Forsythiaside A Regulates Activation of Hepatic Stellate Cells by Inhibiting NOX4-Dependent ROS.

Hepatic stellate cells (HSCs) activation is an important step in the process of hepatic fibrosis. NOX4 and reactive oxygen species expressed in HSCs play an important role in liver fibrosis. Forsythiaside A (FA), a phenylethanoid glycoside extracted and isolated from Forsythiae Fructus, has significant antioxidant activities. However, it is not clear whether FA can play a role in inhibiting the HSCs activation through regulating NOX4/ROS pathway. Therefore, our purpose is to explore the effect and mechanism of FA on HSCs activation to alleviate liver fibrosis. LX2 cells were activated by TGF-ß1 in vitro. MTT assay and Wound Healing assay were used to investigate the effect of FA on TGF-ß1-induced LX2 cell proliferation and migration. Elisa kit was used to measure the expression of MMP-1 and TIMP-1. Western blot and RT-qPCR were used to investigate the expression of fibrosis-related COLI, α-SMA, MMP-1 and TIMP-1, and inflammation-related TNF-α, IL-6 and IL-1ß. The hydroxyproline content was characterized using a biochemical kit. The mechanism of FA to inhibit HSCs activation and apoptosis was detected by DCF-DA probe, RT-qPCR, western blot and flow cytometry. NOX4 siRNA was used to futher verify the effect of FA on NOX4/ROS pathway. The results showed that FA inhibited the proliferation and migration of LX2 cells and adjusted the expression of MMP-1, TIMP-1, COLI, α-SMA, TNF-α, IL-6 and IL-1ß as well as promoted collagen metabolism to show potential in anti-hepatic fibrosis. Mechanically, FA down-regulated NOX4/ROS signaling pathway to improve oxidation imbalances, and subsequently inhibited PI3K/Akt pathway to suppress proliferation. FA also promoted the apoptosis of LX2 cells by Bax/Bcl2 pathway. Furthermore, the effects of FA on TGF-ß1-induced increased ROS levels and α-SMA and COLI expression were weaken by silencing NOX4. In conclusion, FA had potential in anti-hepatic fibrosis at least in part by remolding of extracellular matrix and improving oxidation imbalances to inhibit the activation of HSCs and promote HSCs apoptosis.

Ginsenoside Rg1 ameliorates aging­induced liver fibrosis by inhibiting the NOX4/NLRP3 inflammasome in SAMP8 mice.

Aging is often accompanied by liver injury and fibrosis, eventually leading to the decline in liver function. However, the mechanism of aging­induced liver injury and fibrosis is still not fully understood, to the best of our knowledge, and there are currently no effective treatment options available for liver aging. Ginsenoside Rg1 (Rg1) has been reported to exert potent anti­aging effects due to its potential antioxidant and anti­inflammatory activity. The present study aimed to investigate the protective effect and underlying mechanism of action of Rg1 in aging­induced liver injury and fibrosis in senescence­accelerated mouse prone 8 (SAMP8) mice treated for 9 weeks. The histopathological results showed that the arrangement of hepatocytes was disordered, vacuole­like degeneration occurred in the majority of cells, and collagen IV and TGF­ß1 expression levels, that were detected via immunohistochemistry, were also significantly upregulated in the SAMP8 group. Rg1 treatment markedly improved aging­induced liver injury and fibrosis, and significantly downregulated the expression levels of collagen IV and TGF­ß1. In addition, the dihydroethylene staining and western blotting results showed that Rg1 treatment significantly reduced the levels of reactive oxygen species (ROS) and IL­1ß, and downregulated the expression levels of NADPH oxidase 4 (NOX4), p47phox, p22phox, phosphorylated­NF­κB, caspase­1, apoptosis­associated speck­like protein containing a C­terminal caspase recruitment domain and the NLR family pyrin domain containing 3 (NLRP3) inflammasome, which were significantly upregulated in the liver tissues of elderly SAMP8 mice. In conclusion, the findings of the present study suggested that Rg1 may attenuate aging­induced liver injury and fibrosis by reducing NOX4­mediated ROS oxidative stress and inhibiting NLRP3 inflammasome activation.

Overlapping roles of NADPH oxidase 4 for diabetic and gadolinium-based contrast agent-induced systemic fibrosis.

Dozens of millions of people are exposed to gadolinium-based contrast agents annually for enhanced magnetic resonance imaging. Gadolinium-based contrast agents are known nephrotoxins and can trigger the potentially fatal condition of systemic fibrosis. Risk factors are practically entirely undefined. We examined the role of NADPH oxidase 4 (Nox4) in gadolinium-induced systemic disease. Age- and weight-matched mice were randomized to experimental diabetes (streptozotocin) and control groups followed by systemic gadolinium-based contrast agent treatment. Nox4-deficient mice were randomized to experimental diabetes and gadolinium-based contrast agent treatment. Skin fibrosis and cellular infiltration were apparent in both gadolinium-based contrast agent-treated and experimental diabetes groups. Similarly, both groups demonstrated renal pathologies with evidence of reactive oxygen species generation. Deletion of Nox4 abrogated both skin and renal pathology, whether from diabetes or gadolinium-based contrast agent treatment. These discoveries demonstrate the importance of Nox4 in gadolinium-based contrast agent- and diabetes-induced fibrosis.NEW & NOTEWORTHY A mouse model of gadolinium-based contrast agent- and diabetes-induced fibrosis was used to demonstrate the role of NADPH oxidase 4 (Nox4) in gadolinium-induced systemic disease. Using these models, we established the role of Nox4 as a mediator of reactive oxygen species generation and subsequent skin and kidney fibrosis. These novel findings have defined Nox-4-mediated mechanisms by which gadolinium-based contrast agents induce systemic diseases.

Thrombin Signaling Contributes to High Glucose-Induced Injury of Human Brain Microvascular Endothelial Cells.

BACKGROUND: Diabetes is one of the strongest disease-related risk factors for Alzheimer's disease (AD). In diabetics, hyperglycemia-induced microvascular complications are the major cause of end-organ injury, contributing to morbidity and mortality. Microvascular pathology is also an important and early feature of AD. The cerebral microvasculature may be a point of convergence of both diseases. Several lines of evidence also implicate thrombin in AD as well as in diabetes. OBJECTIVE: Our objective was to investigate the role of thrombin in glucose-induced brain microvascular endothelial injury. METHODS: Cultured Human brain microvascular endothelial cells (HBMVECs) were treated with 30 mM glucose±100 nM thrombin and±250 nM Dabigatran or inhibitors of PAR1, p38MAPK, MMP2, or MMP9. Cytotoxicity and thrombin activity assays on supernatants and western blotting for protein expression in lysates were performed. RESULTS: reatment of HBMVECs with 30 mM glucose increased thrombin activity and expression of inflammatory proteins TNFα, IL-6, and MMPs 2 and 9; this elevation was reduced by the thrombin inhibitor dabigatran. Direct treatment of brain endothelial cells with thrombin upregulated p38MAPK and CREB, and induced TNFα, IL6, MMP2, and MMP9 as well as oxidative stress proteins NOX4 and iNOS. Inhibition of thrombin, thrombin receptor PAR1 or p38MAPK decrease expression of inflammatory and oxidative stress proteins, implying that thrombin may play a central role in glucose-induced endothelial injury. CONCLUSION: Since preventing brain endothelial injury would preserve blood-brain barrier integrity, prevent neuroinflammation, and retain intact functioning of the neurovascular unit, inhibiting thrombin, or its downstream signaling effectors, could be a therapeutic strategy for mitigating diabetes-induced dementia.

From single drug targets to synergistic network pharmacology in ischemic stroke.

Drug discovery faces an efficacy crisis to which ineffective mainly single-target and symptom-based rather than mechanistic approaches have contributed. We here explore a mechanism-based disease definition for network pharmacology. Beginning with a primary causal target, we extend this to a second using guilt-by-association analysis. We then validate our prediction and explore synergy using both cellular in vitro and mouse in vivo models. As a disease model we chose ischemic stroke, one of the highest unmet medical need indications in medicine, and reactive oxygen species forming NADPH oxidase type 4 (Nox4) as a primary causal therapeutic target. For network analysis, we use classical protein-protein interactions but also metabolite-dependent interactions. Based on this protein-metabolite network, we conduct a gene ontology-based semantic similarity ranking to find suitable synergistic cotargets for network pharmacology. We identify the nitric oxide synthase (Nos1 to 3) gene family as the closest target to Nox4 Indeed, when combining a NOS and a NOX inhibitor at subthreshold concentrations, we observe pharmacological synergy as evidenced by reduced cell death, reduced infarct size, stabilized blood-brain barrier, reduced reoxygenation-induced leakage, and preserved neuromotor function, all in a supraadditive manner. Thus, protein-metabolite network analysis, for example guilt by association, can predict and pair synergistic mechanistic disease targets for systems medicine-driven network pharmacology. Such approaches may in the future reduce the risk of failure in single-target and symptom-based drug discovery and therapy.

Protein kinase B/AKT mediates insulin-like growth factor 1-induced phosphorylation and nuclear export of histone deacetylase 5 via NADPH oxidase 4 activation in vascular smooth muscle cells.

Insulin-like growth factor 1 (IGF-1) mediates the generation of reactive oxygen species (ROS) and the activation of growth promoting signaling pathways. Histone deacetylases (HDACs) regulate gene transcription by deacetylating lysine residues in histone and nonhistone proteins and a heightened HDAC activation, notably of HDAC5, is associated with vascular disorders, such as atherosclerosis. Although the contribution of IGF-1 in these pathologies is well documented, its role in HDAC phosphorylation and activation remains unexplored. Here, we examined the effect of IGF-1 on HDAC5 phosphorylation in vascular smooth muscle cells (VSMCs) and identified the signaling pathways involved in controlling HDAC5 phosphorylation and nuclear export. Treatment of A10 VSMCs with IGF-1 enhanced HDAC5 phosphorylation. Blockade of the IGF-1 receptor tyrosine kinase (TK) activity with the specific pharmacological inhibitor, AG1024, significantly inhibited IGF-1-induced HDAC5 phosphorylation, whereas the epidermal growth factor receptor (EGFR) TK antagonist, AG1478, had no effect. Inhibition of the mitogen-activated protein kinase pathway with U0126, SP600125, or SB203580, did not affect HDAC5 phosphorylation, whereas two inhibitors of the phosphoinositide 3-kinase (PI3K)/AKT pathways, wortmannin and SC66, almost completely attenuated IGF-1-induced responses as confirmed by immunoblotting of phospho-HDAC5 and by small interfering RNA (siRNA)-induced AKT silencing. Moreover, the NAD(P)H oxidase (Nox) inhibitor, diphenyleneiodonium (DPI), and Nox4 siRNA, attenuated IGF-1-induced phosphorylation of HDAC5 and AKT. The HDAC5 phosphorylation resulted in its nuclear export, which was reversed by SC66 and DPI. Our results indicate that IGF-1-induced phosphorylation and nuclear export of HDAC5 involve Nox4-dependent ROS generation and PI3K/AKT signaling pathways.

Sevoflurane Effect on Cognitive Function and the Expression of Oxidative Stress Response Proteins in Elderly Patients undergoing Radical Surgery for Lung Cancer.

OBJECTIVE: To investigate the effects of sustained inhalation of sevoflurane on cognitive function and the expression of oxidative stress response proteins such as NADPH oxidase subunits NOX2 and NOX4 in elderly patients undergoing radical surgery for lung cancer. STUDY DESIGN: An experimental study. PLACE AND DURATION OF STUDY: Department of Anesthesiology, Suzhou Kowloon Hospital, Shanghai Jiao Tong University School of Medicine, China, from February 2016 to October 2017. METHODOLOGY: Elderly patients who underwent radical surgery for lung cancer were divided into the sevoflurane group and the propofol group, with 52 cases in each group. Sustained inhalation of sevoflurane and propofol was administered to maintain anesthesia in the respective groups. Cognitive function and lung function parameters were compared between the two groups. Serum S100 β levels and expression of NOX2 and NOX4 proteins in peripheral blood mononuclear cells of the two groups were determined. RESULTS: At 24 hours after surgery, the lung function indices of the sevoflurane group such as FEV1, FVC and VC were higher than those of the propofol group (p<0.001, p=0.008 and p=0.002, respectively). At the end of the surgery and at 24 hours after surgery, the MMSE scores of the sevoflurane group were higher than the propofol group (all p<0.001). S100 levels were lower than the propofol group (p=0.003 and p<0.001, respectively). Levels of NADPH oxidase subunits NOX2 and NOX4 proteins in peripheral blood mononuclear cells of the sevoflurane group were lower than the propofol group (p=0.033, p<0.001, p<0.001and p<0.001, respectively). CONCLUSION: Compared with intravenous anesthesia with propofol, general anesthesia with sevoflurane inhalation has little effect on the short-term cognitive function in elderly patients undergoing radical surgery for lung cancer, and can effectively improve lung function. The mechanism may be related to the reduction of the expression of NOX2 and NOX4 proteins.

Increased Muscle Mass Protects Against Hypertension and Renal Injury in Obesity.

Background Obesity compromises cardiometabolic function and is associated with hypertension and chronic kidney disease. Exercise ameliorates these conditions, even without weight loss. Although the mechanisms of exercise's benefits remain unclear, augmented lean body mass is a suspected mechanism. Myostatin is a potent negative regulator of skeletal muscle mass that is upregulated in obesity and downregulated with exercise. The current study tested the hypothesis that deletion of myostatin would increase muscle mass and reduce blood pressure and kidney injury in obesity. Methods and Results Myostatin knockout mice were crossed to db/db mice, and metabolic and cardiovascular functions were examined. Deletion of myostatin increased skeletal muscle mass by ≈50% to 60% without concomitant weight loss or reduction in fat mass. Increased blood pressure in obesity was prevented by the deletion of myostatin, but did not confer additional benefit against salt loading. Kidney injury was evident because of increased albuminuria, which was abolished in obese mice lacking myostatin. Glycosuria, total urine volume, and whole kidney NOX-4 levels were increased in obesity and prevented by myostatin deletion, arguing that increased muscle mass provides a multipronged defense against renal dysfunction in obese mice. Conclusions These experimental observations suggest that loss of muscle mass is a novel risk factor in obesity-derived cardiovascular dysfunction. Interventions that increase muscle mass, either through exercise or pharmacologically, may help limit cardiovascular disease in obese individuals.

Protein Kinase G Activation Reverses Oxidative Stress and Restores Osteoblast Function and Bone Formation in Male Mice With Type 1 Diabetes.

Bone loss and fractures are underrecognized complications of type 1 diabetes and are primarily due to impaired bone formation by osteoblasts. The mechanisms leading to osteoblast dysfunction in diabetes are incompletely understood, but insulin deficiency, poor glycemic control, and hyperglycemia-induced oxidative stress likely contribute. Here we show that insulin promotes osteoblast proliferation and survival via the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) signal transduction pathway and that PKG stimulation of Akt provides a positive feedback loop. In osteoblasts exposed to high glucose, NO/cGMP/PKG signaling was reduced due in part to the addition of O-linked N-acetylglucosamine to NO synthase-3, oxidative inhibition of guanylate cyclase activity, and suppression of PKG transcription. Cinaciguat-an NO-independent activator of oxidized guanylate cyclase-increased cGMP synthesis under diabetic conditions and restored proliferation, differentiation, and survival of osteoblasts. Cinaciguat increased trabecular and cortical bone in mice with type 1 diabetes by improving bone formation and osteocyte survival. In bones from diabetic mice and in osteoblasts exposed to high glucose, cinaciguat reduced oxidative stress via PKG-dependent induction of antioxidant genes and downregulation of excess NADPH oxidase-4-dependent H2O2 production. These results suggest that cGMP-elevating agents could be used as an adjunct treatment for diabetes-associated osteoporosis.

Hydrogen Peroxide-Responsive Nanoparticle Reduces Myocardial Ischemia/Reperfusion Injury.

BACKGROUND: During myocardial ischemia/reperfusion (I/R), a large amount of reactive oxygen species (ROS) is produced. In particular, overproduction of hydrogen peroxide (H2O2) is considered to be a main cause of I/R-mediated tissue damage. We generated novel H2O2-responsive antioxidant polymer nanoparticles (PVAX and HPOX) that are able to target the site of ROS overproduction and attenuate the oxidative stress-associated diseases. In this study, nanoparticles were examined for their therapeutic effect on myocardial I/R injury. METHODS AND RESULTS: The therapeutic effect of nanoparticles during cardiac I/R was evaluated in mice. A single dose of PVAX (3 mg/kg) showed a significant improvement in both cardiac output and fraction shortening compared with poly(lactic-coglycolic acid) (PLGA) particle, a non-H2O2-activatable nanoparticle. PVAX also significantly reduced the myocardial infarction/area compared with PLGA (48.7±4.2 vs 14.5±2.1). In addition, PVAX effectively reduced caspase-3 activation and TUNEL-positive cells compared with PLGA. Furthermore, PVAX significantly decreased TNF-α and MCP-1 mRNA levels. To explore the antioxidant effect of PVAX by scavenging ROS, dihydroethidium staining was used as an indicator of ROS generation. PVAX effectively suppressed the generation of ROS caused by I/R, whereas a number of dihydroethidium-positive cells were observed in a group with PLGA I/R. In addition, PVAX significantly reduced the level of NADPH oxidase (NOX) 2 and 4 expression, which favors the reduction in ROS generation after I/R. CONCLUSIONS: Taken together, these results suggest that H2O2-responsive antioxidant PVAX has tremendous potential as a therapeutic agent for myocardial I/R injury.