Critical roles of tubular mitochondrial ATP synthase dysfunction in maleic acid-induced acute kidney injury.

Maleic acid (MA) induces renal tubular cell dysfunction directed to acute kidney injury (AKI). AKI is an increasing global health burden due to its association with mortality and morbidity. However, targeted therapy for AKI is lacking. Previously, we determined mitochondrial-associated proteins are MA-induced AKI affinity proteins. We hypothesized that mitochondrial dysfunction in tubular epithelial cells plays a critical role in AKI. In vivo and in vitro systems have been used to test this hypothesis. For the in vivo model, C57BL/6 mice were intraperitoneally injected with 400 mg/kg body weight MA. For the in vitro model, HK-2 human proximal tubular epithelial cells were treated with 2 mM or 5 mM MA for 24 h. AKI can be induced by administration of MA. In the mice injected with MA, the levels of blood urea nitrogen (BUN) and creatinine in the sera were significantly increased (p < 0.005). From the pathological analysis, MA-induced AKI aggravated renal tubular injuries, increased kidney injury molecule-1 (KIM-1) expression and caused renal tubular cell apoptosis. At the cellular level, mitochondrial dysfunction was found with increasing mitochondrial reactive oxygen species (ROS) (p < 0.001), uncoupled mitochondrial respiration with decreasing electron transfer system activity (p < 0.001), and decreasing ATP production (p < 0.05). Under transmission electron microscope (TEM) examination, the cristae formation of mitochondria was defective in MA-induced AKI. To unveil the potential target in mitochondria, gene expression analysis revealed a significantly lower level of ATPase6 (p < 0.001). Renal mitochondrial protein levels of ATP subunits 5A1 and 5C1 (p < 0.05) were significantly decreased, as confirmed by protein analysis. Our study demonstrated that dysfunction of mitochondria resulting from altered expression of ATP synthase in renal tubular cells is associated with MA-induced AKI. This finding provides a potential novel target to develop new strategies for better prevention and treatment of MA-induced AKI.

miR-26a attenuates cardiac apoptosis and fibrosis by targeting ataxia-telangiectasia mutated in myocardial infarction.

Apoptosis and fibrosis play a vital role in myocardial infarction (MI) induced tissue injury. Although microRNAs have been the focus of many studies on cardiac apoptosis and fibrosis in MI, the detailed effects of miR-26a is needed to further understood. The present study demonstrated that miR-26a was downregulated in ST-elevation MI (STEMI) patients and oxygen-glucose deprivation (OGD)-treated H9c2 cells. Downregulation of miR-26a was closely correlated with the increased expression of creatine kinase, creatine kinase-MB and troponin I in STEMI patients. Further analysis identified that ataxia-telangiectasia mutated (ATM) was a target gene for miR-26a based on a bioinformatics analysis. miR-26a overexpression effectively reduced ATM expression, apoptosis, and apoptosis-related proteins in OGD-treated H9c2 cells. In a mouse model of MI, the expression of miR-26a was significantly decreased in the infarct zone of the heart, whereas apoptosis and ATM expression were increased. miR-26a overexpression effectively reduced ATM expression and cardiac apoptosis at Day 1 after MI. Furthermore, we demonstrated that overexpression of miR-26a improved cardiac function and reduced cardiac fibrosis by the reduced expression of collagen type I and connective tissue growth factor (CTGF) in mice at Day 14 after MI. Overexpression of miR-26a or ATM knockdown decreased collagen I and CTGF expression in cultured OGD-treated cardiomyocytes. Taken together, these data demonstrate a prominent role for miR-26a in linking ATM expression to ischemia-induced apoptosis and fibrosis, key features of MI progression. miR-26a reduced MI development by affecting ATM expression and could be targeted in the treatment of MI.

Role of Low-Density Lipoprotein in Early Vascular Aging Associated With Systemic Lupus Erythematosus.

OBJECTIVE: Patients with systemic lupus erythematosus (SLE) often have atherosclerotic complications at a young age but normal low-density lipoprotein (LDL) levels. This study was undertaken to investigate the role of LDL composition in promoting early vascular aging in SLE patients. METHODS: Plasma LDL from 45 SLE patients (SLE-LDL) and from 37 normal healthy controls (N-LDL) was chromatographically divided into 5 subfractions (L1-L5), and the subfraction composition was analyzed. Correlations between subfraction levels and signs of early vascular aging were assessed. Mechanisms of lipid-mediated endothelial dysfunction were explored using in vitro assays and experiments in apoE-/- mice. RESULTS: The L5 percentage was increased 3.4 times in the plasma of SLE patients compared with normal controls. This increased percentage of SLE-L5 was positively correlated with the mean blood pressure (r = 0.27, P = 0.04), carotid intima-media thickness (IMT) (right carotid IMT, r = 0.4, P = 0.004; left carotid IMT, r = 0.36, P = 0.01), pulse wave velocity (r = 0.29, P = 0.04), and blood levels of CD16+ monocytes (r = 0.35, P = 0.004) and CX3CL1 cytokines (r = 0.43, P < 0.001) in SLE patients. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis revealed that plasma levels of lysophosphatidylcholine (LPC) and platelet-activating factor (PAF) were increased in SLE-LDL and in the SLE-L5 plasma subfraction. Injecting SLE-LDL, SLE-L5, or LPC into young, male apoE-/- mice caused increases in plasma CX3CL1 levels, aortic fatty-streak areas, aortic vascular aging, and macrophage infiltration into the aortic wall, whereas injection of N-LDL or SLE-L1 had negligible effects (n = 3-8 mice per group). In vitro, SLE-L5 lipid extracts induced increases in CX3CR1 and CD16 expression in human monocytes; synthetic PAF and LPC had similar effects. Furthermore, lipid extracts of SLE-LDL and SLE-L5 induced the expression of CX3CL1 and enhanced monocyte-endothelial cell adhesion in assays with bovine aortic endothelial cells. CONCLUSION: An increase in plasma L5 levels, not total LDL concentration, may promote early vascular aging in SLE patients, leading to premature atherosclerosis.

O-GlcNAcylation disrupts STRA6-retinol signals in kidneys of diabetes.

BACKGROUND: O-GlcNAcylation is an important mechanism of diabetic complication. Retinoid homeostasis regulates cell-physiological functions through STRA6-retinol signaling. Therefore, we investigated whether O-GlcNAcylation disrupted STRA6-retinol signals in diabetes. METHODS: Immunoprecipitation and proximity ligation assay were used to investigate O-GlcNAcylation of STRA6-retinol signals in kidneys of db/db and ob/ob mice. Western blot and immunohistochemistry were done for STRA6/CRBP1/LRAT/RALDH1/RARs pathway, GFAT, OGT, TGFß1 and collagen 1 level. HPLC and ELISA for retinol, retinal, and retinoic acid concentrations were performed in vivo and vitro. RBP4 binding with STRA6 was measured in vitro. To verify whether O-GlcNAcylation disrupted STRA6-retinol signals, treatment of TMG and OSMI-1, transfection of OGA and OGT, and OGT siRNA were performed in HK-2 cells. RESULTS: STRA6 and RALDH1 were highly O-GlcNAc-modified in glomeruli and tubules of db/db and ob/ob mice. RBP4, p-Try, p-JAK2, and p-STAT5 on STRA6 immunoprecipitate were reduced. Cellular retinol signals (CRBP1, LRAT, ADH, retinol, retinal, RA, RARα, RARγ and RXRα) remarkably decreased in kidneys of db/db, ob/ob mice and HG-cultured cells. TMG and OGT transfection induced O-GlcNAcylation of STRA6 and RALDH1, repressed RBP4-bound STRA6, and retinol signals in NG-cultured cells. OSMI-1, OGA transfection, and OGT silence reversed O-GlcNAc-modification of STRA6 and RALDH1, and rescued the decrease of retinol signals, and reversed the increase of TGFß1 and collagen 1 in HG-treated cells. CONCLUSIONS: O-GlcNAcylation significantly modified STRA6 and RALDH1, suppressed RBP4 binding activity, and disrupted retinol signals in the kidney of diabetes. GENERAL SIGNIFICANCE: This study first indicates that STRA6-retinol signals were directly disrupted by O-GlcNAcylation in diabetic kidney.

The use of chemical probes to detect the proteomics of renal tubular injury induced by maleic acid.

Maleic acid (MA), an industrial raw material, was found to be illegally added to edible starch-based food products in Taiwan in 2013, a practice unheard of in most of the world. MA has been associated with renal dysfunction in many experimental animal studies. In this study, we developed chemical probes to investigate protein-protein interactions between MA and renal proteins. In the fabrication of the MA probes, we used silicon dioxide (SiO2) modified with a silanized linker (3-aminopropyl triethoxyslane, APTES) to generate MA with APTES-SiO2 particles. The probes were then incubated with the cell lysates of normal human kidney cell lines (HK-2) and subjected to MS/MS for identifying several MA-related proteins, including nucleophosmin, neutral alpha-glucosidase AB, translocon-associated protein subunit alpha, elongation factor 1-gamma, 60S acidic ribosomal protein P0-like, and heat shock protein (HSP 90-alpha and beta). Based on our findings, we believed that the probe can potentially be used to identify and detect the target proteins and help characterize a network of MA protein-protein interactions.

Curcumin accelerates cutaneous wound healing via multiple biological actions: The involvement of TNF-α, MMP-9, α-SMA, and collagen.

Curcumin, a constituent of the turmeric plant, has antitumor, anti-inflammatory, and antioxidative effects, but its effects on wound healing are unclear. We created back wounds in 72 mice and treated them with or without topical curcumin (0.2 mg/mL) in Pluronic F127 gel (20%) daily for 3, 5, 7, 9, and 12 days. Healing in wounds was evaluated from gross appearance, microscopically by haematoxylin and eosin staining, by immunohistochemistry for tumour necrosis factor alpha and alpha smooth muscle actin, and by polymerase chain reaction amplification of mRNA expression levels. Treatment caused fast wound closure with well-formed granulation tissue dominated by collagen deposition and regenerating epithelium. Curcumin increased the levels of tumour necrosis factor alpha mRNA and protein in the early phase of healing, which then decreased significantly. However, these levels remained high in controls. Levels of collagen were significantly higher in curcumin-treated wounds. Immunohistochemical staining for alpha smooth muscle actin was increased in curcumin-treated mice on days 7 and 12. Curcumin treatment significantly suppressed matrix metallopeptidase-9 and stimulated alpha smooth muscle levels in tumour necrosis factor alpha-treated fibroblasts via nuclear factor kappa B signalling. Thus, topical curcumin accelerated wound healing in mice by regulating the levels of various cytokines.

The effects of wild bitter gourd fruit extracts on ICAM-1 expression in pulmonary epithelial cells of C57BL/6J mice and microRNA-221/222 knockout mice: Involvement of the miR-221/-222/PI3K/AKT/NF-κB pathway.

BACKGROUND: The extracts from wild bitter gourd fruit (WBGE) were reported to possess numerous pharmacological activities. However, the anti-inflammatory effects of WBGE on human lung epithelial cells and the underlying mechanisms have not been determined. PURPOSE: To evaluate the molecular basis of the effects of WBGE on intercellular adhesion molecule-1 (ICAM-1) expression in alveolar epithelial (A549) cells, C57BL/6 wild-type (WT) mice and microRNA (miR)-221/-222 knockout (KO) mice with or without tumor necrosis factor (TNF-α; 3 ng/ml) treatment. STUDY DESIGN/METHODS: WT mice and miR-221/-222 KO mice were fed a control diet and divided into four groups (C: control mice; T: treated with TNF-α alone; WBGE/T: pretreated with WBGE and then stimulated with TNF-α; WBGE: treated with WBGE alone). The effects of WBGE on ICAM-1 expression and the related signals in A549 cells and mice with or without TNF-α treatment were examined by Western blot and immunofluorescent staining. RESULTS: WBGE significantly decreased the TNF-α-induced ICAM-1 expression in A549 cells through the inhibition of phosphoinositide 3-kinase (PI3K)/ protein kinase B (AKT)/ nuclear factor- kappa B (NF-κB)/ inhibitor of NF-κB (IκB) phosphorylation and decreased leukocyte adhesion. In addition, WBGE reduced endogenous ICAM-1 expression and upregulated miR-221/-222 expression. The overexpression of miR-222 decreased PI3K/AKT/NF-κB/IκB and ICAM-1 expression, which resulted in reducing monocyte adhesion. Moreover, WBGE reduced ICAM-1 expression in lung tissues of WT mice with or without TNF-α treatment and upregulated miR-221/222. WBGE did not affect the miR-221/-222 level and had little effect on ICAM-1 expression in miR-221/-222 KO mice. CONCLUSIONS: These results suggest that WBGE reduced ICAM-1 expression both under in vitro and in vivo conditions. The protective effects were mediated partly through the miR-221/-222/PI3K/AKT/NF-κB pathway.

PM2.5-induced oxidative stress increases intercellular adhesion molecule-1 expression in lung epithelial cells through the IL-6/AKT/STAT3/NF-κB-dependent pathway.

BACKGROUND: Epidemiological studies have shown that ambient air pollution is closely associated with increased respiratory inflammation and decreased lung function. Particulate matters (PMs) are major components of air pollution that damages lung cells. However, the mechanisms remain to be elucidated. This study examines the effects of PMs on intercellular adhesion molecule-1 (ICAM-1) expression and the related mechanisms in vitro and in vivo. RESULT: The cytotoxicity, reactive oxygen species (ROS) generation, and monocyte adherence to A549 cells were more severely affected by treatment with O-PMs (organic solvent-extractable fraction of SRM1649b) than with W-PMs (water-soluble fraction of SRM1649b). We observed a significant increase in ICAM-1 expression by O-PMs, but not W-PMs. O-PMs also induced the phosphorylation of AKT, p65, and STAT3. Pretreating A549 cells with N-acetyl cysteine (NAC), an antioxidant, attenuated O-PMs-induced ROS generation, the phosphorylation of the mentioned kinases, and the expression of ICAM-1. Furthermore, an AKT inhibitor (LY294002), NF-κB inhibitor (BAY11-7082), and STAT3 inhibitor (Stattic) significantly down-regulated O-PMs-induced ICAM-1 expression as well as the adhesion of U937 cells to epithelial cells. Interleukin-6 (IL-6) was the most significantly changed cytokine in O-PMs-treated A549 cells according to the analysis of the cytokine antibody array. The IL-6 receptor inhibitor tocilizumab (TCZ) and small interfering RNA for IL-6 significantly reduced ICAM-1 secretion and expression as well as the reduction of the AKT, p65, and STAT3 phosphorylation in O-PMs-treated A549 cells. In addition, the intratracheal instillation of PMs significantly increased the levels of the ICAM-1 and IL-6 in lung tissues and plasma in WT mice, but not in IL-6 knockout mice. Pre-administration of NAC attenuated those PMs-induced adverse effects in WT mice. Furthermore, patients with chronic obstructive pulmonary disease (COPD) had higher plasma levels of ICAM-1 and IL-6 compared to healthy subjects. CONCLUSION: These results suggest that PMs increase ICAM-1 expression in pulmonary epithelial cells in vitro and in vivo through the IL-6/AKT/STAT3/NF-κB signaling pathway.

Aliskiren Improves Ischemia- and Oxygen Glucose Deprivation-Induced Cardiac Injury through Activation of Autophagy and AMP-Activated Protein Kinase.

Aliskiren is a direct renin inhibitor that has been effective in anti-hypertension. We investigated whether aliskiren could improve the ischemia-induced cardiac injury and whether the autophagy was involved in this effect. A myocardial infarction (MI) model was created by the ligation of the left anterior coronary artery in C57J/BL6 mice. They were treated for 1, 3, 7, and 14 days with vehicle or aliskiren (25 mg/kg/day via subcutaneous injection). In vivo, the MI mice exhibited worse cardiac function by echocardiographic assessment and showed larger myocardial scarring by light microscopy, whereas aliskiren treatment reversed these effects, which were also associated with the changes in caspase-3 and Bcl-2 expression as well as in the number of apoptotic cells. Aliskiren increased autophagy, as demonstrated by LC3B-II expression and transmission electron microscopy. Furthermore, H9c2 cardiomyocytes were employed as an in vitro model to examine the effects of aliskiren on apoptosis and autophagy under oxygen glucose deprivation (OGD)-induced injury. Aliskiren significantly increased cell viability in a dose-dependent manner. The beneficial effects of aliskiren were associated with decreased apoptosis and mitochondrial membrane potential as well as increased autophagy via increased autophagosome formation. We also found that aliskiren-induced cardiomyocyte survival occurred via AMP-activated protein kinase (AMPK)-dependent autophagy. Taken together, these results indicated that aliskiren increased cardiomyocyte survival through increased autophagosomal formation and decreased apoptosis and necrosis via modulating AMPK expression. AMPK-dependent autophagy may represent a novel mechanism for aliskiren in ischemic cardiac disease therapy.

Resveratrol attenuates ICAM-1 expression and monocyte adhesiveness to TNF-α-treated endothelial cells: evidence for an anti-inflammatory cascade mediated by the miR-221/222/AMPK/p38/NF-κB pathway.

Resveratrol, an edible polyphenolic phytoalexin, improves endothelial dysfunction and attenuates inflammation. However, the mechanisms have not been thoroughly elucidated. Therefore, we investigated the molecular basis of the effects of resveratrol on TNF-α-induced ICAM-1 expression in HUVECs. The resveratrol treatment significantly attenuated the TNF-α-induced ICAM-1 expression. The inhibition of p38 phosphorylation mediated the reduction in ICAM-1 expression caused by resveratrol. Resveratrol also decreased TNF-α-induced IκB phosphorylation and the phosphorylation, acetylation, and translocation of NF-κB p65. Moreover, resveratrol induced the AMPK phosphorylation and the SIRT1 expression in TNF-α-treated HUVECs. Furthermore, TNF-α significantly suppressed miR-221/-222 expression, which was reversed by resveratrol. miR-221/-222 overexpression decreased p38/NF-κB and ICAM-1 expression, which resulted in reduced monocyte adhesion to TNF-α-treated ECs. In a mouse model of acute TNF-α-induced inflammation, resveratrol effectively attenuated ICAM-1 expression in the aortic ECs of TNF-α-treated wild-type mice. These beneficial effects of resveratrol were lost in miR-221/222 knockout mice. Our data showed that resveratrol counteracted the TNF-α-mediated reduction in miR-221/222 expression and decreased the TNF-α-induced activation of p38 MAPK and NF-κB, thereby suppressing ICAM-1 expression and monocyte adhesion. Collectively, our results show that resveratrol attenuates endothelial inflammation by reducing ICAM-1 expression and that the protective effect was mediated partly through the miR-221/222/AMPK/p38/NF-κB pathway.

Adipose-Derived Stem Cells Protect Skin Flaps against Ischemia/Reperfusion Injury via IL-6 Expression.

Flap necrosis is the most frequent postoperative complication encountered in reconstructive surgery. We elucidated whether adipose-derived stem cells (ADSCs) and their derivatives might induce neovascularization and protect skin flaps during ischemia/reperfusion (I/R) injury. Flaps were subjected to 3 hours of ischemia by ligating long thoracic vessels and then to blood reperfusion. Qtracker-labeled ADSCs, ADSCs in conditioned medium (ADSC-CM), or ADSC exosomes (ADSC-Exo) were injected into the flaps. These treatments led to significantly increased flap survival and capillary density compared with I/R on postoperative day 5. IL-6 levels in the cell lysates or in conditioned medium were significantly higher in ADSCs than in Hs68 fibroblasts. ADSC-CM and ADSC-Exo increased tube formation. This result was corroborated by a strong decrease in skin repair after adding IL-6-neutralizing antibodies or small interfering RNA for IL-6 ADSCs. ADSC transplantation also increased flap recovery in I/R injury of IL-6-knockout mice. IL-6 was secreted from ADSCs through signal transducer and activator of transcription phosphorylation, and then IL-6 stimulated angiogenesis and enhanced recovery after I/R injury by the classic signaling pathway. The mechanism of skin recovery includes the direct differentiation of ADSCs into endothelial cells and the indirect effect of IL-6 released from ADSCs. ADSC-CM and ADSC-Exo could be used as off-the-shelf products for this therapy.

Magnolol Nanoparticles Exhibit Improved Water Solubility and Suppress TNF-α-Induced VCAM-1 Expression in Endothelial Cells.

The expression of the adhesion molecule vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells enables the attachment of leukocytes to the endothelium, which may lead to inflammation and the development of atherosclerosis. Magnolol is a major bioactive compound derived from the plant species Magnolia officinalis. In this study, we synthesized a novel nanoparticle formulation of magnolol to improve its water solubility and physicochemical properties, evaluated its effects on TNF-α-induced VCAM-1 expression in endothelial cells, and determined the signal transduction pathways involved. Our findings demonstrated that the magnolol nanoparticle system showed great improvements in physicochemical properties and water solubility owing to a reduction in particle size, transformation from a crystalline to amorphous structure, and the formation of hydrogen bonds with the nanoparticle carriers. In terms of its biological actions, magnolol nanoparticles attenuated TNF-α-induced VCAM-1 protein expression, promoter activity, and mRNA expression in endothelial cells in vitro. This was found to be mediated by the ERK, AKT, and NF-κB signaling pathways. In addition, magnolol nanoparticles inhibited TNF-α-induced leukocyte adhesion to endothelial cells, and suppressed TNF-α-induced VCAM-1 expression in the aortic endothelium of mice. In summary, since magnolol nanoparticles inhibit endothelial VCAM-1 expression and leukocyte adhesion to endothelial cells, this novel drug formulation may be a potentially useful therapeutic formulation to prevent the development of atherosclerosis and inflammatory diseases.

Viscolin Inhibits In Vitro Smooth Muscle Cell Proliferation and Migration and Neointimal Hyperplasia In Vivo.

Viscolin, an extract of Viscum coloratum, has anti-inflammatory and anti-proliferative properties against harmful stimuli. The aim of the study was to examine the anti-proliferative effects of viscolin on platelet derived growth factor-BB (PDGF)-treated human aortic smooth muscle cells (HASMCs) and identify the underlying mechanism responsible for these effects. Viscolin reduced the PDGF-BB-induced HASMC proliferation and migration in vitro; it also arrested HASMCs in the G0/G1 phase by decreasing the protein expression of Cyclin D1, CDK2, Cyclin E, CDK4, and p21Cip1 as detected by Western blot analysis. These effects may be mediated by reduced PDGF-induced phosphorylation of ERK1/2, JNK, and P38, but not AKT as well as inhibition of PDGF-mediated nuclear factor (NF)-κB p65 and activator protein 1 (AP-1)/c-fos activation. Furthermore, viscolin pre-treatment significantly reduced neointimal hyperplasia of an endothelial-denuded femoral artery in vivo. Taken together, viscolin attenuated PDGF-BB-induced HASMC proliferation in vitro and reduced neointimal hyperplasia in vivo. Thus, viscolin may represent a therapeutic candidate for the prevention and treatment of vascular proliferative diseases.

Eupafolin nanoparticles protect HaCaT keratinocytes from particulate matter-induced inflammation and oxidative stress.

Exposure to particulate matter (PM), a major form of air pollution, can induce oxidative stress and inflammation and may lead to many diseases in various organ systems including the skin. Eupafolin, a flavonoid compound derived from Phyla nodiflora, has been previously shown to exhibit various pharmacological activities, including antioxidant and anti-inflammatory effects. Unfortunately, eupafolin is characterized by poor water solubility and skin penetration, which limits its clinical applications. To address these issues, we successfully synthesized a eupafolin nanoparticle delivery system (ENDS). Our findings showed that ENDS could overcome the physicochemical drawbacks of raw eupafolin with respect to water solubility and skin penetration, through reduction of particle size and formation of an amorphous state with hydrogen bonding. Moreover, ENDS was superior to raw eupafolin in attenuating PM-induced oxidative stress and inflammation in HaCaT keratinocytes, by mediating the antioxidant pathway (decreased reactive oxygen species production and nicotinamide adenine dinucleotide phosphate oxidase activity) and anti-inflammation pathway (decreased cyclooxygenase-2 expression and prostaglandin E2 production through downregulation of mitogen-activated protein kinase and nuclear factor-κB signaling). In summary, ENDS shows better antioxidant and anti-inflammatory activities than raw eupafolin through improvement of water solubility and skin penetration. Therefore, ENDS may potentially be used as a medicinal drug and/or cosmeceutical product to prevent PM-induced skin inflammation.

The Anti-Inflammatory Effects and Mechanisms of Eupafolin in Lipopolysaccharide-Induced Inflammatory Responses in RAW264.7 Macrophages.

Eupafolin is a flavone isolated from Artemisia princeps Pampanini (family Asteraceae). The aim of this study was to examine the anti-inflammatory effects of eupafolin in lipopolysaccharide (LPS)-treated RAW264.7 macrophages and LPS-induced mouse skin and lung inflammation models and to identify the mechanism underlying these effects. Eupafolin decreased the LPS-induced release of inflammatory mediators (iNOS, COX-2 and NO) and proinflammatory cytokines (IL-6 and TNF-α) from the RAW264.7 macrophages. Eupafolin inhibited the LPS-induced phosphorylation of p38 MAPK, ERK1/2, JNK, AKT and p65 and the nuclear translocation of p65 and c-fos. These effects were mainly mediated by the inhibition of JNK. In the mouse paw and lung models, eupafolin effectively suppressed the LPS-induced edema formation and down-regulated iNOS and COX-2 expression. These results demonstrated that eupafolin exhibits anti-inflammatory properties and suggested that eupafolin can be developed as an anti-inflammatory agent.

Nonapnea Sleep Disorders and the Risk of Acute Kidney Injury: A Nationwide Population-Based Study.

Nonapnea sleep disorders (NASDs) and associated problems, which are highly prevalent in patients with kidney diseases, are associated with unfavorable medical sequelae. Nonetheless, whether NASDs are associated with acute kidney injury (AKI) development has not been thoroughly analyzed. We examined the association between NASD and AKI. We conducted a population-based study by using 1,000,000 representative data from the Taiwan National Health Insurance Research Database for the period from January 1, 2000, to December 31, 2010. We studied the incidence and risk of AKI in 9178 newly diagnosed NASD patients compared with 27,534 people without NASD matched according to age, sex, index year, urbanization level, region of residence, and monthly income at a 1:3 ratio. The NASD cohort had an adjusted hazard ratio (hazard ratio [HR]; 95% confidence interval [CI] = 1.15-2.63) of subsequent AKI 1.74-fold higher than that of the control cohort. Older age and type 2 diabetes mellitus were significantly associated with an increased risk of AKI (P < 0.05). Among different types of NASDs, patients with insomnia had a 120% increased risk of developing AKI (95% CI = 1.38-3.51; P = 0.001), whereas patients with other sleep disorders had a 127% increased risk of subsequent AKI (95% CI = 1.07-4.80; P = 0.033). Men with NASDs were at a high risk of AKI (P < 0.05). This nationwide population-based cohort study provides evidence that patients with NASDs are at higher risk of developing AKI than people without NASDs.

Indoxyl sulfate enhances IL-1ß-induced E-selectin expression in endothelial cells in acute kidney injury by the ROS/MAPKs/NFκB/AP-1 pathway.

Uremic toxins are considered a risk factor for cardiovascular disorders in kidney diseases, but it is not known whether, under inflammatory conditions, they affect adhesion molecule expression on endothelial cells, which may play a critical role in acute kidney injury (AKI). In the present study, in cardiovascular surgery-related AKI patients, who are known to have high plasma levels of the uremic toxin indoxyl sulfate (IS), plasma levels of IL-1ß were found to be positively correlated with plasma levels of the adhesion molecule E-selectin. In addition, high E-selectin and IL-1ß expression were seen in the kidney of ischemia/reperfusion mice in vivo. We also examined the effects of IS on E-selectin expression by IL-1ß-treated human umbilical vein endothelial cells (HUVECs) and the underlying mechanism. IS pretreatment of HUVECs significantly increased IL-1ß-induced E-selectin expression, monocyte adhesion, and the phosphorylation of mitogen-activated protein kinases (ERK, p38, and JNK) and transcription factors (NF-κB and AP-1), and phosphorylation was decreased by pretreatment with inhibitors of ERK1/2 (PD98059), p38 MAPK (SB202190), and JNK (SP600125). Furthermore, IS increased IL-1ß-induced reactive oxygen species (ROS) production and this effect was inhibited by pretreatment with N-acetylcysteine (a ROS scavenger) or apocynin (a NADPH oxidase inhibitor). Gel shift assays and ChIP-PCR demonstrated that IS enhanced E-selectin expression in IL-1-treated HUVECs by increasing NF-κB and AP-1 DNA-binding activities. Moreover, IS-enhanced E-selectin expression in IL-1ß-treated HUVECs was inhibited by Bay11-7082, a NF-κB inhibitor. Thus, IS may play an important role in the development of cardiovascular disorders in kidney diseases during inflammation by increasing endothelial expression of E-selectin.

The protective effect of eupafolin against TNF-α-induced lung inflammation via the reduction of intercellular cell adhesion molecule-1 expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Eupafolin, a major bioactive compound found in Phyla nodiflora, has the anti-inflammatory property. Upregulation of cell adhesion molecules in the lung airway epithelium is associated with the epithelium-leukocyte interaction and plays a critical role in the pathogenesis of lung airway inflammatory disorders. To investigate the effects of eupafolin on tumor necrosis factor-α (TNF-α)-induced intercellular cell adhesion molecule-1 (ICAM-1) expression in A549 human lung airway epithelial cells and the underlying mechanisms. MATERIALS AND METHODS: The effect of eupafolin on ICAM-1 expression in A549 cells were examined by Western blotting and immunofluorescent staining. The mice were injected intraperitoneally with or without eupafolin and then were left untreated or were injected intratracheally with TNF-α. To detect the effect of eupafolin on ICAM-1 expression, the lung tissues were also examined by Western blotting and immunohistochemical staining. RESULTS: Eupafolin pretreatment reduced the TNF-α-induced ICAM-1 expression and also the ERK1/2, JNK, p38, and AKT/PI3K phosphorylation. However, the increase in ICAM-1 expression with TNF-α treatment was unaffected by p38 and PI3K inhibitors. Eupafolin decreased the TNF-α-induced NF-κB p65 activation and its nuclear translocation. Furthermore, eupafolin reduced ICAM-1 expression in the lung tissues of TNF-α-treated mice. CONCLUSIONS: Eupafolin exerts its anti-inflammatory activity by suppressing the TNF-α-induced ICAM-1 expression and subsequent monocyte adhesion via AKT/ERK1/2/JNK phosphorylation and nuclear translocation of NF-κB p65. These results suggest that eupafolin may represent a novel therapeutic agent targeting epithelial activation in lung inflammation.

Endothelial Progenitor Cells Derived From Wharton's Jelly of Human Umbilical Cord Attenuate Ischemic Acute Kidney Injury by Increasing Vascularization and Decreasing Apoptosis, Inflammation, and Fibrosis.

Ischemia-reperfusion (I/R) injury to the kidney, a major cause of acute renal failure in humans, is associated with a high mortality, and the development of a new therapeutic strategy is therefore highly desirable. In this study, we examined the therapeutic potential of implantation of endothelial progenitor cells (EPCs) isolated from Wharton's jelly of human umbilical cords in the treatment of renal I/R injury in mice. To visualize the localization of the transplanted EPCs, the cells were labeled with Q-tracker before injection into the renal capsule. Mice with renal I/R injury showed a significant increase in blood urea nitrogen and creatinine levels, and these effects were decreased by EPC transplantation. The kidney injury score in the mice with I/R injury was also significantly decreased by EPC transplantation. EPC transplantation increased the microvascular density, and some of the EPCs surrounded and were incorporated into microvessels. In addition, EPC transplantation inhibited the I/R-induced cell apoptosis of endothelial, glomerular, and renal tubular cells, as demonstrated by TUNEL staining, and significantly reduced reactive oxygen species production and the expression of the inflammatory chemokines macrophage inflammatory protein-2 and keratinocyte-derived cytokine, as shown by immunostaining and ELISA. Moreover, EPC transplantation reduced I/R-induced fibrosis, as demonstrated by immunostaining for S100A4, a fibroblast marker, and by Jones silver staining. To our knowledge, this is the first report that transplantation of EPCs from Wharton's jelly of human umbilical cords might provide a novel therapy for ischemic acute kidney injury by promoting angiogenesis and inhibiting apoptosis, inflammation, and fibrosis.