Oxidative Medicine and Cellular Longevity

Intraplaque hemorrhage frequently occurs in atherosclerotic plaques resulting in cell-free hemoglobin, which is oxidized to ferryl hemoglobin (FHb) in the highly oxidative environment. Osteoclast-like cells (OLCs) derived from macrophages signify a counterbalance mechanism for calcium deposition in atherosclerosis. Our aim was to investigate whether oxidized hemoglobin alters osteoclast formation, thereby affecting calcium removal from mineralized atherosclerotic lesions. RANKL- (receptor activator of nuclear factor kappa-Β ligand-) induced osteoclastogenic differentiation and osteoclast activity of RAW264.7 cells were studied in response to oxidized hemoglobin via assessing bone resorption activity, expression of osteoclast-specific genes, and the activation of signalization pathways. OLCs in diseased human carotid arteries were assessed by immunohistochemistry. FHb, but not ferrohemoglobin, decreased bone resorption activity and inhibited osteoclast-specific gene expression (tartrate-resistant acid phosphatase, calcitonin receptor, and dendritic cell-specific transmembrane protein) induced by RANKL. In addition, FHb inhibited osteoclastogenic signaling pathways downstream of RANK (receptor activator of nuclear factor kappa-Β). It prevented the induction of TRAF6 (tumor necrosis factor (TNF) receptor-associated factor 6) and c-Fos, phosphorylation of p-38 and JNK (c-Jun N-terminal kinase), and nuclear translocation of NFκB (nuclear factor kappa-Β) and NFATc1 (nuclear factor of activated T-cells, cytoplasmic 1). These effects were independent of heme oxygenase-1 demonstrated by knocking down HO-1 gene in RAW264.7 cells and in mice. Importantly, FHb competed with RANK for RANKL binding suggesting possible mechanisms by which FHb impairs osteoclastic differentiation. In diseased human carotid arteries, OLCs were abundantly present in calcified plaques and colocalized with regions of calcium deposition, while the number of these cells were lower in hemorrhagic lesions exhibiting accumulation of FHb despite calcium deposition. We conclude that FHb inhibits RANKL-induced osteoclastic differentiation of macrophages and suggest that accumulation of FHb in a calcified area of atherosclerotic lesion with hemorrhage retards the formation of OLCs potentially impairing calcium resorption.

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Vitamin D deficiency has been reported in alcoholics. This study is aimed at evaluating the effects of vitamin D deficiency on chronic alcohol-induced liver injury in mice. Mice were fed with modified Lieber-DeCarli liquid diets for 6 weeks to establish an animal model of chronic alcohol-induced liver injury. In the VDD+EtOH group, mice were fed with modified diets, in which vitamin D was depleted. Vitamin D deficiency aggravated alcohol-induced liver injury. Furthermore, vitamin D deficiency aggravated hepatocyte apoptosis during alcohol-induced liver injury. Although it has a little effect on hepatic TG content, vitamin D deficiency promoted alcohol-induced hepatic GSH depletion and lipid peroxidation. Further analysis showed that vitamin D deficiency further increased alcohol-induced upregulation of hepatic inducible nitric oxide synthase (inos), two NADPH oxidase subunits p47phox and gp91phox, and heme oxygenase- (HO-) 1. By contrast, vitamin D deficiency attenuated alcohol-induced upregulation of hepatic antioxidant enzyme genes, such as superoxide dismutase (sod) 1 and gshpx. In addition, vitamin D deficiency significantly elevated alcohol-induced upregulation of hepatic proinflammatory cytokines and chemokines. Taken together, these results suggest that vitamin D deficiency aggravates hepatic oxidative stress and inflammation during chronic alcohol-induced liver injury.

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Currently, one of the central problems in cancer management is the relapse of disease following conventional treatments, yet few therapeutic agents targeting resistance and tolerance exist. Propolis is known as a healing agent since ancient times. Therefore, over time, its curative properties have kept the interest of scientists, thus leading permanently to investigations of its other possible undiscovered effects. In this context, current experiments were performed to establish the chemopreventive potential of propolis extract (PE) (1.05 mg/kg BW/day) in N-methyl-N-nitrosourea- (MNU-) induced rat mammary tumors. MNU-inoculated/PE-treated rats had tumors of different physical attributes compared with control rats MNU-inoculated. The number of developed tumors (mean 49% versus 100%), incidence (mean 49% versus 100%), multiplicity (1.8 versus 3.7 ()), tumor volume (mean 10 cm³ versus 16 cm³ ()), and weight of the tumor mass (mean 7.42 g versus 9.00 g ()) were noted. The numbers of grade I tumors recorded for MNU-inoculated rats were 24 (Group 1) and 7 (Group 2) for MNU-induced/PE-treated rats. In the serum of rats MNU-inoculated/PE-treated were found higher levels of antioxidative enzymes (SOD, CAT, and GPx) than in MNU-induced. Taken together, these data indicate that propolis could be a chemopreventive agent against MNU-induced mammary carcinogenesis.

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Oxidative stress has been recognized as the contributor to diabetic peripheral neuropathy (DPN). Antioxidant strategies have been most widely explored; nevertheless, whether antioxidants alone prevent DPN still remains inconclusive. In the present study, we established an in vitro DPN cell model for drug screening using Schwann RSC96 cells under high glucose (HG) stimulation, and we found that salvianolic acid A (SalA) mitigated HG-induced injury evidenced by cell viability and myelination. Mechanistically, SalA exhibited strong antioxidative effects by inhibiting 1,1-diphenyl-2-picrylhydrazyl (DPPH) and reducing reactive oxygen species (ROS), malondialdehyde (MDA), and oxidized glutathione (GSSG) content, as well as upregulating antioxidative enzyme mRNA expression. In addition, SalA significantly extenuated neuroinflammation with downregulated inflammatory factor mRNA expression. Furthermore, SalA improved the mitochondrial function of HG-injured Schwann cells by scavenging mitochondrial ROS, decreasing mitochondrial membrane potential (MMP), and enhancing ATP production, as well as upregulating oxidative phosphorylation gene expression. More importantly, we identified nuclear factor-E2-related factor 2 (Nrf2) as the upstream regulator which mediated protective effects of SalA on DPN. SalA directly bound to the Kelch domain of Kelch-like ECH-associated protein 1 (Keap1) and thus disrupted the interaction of Nrf2 and Keap1 predicted by LibDock of Discovery Studio. Additionally, SalA significantly inhibited Nrf2 promoter activity and downregulated Nrf2 mRNA expression but without affecting Nrf2 protein expression. Interestingly, SalA upregulated the nuclear Nrf2 expression and promoted Nrf2 nuclear translocation by high content screening assay, which was confirmed to be involved in its antiglucotoxicity effect by the knockdown of Nrf2 in RSC96 cells. In KK-Ay mice, we demonstrated that SalA could effectively improve the abnormal glucose and lipid metabolism and significantly protect against DPN by increasing the mechanical withdrawal threshold and sciatic nerve conduction velocity and restoring the ultrastructural impairment of the injured sciatic nerve induced by diabetes. Hence, SalA protected against DPN by antioxidative stress, attenuating neuroinflammation, and improving mitochondrial function via Nrf2. SalA may be prospective therapeutics for treating DPN.

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It is generally accepted that the amyloid β (Aβ) peptide toxicity contributes to neuronal loss and is involved in the initiation and progression of Alzheimer’s disease (AD). Cold-inducible RNA-binding protein (CIRBP) is reported to be a general stress-response protein, which is induced by different stress conditions. Previous reports have shown the neuroprotective effects of CIRBP through the suppression of apoptosis via the Akt and ERK pathways. The objective of this study is to examine the effect of CIRBP against Aβ-induced toxicity in cultured rat primary cortical neurons and attempt to uncover its underlying mechanism. Here, MTT, LDH release, and TUNEL assays showed that CIRBP overexpression protected against both intracellular amyloid β- (iAβ-) induced and Aβ_25-35-induced cytotoxicity in rat primary cortical neurons. Electrophysiological changes responsible for iAβ-induced neuronal toxicity, including an increase in neuronal resting membrane potentials and a decrease in K⁺ currents, were reversed by CIRBP overexpression. Western blot results further showed that Aβ_25-35 treatment significantly increased the level of proapoptotic protein Bax, cleaved caspase-3, and cleaved caspase-9 and decreased the level of antiapoptotic factor Bcl-2, but were rescued by CIRBP overexpression. Furthermore, CIRBP overexpression prevented the elevation of ROS induced by Aβ_25-35 treatment by decreasing the activities of oxidative biomarker and increasing the activities of key enzymes in antioxidant system. Taken together, our findings suggested that CIRBP exerted protective effects against neuronal amyloid toxicity via antioxidative and antiapoptotic pathways, which may provide a promising candidate for amyloid-based AD prevention or therapy.

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Mitophagy is involved in sepsis-induced acute lung injury (ALI). Bcl-2 family proteins play an important role in mitochondrial homeostasis. However, whether targeting Bcl-2 proteins (Bcl-2 and Bad) could influence mitophagy in ALI remains unclear. In this study, lipopolysaccharide (LPS) was used to induce injury in A549 cells and ALI in mice. LPS treatment resulted in elevated cell apoptosis, enhanced mitophagy, decreased Bcl-2 expression, increased Bad expression, and activation of PINK1/Parkin signaling in cells and lung tissues. Both Bcl-2 overexpression and Bad knockdown attenuated LPS-induced injury, inhibited cell apoptosis and mitophagy, and improved survival. Atg5 knockout (KO) inhibited LPS-induced cell apoptosis. Furthermore, Bcl-2 proteins regulated mitophagy by modulating the recruitment of Parkin from the cytoplasm to mitochondria via direct protein-protein interactions. These results were further confirmed in Park2 KO cells and Park2^-/- mice. This is the first study to demonstrate that Bcl-2 proteins regulated mitophagy in LPS-induced ALI via modulating the PINK1/Parkin signaling pathway, promoting new insights into the mechanisms and investigation of therapeutic strategies for a septic patient with ALI.

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