Grzegorz Machnik

Gene expression and activity of antioxidant enzymes in the blood cells of workers who were occupationally exposed to lead

In this study, we sought to understand the influence of occupational lead-exposure on the gene expression (Sod1) and activity (SOD) of superoxide dismutase, catalase and glutathione peroxidase (GPx, Gpx1) in leukocytes and erythrocytes. The study group consisted of 45 healthy male employees of a lead-zinc works and was divided into two subgroups: those with low exposure to lead (LE) and those with high exposure to lead (HE). In addition, 17 healthy male administrative workers participated in the study as the control group. The gene expression levels of both Sod1 and Gpx1 were significantly increased in the LE group as compared to the control group. By contrast, we noted only an insignificant tendency for increased gene expression of both Sod1 and Gpx1 in the HE group. The expression and activity of catalase were unchanged. Nevertheless, SOD and GPx activities in erythrocytes was significantly elevated in both examined subgroups, whereas SOD activity in leukocytes was raised only in the LE group. The results of this study led us to conclude that lead has a significant influence not only on the activities of antioxidant enzymes but also on the dose-dependent expression in their genes.

Metformin affects macrophages' phenotype and improves the activity of glutathione peroxidase, superoxide dismutase, catalase and decreases malondialdehyde concentration in a partially AMPK-independent manner in LPS-stimulated human monocytes/macrophages.

BACKGROUND Diabetic patients experience accelerated atherosclerosis. Metformin is a cornerstone of the current therapy of type 2 diabetes. Macrophages are the key cells associated with the development of atherosclerotic plaques. Therefore, our aim was to assess the in vitro effects of metformin on macrophages and its influence on the mechanisms involved in the development of atherosclerosis. MATERIALS AND METHODS Peripheral blood mononuclear cells were obtained from the group including 16 age-matched healthy non-smoking volunteers aged 18-40 years. Monocytes were further incubated with metformin, LPS and compound C--a pharmacological inhibitor of AMPK. The impact of metformin on oxidative stress markers, antioxidative properties, inflammatory cytokines and phenotypical markers of macrophages was studied. RESULTS We showed that macrophages treated with metformin expressed less reactive oxygen species (ROS), which resulted from increased antioxidative potential. Furthermore, a reduction in inflammatory cytokines was observed. We also observed a phenotypic shift toward the alternative activation of macrophages that was induced by metformin. All the aforementioned results resulted from AMPK activation, but a residual activity of metformin after AMPK blockade was still noticeable even after inhibition of AMPK by compound C. CONCLUSIONS Authors believe that metformin-based therapy, a cornerstone in diabetes therapy, not only improves the prognosis of diabetics by reducing blood glucose but also by reducing oxidative stress, inflammatory cytokine production and the shift toward alternative activation of macrophages.

Transforming growth factor-β1 and its receptors in patients with ulcerative colitis

Transforming growth factor-beta1 (TGF-beta1) plays a role in the pathogenesis of ulcerative colitis (UC) by activating its specific receptors (T beta RI-T beta RIII). We investigated the expression of genes encoding for TGF-beta1 and T beta RI-III using RT-QPCR in patients with active and inactive UC and non-IBD controls. The localization and level of TGF-beta1 protein in intestinal tissue was estimated by immunohistochemistry, and serum TGF-beta1 concentrations were determined using ELISA. We found a significant increase in TGF-beta1 gene expression and increase in the expression of genes encoding receptor T beta RI in patients with active UC when compared with controls. The expression of genes encoding T beta RII was found to be higher in patients with both active and inactive UC when compared to controls. Specific staining for TGF-beta1 in fibroblasts was significantly greater in both active and inactive UC as compared to controls. The serum concentration of TGF-beta1 was significantly higher in patients with active UC when compared with controls as well as in UC patients with left side/total colonic extension when compared with those with disease limited to rectum/rectosigmoid area. However, no correlation between TGF-beta1 serum concentrations and UC activity index was found. Increases in TGF-beta1 gene expression and its protein level, associated with altered TGF-beta1 receptor profile indicate a functional role for TGF-beta1 in intestinal inflammatory/repair processes in UC. Increases in TGF-beta1 serum concentrations correlate with extension of disease.

Effect of N-acetylcysteine administration on the expression and activities of antioxidant enzymes and the malondialdehyde level in the blood of lead-exposed workers

We investigated whether treatment with N-acetylcysteine (NAC) reduces oxidative stress intensity and restores the expression and activities of superoxide dismutase (Sod1, SOD), catalase (Cat, CAT) and glutathione peroxidase (Gpx1, GPx) in lead-exposed workers. The exposed population was divided randomly into two groups. Workers in the first group (reference group, n=49) were not administered any drugs, while workers in the second group (n=122) were treated with NAC at three doses for 12 weeks (200 mg, 400 mg, 800 mg/day). NAC administered orally to lead-exposed workers normalized antioxidant enzyme activities in blood cells. Oxidative stress intensity measured as malondialdehyde (MDA) levels in serum, leukocytes and erythrocytes significantly decreased after NAC administration. NAC may be an alternative therapy for chronic lead intoxication.

AMP‐activated protein kinase is involved in induction of protective autophagy in astrocytes exposed to oxygen‐glucose deprivation

AMP‐activated kinase (AMPK) acts as the intracellular ATP depletion sensor, which detects and limits increases in the AMP/ATP ratio. AMPK may be significantly activated under stress conditions that deplete cellular ATP levels such as ischemia/hypoxia or glucose deprivation. Recent studies strongly suggest that AMPK participates in autophagy regulation, but it is not known whether AMPK activated by ischemia regulates autophagy in astrocytes and the consequence of autophagy activation in ischemic astrocytes are unclear. We have investigated the contribution of AMPK to autophagy activation in rat primary astrocyte cultures subjected to ischemia‐simulating conditions (combined oxygen glucose deprivation, OGD) and its potential effects on astrocyte damage induced by OGD (1–12 h). The evidence supports the conclusion that AMPK activation at early stages of OGD is involved in induction of protective autophagy in astrocytes. Inhibition of AMPK, either by siAMPKα1 or by compound C, significantly attenuated the expression of autophagy‐related proteins and decrease of astrocyte viability following OGD. The findings provide additional data about the role of AMPK in ischemic astrocytes and downstream responses that may be involved in OGD‐induced protective autophagy.

Eplerenone promotes alternative activation in human monocyte-derived macrophages

BACKGROUND In this study, we have analyzed the response of human monocyte-derived macrophages to mineralocorticoid axis modulators. METHODS Human monocyte-derived macrophages were incubated with aldosterone alone, eplerenone alone, and the combination of aldosterone and eplerenone. The analyzed variables were nitric oxide and reactive oxygen species production, and the gene and protein expression of inducible nitric oxide synthase, arginase I, and mannose receptor. RESULTS We showed that aldosterone promotes a classic inflammatory response in macrophages, whereas its antagonist, eplerenone, attenuates aldosterone-induced activity. CONCLUSION Eplerenone did not quantitatively weaken the response of macrophages to aldosterone but instead qualitatively changed their behavior.

SOCS and diabetes—ups and downs of a turbulent relationship

Diabetes mellitus is one of the most emerging diseases threatening the present world. Thus, intensive investigations are carried out to better understand the mechanisms occurring in type 1 (T1D) and 2 (T2D) diabetes, and to elaborate more potent methods to fight the disease. In this aspect, the suppressors of cytokine signalling (SOCS) are one of the most studied factors of recent years. SOCS proteins have been discovered as cytokine pathway inhibitors; however, presently, their influence seems wider. Most of the known SOCS proteins are involved in the modulation of the development of insulin resistance, β‐cell failure and eventually T1D and T2D. They are also involved in complications related with diabetes, such as retinopathy, nephropathy and cardiomyopathy. In T1D, SOCS proteins regulate β‐cell mass, mediate resistance to damaging factors and improve pancreatic islet graft survival. Regarding insulin resistance and T2D, SOCS proteins take part in mediating signals produced by diabetogenic substances and regulate insulin receptor functioning, affecting insulin sensitivity. However, not all of the present data are consistent, and thus, further studies are required. Finally, for several pharmacologically active substances of importance regarding the treatment of diabetes, SOCS‐modulating properties have already been described. Here, we review the findings of SOCS–diabetes relations of the last decade. Copyright

Exenatide (a GLP-1 agonist) improves the antioxidative potential of in vitro cultured human monocytes/macrophages

Macrophages are dominant cells in the pathogenesis of atherosclerosis. They are also a major source of reactive oxygen species (ROS). Oxidative stress, which is particularly high in subjects with diabetes, is responsible for accelerated atherosclerosis. Novel antidiabetic drugs (e.g., glucagon-like peptide-1 (GLP-1) agonists) were shown to reduce ROS level. Therefore, we conceived a study to evaluate the influence of exenatide, a GLP-1 agonist, on redox status in human monocytes/macrophages cultured in vitro, which may explain the beneficial effects of incretin-based antidiabetic treatment. Human macrophages obtained from 10 healthy volunteers were in vitro subjected to the treatment with GLP-1 agonist (exenatide) in the presence of lipopolysaccharide (LPS), antagonist of GLP-1 receptors (exendin 9-39), or protein kinase A inhibitor (H89). Afterwards, reactive oxygen species, malondialdehyde level, NADPH oxidase, and antioxidative enzymes [superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase] expression was evaluated. Finally, we estimated the activity of the abovementioned enzymes in the presence of H89. According to our findings, exenatide reduced ROS and malondialdyhyde (MDA) level by decreasing the expression of ROS-generating NADPH oxidase and by increasing the expression and activities of SOD and GSH-Px. We also showed that this effect was significantly inhibited by exendin 9-39 (a GLP-1 antagonist) and blocked by H89. Exenatide improved the antioxidative potential and reduced oxidative stress in cultured human monocytes/macrophages, and this finding may be responsible for the pleiotropic effects of incretin-based therapies. This effect relied on the stimulation of GLP-1 receptor.

Exenatide and metformin express their anti-inflammatory effects on human monocytes/macrophages by the attenuation of MAPKs and NFκB signaling.

Metformin and exenatide are effective antidiabetic drugs, and they seem to have pleiotropic properties improving cardiovascular outcomes. Macrophages’ phenotype is essential in the development of atherosclerosis, and it can be modified during antidiabetic therapy, resulting in attenuated atherogenesis. The mechanism orchestrating this phenomenon is not fully clear. We examined the impact of exenatide and metformin on the level of TNF alpha, MCP-1, reactive oxygen species (ROS), and the activation of mitogen-activated protein kinases (MAPK), nuclear factor kappa B (NFκB), and CCAAT/enhancer-binding protein beta (C/EBP beta) in human monocytes/macrophages. We found that both drugs reduced levels of TNF alpha, ROS, and NFκB binding activity to a similar extent. Compared to metformin, exenatide was more effective in reducing MCP-1 levels. We noted that Compound C (AMPK inhibitor) reduced the impact of exenatide on cytokines, ROS, and NFκB in cultures. Both drugs elevated the C/EBP beta phosphorylation level. Experiments on MAPKs showed effective inhibitory potential of exenatide toward p38, JNK, and ERK, whereas metformin inhibited JNK and ERK only. Exenatide was more effective in the inhibition of JNK than metformin. Interestingly, an in vitro setting additive effect of drugs was absent. In conclusion, here, we report that metformin and exenatide inhibit the proinflammatory phenotype of human monocytes/macrophages via influence on MAPK, C/EBP beta, and NFκB. Exenatide was more effective than metformin in reducing MCP-1 expression and JNK activity. We also showed that some effects of exenatide relied on AMPK activation. This shed light on the possible mechanisms responsible for pleiotropic effects of metformin and exenatide.

Superoxide dismutase 1 and glutathione peroxidase 1 are involved in the protective effect of sulodexide on vascular endothelial cells exposed to oxygen-glucose deprivation.

Sulodexide (SDX) is widely used in the treatment of both arterial and venous thrombotic disorders. In addition to its recognized antithrombotic action, SDX has endothelial protective potential, which is independent of the coagulation/fibrinolysis system. However, the detailed molecular mechanisms of the endothelioprotective action of the drug are still unresolved. The aim of the present study was to determine whether treatment with SDX at concentrations of 0.125-0.5 lipase releasing unit (LRU)/ml have on the expression and activity of antioxidant enzymes in ischemic endothelial cells and how these effects might be related to the antiapoptotic properties of SDX. In the present study, human umbilical vein endothelial cells (HUVECs) were subjected to ischemia-simulating conditions (combined oxygen and glucose deprivation, OGD) for 6h to determine the protective effects of SDX. SDX (0.25 and 0.5LRU/ml) in OGD significantly increased the cell viability and prevented mitochondrial depolarization in the HUVECs. Moreover, SDX protected the HUVECs against OGD-induced apoptosis. At concentrations of 0.25 and 0.5LRU/ml, the drug increased both superoxide dismutase 1 (SOD1) and glutathione peroxidase 1 (GPx1) mRNA/protein expression together with a significant attenuation of oxidative stress in ischemic HUVECs. Our findings also demonstrate that an increase in both SOD and GPx activity is involved in the protective effect of SDX on ischemic endothelial cells. Altogether, these results suggest that SDX has a positive effect on ischemia-induced endothelial damage because of its antioxidant and antiapoptotic properties.