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.

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.

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.

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.

Eupafolin inhibits PGE2 production and COX2 expression in LPS-stimulated human dermal fibroblasts by blocking JNK/AP-1 and Nox2/p47(phox) pathway.

Eupafolin, a major active component found in the methanol extracts of Phyla nodiflora, has been used to treat inflammation of skin. We examined its effects on cyclooxygenase-2 (COX-2) expression in LPS-treated human dermal fibroblasts. Lipopolysaccharide (LPS) significantly increased prostaglandin-E2 (PGE2) production associated with increased COX-2 expression in Hs68 cells. This effect was blocked by eupafolin, TLR-4 antibody, antioxidants (APO and NAC), as well as inhibitors, including U0126 (ERK1/2), SB202190 (p38), SP600125 (JNK1/2), and Tanshinone IIA (AP-1). In gene regulation level, qPCR and promoter assays revealed that COX-2 expression was attenuated by eupafolin. In addition, eupafolin also ameliorated LPS-induced p47 phox activation and decreased reactive oxygen species (ROS) generation and NADPH oxidase (Nox) activity. Moreover, pretreatment with eupafolin and APO led to reduced LPS-induced phosphorylation of ERK1/2, JNK, and p38. Further, eupafolin attenuated LPS-induced increase in AP-1 transcription factor binding activity as well as the increase in the phosphorylation of c-Jun and c-Fos. In vivo studies have shown that in dermal fibroblasts of LPS treated mice, eupafolin exerted anti-inflammation effects by decreasing COX-2 protein levels. Our results reveal a novel mechanism for anti-inflammatory and anti-oxidative effects of eupafolin that involved inhibition of LPS-induced ROS generation, suppression of MAPK phosphorylation, diminished DNA binding activity of AP-1 and attenuated COX-2 expression leading to reduced production of prostaglandin E2 (PGE2). Our results demonstrate that eupafolin may be used to treat inflammatory responses associated with dermatologic diseases.

Troglitazone and Δ2Troglitazone enhance adiponectin expression in monocytes/macrophages through the AMP-activated protein kinase pathway.

Accumulating evidence indicates that the regimen to increase adiponectin will provide a novel therapeutic strategy for inflammation and cardiovascular disorders. Here, we tested the effect of troglitazone (TG) and its newly synthesized derivative, 5-[4-(6-hydroxy-2,5,7,8-tetramethyl-chroman-2-yl-methoxy)-benzylidene]-2,4-thiazolidinedione (Δ2troglitazone, (Δ2TG)), on the adiponectin expression in monocytes/macrophages and the relative mechanisms. The expression of adiponectin was located in macrophages of atherosclerotic lesions from patients and cholesterol-fed rabbits. TG and Δ2TG enhanced adiponectin mRNA and protein expression in THP-1 cells by quantitative real-time PCR, Western blot, and immunocytochemistry. TG induced adiponectin mRNA expression through a PPARγ-dependent pathway whereas Δ2TG enhanced adiponectin mRNA expression through a PPARγ-independent pathway in THP-1 cells. Both TG and Δ2TG enhanced adiponectin mRNA expression through AMP-activated protein kinase (AMPK) activation. TG and Δ2TG decreased the adhesion of THP-1 cells to TNF-α-treated HUVECs and the inhibitory effect was abolished by specific antiadiponectin antibodies. TG- and Δ2TG-induced suppression on monocyte adhesion were inhibited by a selective AMPK inhibitor compound C. Our data suggest that the inhibitory effect of TG and Δ2TG on monocyte adhesion might be at least in part through de novo adiponectin expression and activation of an AMPK-dependent pathway, which might play an important role in anti-inflammation and antiatherosclerosis.

In acute kidney injury, indoxyl sulfate impairs human endothelial progenitor cells: modulation by statin.

Renal ischemia rapidly mobilizes endothelial progenitor cells (EPCs), which provides renoprotection in acute kidney injury (AKI). Indoxyl sulfate (IS) is a protein-binding uremic toxin with a potential role in endothelial injury. In this study, we examined the effects and mechanisms of action of IS on EPCs in AKI. Forty-one consecutive patients (26 male; age, 70.1 ± 14.1 years) diagnosed with AKI according to the AKIN criteria were enrolled. The AKI patients had higher serum IS levels than patients with normal kidney function (1.35 ± 0.94 × 10(-4)M vs. 0.02 ± 0.02 × 10(-4)M, P < 0.01). IS levels were negatively correlated to the number of double-labeled (CD34(+)KDR(+)) circulating EPCs (P < 0.001). After IS stimulation, the cells displayed decreased expression of phosphorylated endothelial nitric oxide (NO) synthase, vascular cell adhesion molecule-1, increased reactive oxygen species, decreased proliferative capacity, increased senescence and autophagy, as well as decreased migration and angiogenesis. These effects of IS on EPCs were reversed by atorvastatin. Further, exogenous administration of IS significantly reduced EPC number in Tie2-GFP transgenic mice and attenuated NO signaling in aortic and kidney arteriolar endothelium after kidney ischemia-reperfusion injury in mice, and these effects were restored by atorvastatin. Our results are the first to demonstrate that circulating IS is elevated in AKI and has direct effects on EPCs via NO-dependent mechanisms both in vitro and in vivo. Targeting the IS-mediated pathways by NO-releasing statins such as atorvastatin may preempt disordered vascular wall pathology, and represent a novel EPC-rescued approach to impaired neovascularization after AKI.

Norartocarpetin from a folk medicine Artocarpus communis plays a melanogenesis inhibitor without cytotoxicity in B16F10 cell and skin irritation in mice.

BACKGROUND: Many natural products used in preventive medicine have also been developed as cosmeceutical ingredients in skin care products, such as Scutellaria baicalensis and Gardenia jasminoides. Norartocarpetin is one of the antioxidant and antityrosinase activity compound in Artocarpus communis; however, the cytotoxicity, skin irritation and antimelanogenesis mechanisms of norartocarpetin have not been investigated yet. METHODS: In the present study, cell viability in vitro and skin irritation in vivo are used to determine the safety of norartocarpetin. The melanogenesis inhibition of norartocarpetin was determined by cellular melanin content and tyrosinase in B16F10 melanoma cell. Moreover, we examined the related-melanogenesis protein by western blot analysis for elucidating the antimelanogenesis mechanism of norartocarpin. RESULTS: The result of the present study demonstrated that norartocarpetin not only present non-cytotoxic in B16F10 and human fibroblast cells but also non-skin irritation in mice. Moreover, our result also first found that norartocarpetin downregulated phospho-cAMP response element-binding (phospho-CREB) and microphthalmia-associated transcription factor (MITF) expression, which in turn decreased both synthesis of tyrosinases (TRP-1 and TRP-2) and cellular melanin content. This process is dependent on norartocarpetin phosphorylation by mitogen-activated protein kinases such as phospho-JNK and phospho-p38, and it results in decreased melanogenesis. CONCLUSION: The present study suggests that norartocarpetin could be used as a whitening agent in medicine and/or cosmetic industry and need further clinical study.

Ganoderma lucidum polysaccharides prevent platelet-derived growth factor-stimulated smooth muscle cell proliferation in vitro and neointimal hyperplasia in the endothelial-denuded artery in vivo.

Ganoderma lucidum is used in traditional Chinese medicine to prevent or treat a variety of diseases, including cardiovascular disorders. We previously demonstrated that a glucan-containing extract of Reishi polysaccharides (EORP) has the potent anti-inflammatory action of reducing ICAM-1 expression in lipopolysaccharide (LPS)-treated human aortic smooth muscle cells (HASMCs) and LPS-treated mice. In the present study, we examined whether EORP inhibited platelet-derived growth factor-BB (PDGF)-stimulated HASMC proliferation and the mechanism involved. EORP dose-dependently reduced cell numbers and DNA synthesis of PDGF-treated HASMCs in vitro. EORP also arrested cell cycle progression in the G0/G1 phase, and this was associated with decreased expression of cyclin D1, cyclin E, CDK2, CDK4, and p21(Cip1) and upregulation of the cyclin-dependent kinase inhibitor p27(Kip1). The anti-proliferative effect of EORP was partly mediated by downregulation of PDGF-induced JNK phosphorylation. In in vivo studies, the femoral artery of C57BL/6 mice was endothelial-denuded and the mice were fed a diet containing 100 mg/kg/day of EORP. On day 14, both cell proliferation (proliferating cell nuclear antigen-positive cells) in the neointima and the neointima/media area ratio (0.67 ± 0.03 vs. 1.46 ± 0.30) were significantly reduced. Our data show that EORP interferes with the mitogenic activation of JNK, preventing entry of HASMCs into the cell cycle in vitro and reducing cell proliferation in the neointima and decreasing the neointimal area in vivo. Thus, EORP may represent a safe and effective novel approach to the prevention and treatment of vascular proliferative diseases.

Melanogenesis Inhibitor(s) from Phyla nodiflora Extract.

Overexpression of tyrosinase can cause excessive production of melanin and lead to hyperpigmentation disorders, including melasma and freckles. Recently, agents obtained from plants are being used as alternative medicines to downregulate tyrosinase synthesis and decrease melanin production. Phyla nodiflora Greene (Verbenaceae) is used as a folk medicine in Taiwanese for treating and preventing inflammatory diseases such as hepatitis and dermatitis. However, the antimelanogenesis activity and molecular biological mechanism underlying the activity of the methanolic extract of P. nodiflora (PNM) have not been investigated to date. Our results showed that PNM treatment was not cytotoxic and significantly reduced the cellular melanin content and tyrosinase activity in a dose-dependent manner (P < 0.05). Further, PNM exhibited a significant antimelanogenesis effect (P < 0.05) by reducing the levels of phospho-cAMP response element-binding protein and microphthalmia-associated transcription factor (MITF), inhibiting the synthesis of tyrosinase, tyrosinase-related protein-1 (TRP-1), and TRP-2, and decreasing the cellular melanin content. Moreover, PNM significantly activated the phosphorylation of mitogen-activated protein kinases, including phospho-extracellular signal-regulated kinase, c-Jun N-terminal kinase, and phospho-p38, and inhibited the synthesis of MITF, thus decreasing melanogenesis. These properties suggest that PNM could be used as a clinical and cosmetic skin-whitening agent to cure and/or prevent hyperpigmentation.

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.

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.

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.

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.

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.

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.

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.