values were increased in the hyperthyroid group and the hyperthyroid group fed with 0.5 mg/kg sodium selenite compared to the control group (P<0.001, and P<0.05 respectively). miR-451 expression was increased significantly in the hyperthyroid group compared to the control group (P<0.05). CONCLUSIONS: Our findings showed that MDA, SOD and GSH levels increased, and miR-144 and miR-451 expressions changed in hyperthyroidism. Supplementation of 1 mg/kg sodium selenite was more effective than 0.5 mg/kg sodium selenite for normalizing the MDA, GSH, SOD, and miRNA levels in the hyperthyroid group.

Apelin, Omentin-1, and Vaspin in patients with essential hypertension: association of adipokines with trace elements, inflammatory cytokines, and oxidative damage markers.

BACKGROUND: Hypertension (HT) is a disease associated with endothelial dysfunction which is related to some adipokines and pro- and anti-inflammatory cytokines. AIMS: Our aim was to investigate roles of apelin, omentin-1, and vaspin in essential HT and to evaluate their relationships with other pro- and anti-inflammatory cytokines, trace elements, and oxidative stress. We also investigated these parameters to determine asymptomatic target organ damage period and grading essential hypertension. METHODS: One hundred fifty-three patients diagnosed with essential hypertension and 45 healthy controls were included in the study. Hypertension was defined as a systolic blood pressure > 140 mmHg and/or a diastolic blood pressure > 90 mm Hg or current use of an antihypertensive medication. The patients who had secondary HT, other chronic metabolic, cardiovascular, cerebrovascular diseases were excluded. History and physical exam including detailed cardiovascular examination were performed in all participants. Adipokines, cytokines, trace elements, lipid peroxidation, and ischemia-modified albumin levels were measured in blood samples by biochemical methods. RESULTS: Vaspin, IL-4, IL-8, IL-10, selenium, and zinc levels were significantly lower in the HT group compared to healthy controls while omentin-1, TNF-α, copper, iron, MDA, SOD, and IMA-C levels were significantly higher in HT patients compared to controls. Multiple ordinal regression revealed that TNF-α, IL-10, and body mass index of patients were statistically significant independent predictors (P = 0.024, P = 0.019, and P = 0.032, respectively) for grading of HT. IL-4 and IL-10 were significantly higher in patients with asymptomatic target organ damage, compared to patients without asymptomatic target organ damage (P = 0.032 and P = 0.015, respectively). Our findings suggest that adipokines apelin, omentin, and vaspin may be involved in hypertension by a complex interaction with the anti- and pro-inflammatory cytokines, trace elements, and oxidative stress pathways.

Platelet-rich plasma and platelet-derived lipid factors induce different and similar gene expression responses for selected genes related to wound healing in rat dermal wound environment.

Although platelet-rich plasma (PRP) is the plasma fraction that contains higher levels of platelet-sequestered proteins such as growth factors and chemokines, it is also abundant in bioactive lipids whose role in wound healing has not been well characterized. This study provides a preliminary evaluation for the effect of the lipid component of PRP on selected genes related to wound healing. Sprague-Dawley rats were classified into four groups after induction of full thickness excisional wounds: the lipid fraction (LF) (lipid extract from PRP) group, PRP group, dimethyl sulfoxide group, and sham group. Subsequently, relevant groups were topically treated with test preparations. Healing wounds were collected on 3rd, 7th and 14th days, and expression levels of 12 genes were determined using qPCR. LF treatment-induced gene expression signature distinct from that induced by PRP treatment, although there are some overlaps in LF- and PRP-responsive genes. Differentially expressed all eight genes (Cxcl5, Cxc11, Egfr, Tgfb1, IL10, Tgfa, Mmp1, and Mmp7) to LF response were significantly down-regulated at either 3rd, 7th, or 14th days. Also, the comparison between LF- and PRP-treatment groups showed that the LF significantly decreased expression of Cxcl11, Mmp7, and Tgfa mRNA on day 7 of healing. This study revealed that PRP and its LF induced different and similar gene expression responses of the skin during the repair of full thickness excisional wounds. Identifying mRNA response to LF treatment at whole transcriptome level can be beneficial for comprehensive understanding of the role of platelet-derived lipid factors in wound healing processes.

Increased mitochondrial common deletion in platelets from patients with type 2 diabetes is not associated with abnormal platelet activity or mitochondrial function.

The present study examined the presence and frequency of the 4,977­base pair mitochondrial (mt)DNA (mtDNA4977) deletion in blood platelets, and whether increased mtDNA4977 deletion was associated with abnormal mitochondrial and platelet function in type 2 diabetes mellitus. A total of 66 patients with type 2 diabetes mellitus and 23 healthy subjects were included in the present study. Patients were divided into three subgroups according to glycemic control, and the presence or absence of chronic diabetic complications: i) Good glycemic control [glycated hemoglobin (HbA1c) <7] without complications; ii) poor glycemic control (HbA1c ≥7) without complications; and iii) poor glycemic control (HbA1c ≥7) with complications. mtDNA4977 deletion, mtDNA copy number, adenine nucleotides, mitochondrial membrane potential and P­selectin expression levels were analyzed in platelets. Although the frequency of mtDNA4977 deletion in platelets of the patient (96.9%) and control groups (95.6%) was extremely similar, the deletion level significantly increased in all the diabetic groups, compared with the healthy control group. However, the data from the present study revealed that an increased deletion frequency in platelets was not associated with disease severity, although there was clear interindividual variability. Furthermore, all other parameters were not significantly different among the groups, and there were no correlations between mtDNA4977 deletion frequency and all other studied parameters for any of the case groups. The results indicated that the mtDNA4977 deletion occurred in platelets, and increased deletion in patients with type 2 diabetes did not have a marked influence on mitochondrial and/or platelet dysfunction, when compared with the non­diabetic subjects. Further research is required to elucidate the sources of inter­individual variability observed in certain parameters.

Monitoring of platelet function parameters and microRNA expression levels in patients with prostate cancer treated with volumetric modulated arc radiotherapy.

Radiotherapy (RT) may result in platelet activation and thrombosis development. To the best of our knowledge, the potential effect of volumetric-modulated arc therapy (VMAT), a novel radiotherapy technique, on platelet function and microRNA (miRNA/miR) expression has not been previously investigated. The present study aimed to determine the effect of VMAT on the alterations in platelet function parameters and miRNA expression levels. A total of 25 patients with prostate cancer and 25 healthy subjects were included in the present study. Blood samples were collected from the patient group on the day prior to RT (pre-RT), the day RT was completed (post-RT day 0), and 40 days following the end of therapy (post-RT day 40). Platelet count, mean platelet volume (MPV) value, platelet aggregation, plasma P-selectin, thrombospondin-1, platelet factor 4, plasma miR-223 and miR-126 expression levels were measured. A significant decrease in platelet count in the post-RT day 0 group was measured in comparison with the pre-RT and the post-RT day 40 groups. Pre-RT MPV values were higher than those of the post-RT day 0 and the post-RT day 40 groups. No significant differences were observed in the levels of platelet activation markers or miR-223 and miR-126 expression levels between the RT groups. Although RT may result in a reduction in platelet and MPV counts, the results of the present study indicate that platelet activation markers are not affected by VMAT. Therefore, it is possible that no platelet activation occurs during VMAT, owing to the conformal dose distributions, improved target volume coverage and the sparing of normal tissues from undesired radiation.

The Circulating Levels of Selenium, Zinc, Midkine, Some Inflammatory Cytokines, and Angiogenic Factors in Mitral Chordae Tendineae Rupture.

Chordae tendineae rupture process is associated with increased production of inflammatory and angiogenesis mediators in connective tissues, which contributes to chronic inflammation and pathogenesis of degenerative chordae. A few trace elements are known to possess antioxidant, anti-inflammatory, and antiangiogenic properties. Therefore, the aim of this study was to determine whether zinc, selenium, midkine (MK), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-α), vascular endothelial growth factor-A (VEGF-A), platelet-derived growth factor-BB (PDGF-BB), and reduced glutathione (GSH) levels are associated with inflammation and angiogenesis processes in the context of a potential etiology causing aggravation of mitral regurgitation and/or ruptured chordae tendineae. Seventy-one subjects comprising 34 patients with mitral chordae tendineae rupture (MCTR) and 37 healthy controls diagnosed on the basis of their clinical profile and transthoracic echocardiography were included in this study. The levels of GSH, MK, selenium, and zinc were found to be lower in the patients group when compared to control group. There were no significant difference in plasma TNF-α, IL-1ß, IL-6, IL-8, VEGF-A, and PDGF-BB levels between two groups. There were positive significant correlations between MK and GSH, MK, and selenium levels in patients with MCTR. According to our data in which selenium, zinc, MK, and GSH decreased in MCTR patients, inflammatory response, oxidative stress, and trace element levels may contribute to etiopathogenesis of mitral regurgitation and/or ruptured chordae tendineae.