Klotho protects the heart from hyperglycemia-induced injury by inactivating ROS and NF-κB-mediated inflammation both in vitro and in vivo

Cardiac inflammation and oxidative stress play a key role in the pathogenesis of diabetic cardiomyopathy (DCM). The anti-aging protein Klotho has been found to protect cells from inflammation and oxidative stress. The current study aimed to explore the cardioprotective effects of Klotho on DCM and the underlying mechanisms. H9c2 cells and neonatal cardiomyocytes were incubated with 33 mM glucose in the presence or absence of Klotho. Klotho pretreatment effectively inhibited high glucose-induced inflammation, ROS generation, apoptosis, mitochondrial dysfunction, fibrosis and hypertrophy in both H9c2 cells and neonatal cardiomyocytes. In STZ-induced type 1 diabetic mice, intraperitoneal injection of Klotho at 0.01 mg/kg per 48 h for 3 months completely suppressed cardiac inflammatory cytokines and oxidative stress and prevented cardiac cell death and remodeling, which subsequently improved cardiac dysfunction without affecting hyperglycemia. This study revealed that Klotho may exert its protective effects by augmenting nuclear factor erythroid 2-related factor 2 (Nrf2) expression and inactivating nuclear factor κB (NF-κB) activation both in vitro and in vivo. Thus, this work demonstrated for the first time that the anti-aging protein Klotho may be a potential therapeutic agent to treat DCM by inhibiting oxidative stress and inflammation. We also demonstrated the critical roles of the Nrf2 and NF-κB pathways in diabetes-stimulated cardiac injuries and indicated that they may be key therapeutic targets for diabetic complications.     

Retracted: IRAK3 gene silencing prevents cardiac rupture and ventricular remodeling through negative regulation of the NF-κB signaling pathway in a mouse model of acute myocardial infarction

Cardiac rupture and ventricular remodeling are recognized as the severe complications and major risk factors of acute myocardial infarction (AMI). This study aims to evaluate the regulatory roles of interleukin-1 receptor-associated kinase 3 (IRAK3) and nuclear factor-κB (NF-κB) signaling pathway in cardiac rupture and ventricular remodeling. Microarray analysis was performed to screen AMI-related differentially expressed genes and IRAK3 was identified. The models of AMI were established in male C57BL/6 mice to investigate the functional role of IRAK3. Afterwards, lentivirus recombinant plasmid si-IRAK3 was constructed for IRAK3 silencing. Next, cardiac function parameters were measured in response to IRAK3 silencing. The regulatory effects that IRAK3 had on myocardial infarct size and the content of myocardial interstitial collagen were analyzed. The regulation of IRAK3 silencing on the NF-κB signaling pathway was further assayed. The obtained results indicated that highly expressed IRAK3 and activated NF-κB signaling pathway were observed in myocardial tissues of mouse models of AMI, accompanied by increased expression of matrix metalloproteinase (MMP)-2/9 and tissue inhibitor of metalloproteinase 2 (TIMP-2). Notably, IRAK3 gene silencing inhibited the activation of NF-κB signaling pathway. Furthermore, IRAK3 gene silencing led to the decreased thickness of infarct area and collagen content of myocardial interstitium, alleviated diastolic, and systolic dysfunctions, as well as, facilitated cardiac functions in mice with AMI, corresponding to decreased expression of MMP-2/9 expression and increased expression of TIMP-2. Taken together, silencing of IRAK3 inactivates the NF-κB signaling pathway, and thereby impeding the cardiac rupture and ventricular remodeling, which eventually prevents AMI progression.     

C1q/TNF-related protein-9 promotes macrophage polarization and improves cardiac dysfunction after myocardial infarction

The timely regulation of inflammatory M1 macrophage polarization toward regenerative M2 macrophages suggests the possibility of immunotherapy after myocardial infarction (MI). C1q/TNF-related protein-9 (CTRP9) has anti-inflammatory effects and can ameliorate heart function in mice after long-term myocardial infarction. The role of CTRP9 in macrophage polarization remains completely unclear. This study determined whether CTRP9 can preserve post-MI early cardiac function through the regulation of macrophage polarization. In the present study, an adenovirus-delivered CTRP9 supplement promoted macrophage polarization at Day 3 post MI and improved cardiac function at Day 7 post MI. Pretreatment with gCTRP9 promoted the M1 to M2 polarization transition and attenuated inflammation after lipopolysaccharide + interferon-γ stimulation; the effects were partly abrogated by the adenosine monophosphate kinase (AMPK) inhibitor compound C and were obviously reinforced by pyrrolidine dithiocarbamate, a nuclear factor-κB (NF-κB) inhibitor. Meanwhile, CTPR9 markedly reduced the expression of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and NF-κB p65 phosphorylation by promoting AMPK phosphorylation in vivo and in vitro. Moreover, the competitive binding of gCTRP9 and LPS to the myeloid differentiation protein 2 (MD2)/TLR4 complex was associated with direct binding to MD2, thereby inhibiting the downstream signaling molecule MyD88. Taken together, we demonstrated that CTRP9 improved post-MI early cardiac function, at least in part, by modulating M1/M2 macrophage polarization, largely via the TLR4/MD2/MyD88 and AMPK-NF-κB pathways.     

Pomegranate phytoconstituents blunt the inflammatory cascade in a chemically induced rodent model of hepatocellular carcinogenesis

Liver cancer, predominantly hepatocellular carcinoma (HCC), represents a complex and fatal malignancy driven primarily by oxidative stress and inflammation. Due to dismal prognosis and limited therapeutic intervention, chemoprevention has emerged as a viable approach to reduce the morbidity and mortality of HCC. Pomegranate fruit is a rich source of phytochemicals endowed with potent antioxidant and anti-inflammatory properties. We previously reported that pomegranate phytochemicals inhibit diethylnitrosamine (DENA)-initiated hepatocarcinogenesis in rats though nuclear factor E2-related factor 2 (Nrf2)-mediated antioxidant mechanisms. Since Nrf2 also acts as a key mediator of the nuclear factor-kappaB (NF-κB)-regulated inflammatory pathway, our present study investigated the anti-inflammatory mechanisms of a pomegranate emulsion (PE) during DENA-induced rat hepatocarcinogenesis. Rats were administered with PE (1 or 10 g/kg) 4 weeks before and 18 weeks following DENA initiation. There was a significant increase in hepatic expressions of inducible nitric oxide synthase, 3-nitrotyrosine, heat shock protein 70 and 90, cyclooxygenase-2 and NF-κB in DENA-exposed rat livers. PE dose-dependently suppressed all aforementioned elevated inflammatory markers. A conspicuous finding of this study involves lack of cardiotoxicity of PE as assessed by monitoring cardiac function using noninvasive echocardiography. Our results provide substantial evidence that suppression of the inflammatory cascade through modulation of NF-κB signaling pathway may represent a novel mechanism of liver tumor inhibitory effects of PE against experimental hepatocarcinogenesis. Data presented here coupled with those of our earlier study underline the importance of simultaneously targeting two interconnected molecular circuits, namely, Nrf2-mediated redox signaling and NF-κB-regulated inflammatory pathway, by pomegranate phytoconstituents to achieve chemoprevention of HCC.     

Suppression of NF-κB and NF-κB regulated oxidative stress and neuroinflammation by BAY 11-7082 (IκB phosphorylation inhibitor) in experimental diabetic neuropathy

Inflammation is an emerging patho-mechanism of diabetes and its complications. NF-κB pathway is one of the central machinery initiating and propagating inflammatory responses. The present study envisaged the involvement of NF-κB inflammatory cascade in the pathophysiology of diabetic neuropathy using BAY 11-7082, an IκB phosphorylation inhibitor. Streptozotocin was used to induce diabetes in Sprauge Dawley rats. BAY 11-7082 (1 &; 3 mg/kg) was administered to diabetic rats for 14 days starting from the end of six weeks post diabetic induction. Diabetic rats developed deficits in nerve functions and altered nociceptive parameters and also showed elevated expression of NF-κB (p65), IκB and p-IκB along with increased levels of IL-6 &; TNF-α and inducible enzymes (COX-2 and iNOS). Furthermore, there was an increase in oxidative stress and decrease in Nrf2/HO-1 expression. We observed that BAY 11-7082 alleviated abnormal sensory responses and deficits in nerve functions. BAY 11-7082 also ameliorated the increase in expression of NF-κB, IκB and p-IκB. BAY 11-7082 curbed down the levels of IL-6, TNF-α, COX-2 and iNOS in the sciatic nerve. Lowering of lipid peroxidation and improvement in GSH levels was also seen along with increased expression of Nrf2/HO-1. Thus it can be concluded that NF-κB expression and downstream expression of proinflammatory mediators are prominent features of nerve damage leading to inflammation and oxidative stress and BAY 11-7082 was able to ameliorate experimental diabetic neuropathy by modulating neuroinflammation and improving antioxidant defence.     

Nasunin inhibits the lipopolysaccharide-induced pro-inflammatory mediator production in RAW264 mouse macrophages by suppressing ROS-mediated activation of PI3 K/Akt/NF-κB and p38 signaling pathways

Nasunin is a major anthocyanin in eggplant peel. The purpose of this study was to examine the anti-inflammatory effects of nasunin in lipopolysaccharide (LPS)-stimulated RAW264 macrophages and to identify the molecular mechanisms underlying these effects. We found that nasunin reduced the LPS-induced secretion of tumor necrosis factor-α, interleukin-6 and nitric oxide, and expression of inducible nitric oxide synthase in a dose-dependent manner. Nasunin diminished LPS-induced nuclear factor-κB (NF-κB) activation by suppressing the degradation of inhibitor of κB-α and nuclear translocation of p65 subunit of NF-κB. Nasunin also attenuated the phosphorylation of Akt and p38, signaling molecules involved in pro-inflammatory mediator production. Moreover, nasunin inhibited the intracellular accumulation of ROS, leading to the suppression of NF-κB activation, Akt and p38 phosphorylation, and subsequent pro-inflammatory mediator production. These findings suggest that nasunin exerts an anti-inflammatory effect and this effect is mediated, at least in part, by its antioxidant activity.     

Astragalus polysaccharides alleviates LPS-induced inflammation via the NF-κB/MAPK signaling pathway

Early weaning usually causes intestinal disorders, enteritis, and diarrhea in young animals and human infants. Astragalus polysaccharides (APS) possesses anti-inflammatory activity. To study the anti-inflammatory mechanisms of APS and its potential effects on intestinal health, we performed an RNA sequencing (RNA-seq) study in lipopolysaccharide (LPS)-stimulated porcine intestinal epithelial cells (IPEC-J2) in vitro. In addition, LPS-stimulated BALB/c mice were used to study the effects of APS on intestinal inflammation in vivo. The results from the RNA-seq analysis show that there were 107, 756, and 5 differentially expressed genes in the control versus LPS, LPS versus LPS+APS, and control versus LPS+APS comparison groups, respectively. The results of Kyoto Encyclopedia of Genes and Genomes enrichment analysis indicated that the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways play significant roles in the regulation of inflammatory factors and chemokine expression by APS. Further verification of the above two pathways by using western blot and immunofluorescence analysis revealed that the gene expression levels of the phosphorylated p38 MAPK, ERK1/2, and NF-κB p65 were inhibited by APS, while the expression of IκB-α protein was significantly increased (p < .05), indicating that APS inhibits the production of inflammatory factors and chemokines by the inhibition of activation of the MAPK and NF-κB inflammatory pathways induced by LPS stimulation. Animal experiments further demonstrated that prefeeding APS in BALB/c mice can alleviate the expression of the jejunal inflammatory factors interleukin 6 (IL-6), IL-Iβ, and tumor necrosis factor-α induced by LPS stimulation and improve jejunal villus morphology.     

Renoprotective mechanisms of Astragaloside IV in cisplatin-induced acute kidney injury

Nephrotoxicity remains a serious adverse effect of cisplatin chemotherapy, limiting its clinical usage. Numerous studies show that oxidative stress and inflammation are closely associated with cisplatin-induced renal damage. Astragaloside IV (AS-IV) has been found to possess antioxidant and anti-inflammation functions. Therefore, we investigated the potential curative effects of AS-IV against cisplatin-induced renal injury and the possible cellular mechanism for activity, both in vitro and in vivo. We found that pretreatment of HK-2 cells with AS-IV could mitigate cisplatin-induced cell damage caused by oxygen-free radicals and the inflammatory response, as evidenced by reduced formation of reactive oxygen species (ROS) and inflammatory cytokines. AS-IV improved cisplatin-induced renal dysfunction and histopathological injury in mice. Additionally, AS-IV enhanced the activities of total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), and catalase (CAT). It also inhibited cisplatin-induced overproduction of kidney injury molecule-1 (KIM-1), malondialdehyde (MDA), tumour necrosis factor-α (TNF???α), and interleukin-1β (IL-1β) in kidney tissues. We found that the protective effects of AS-IV occurred via activation of the nuclear factor-erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) proteins and inhibition of nuclear factor-κappaB (NF-κB) activation. Further, small interfering RNA (siRNA) knockdown of Nrf2 abrogated the protective effects of AS-IV against cisplatin-induced oxidative stress and blocked the inhibitory effects of AS-IV on cisplatin-induced NF-κB activation and inflammatory cytokine production. In conclusion, our data suggested that AS-IV attenuated cisplatin-mediated renal injury, and these protective effects might be due to inhibition of both oxidative damage and inflammatory response via activation of Nrf2 system and suppression of NF-κB activation.     

Effects of globin digest and its active ingredient Trp-Thr-Gln-Arg on galactosamine/lipopolysaccharide-induced liver injury in ICR mice

AimsWe investigated the effects of globin digest (GD) and its active ingredient Trp-Thr-Gln-Arg (WTQR) on galactosamine/lipopolysaccharide (GalN/LPS)-induced liver injury in imprinting control region (ICR) mice.Main methodsThe effects of WTQR and GD on the liver injury were examined by measuring the survival rate, serum aminotransferase activities, hepatic components, antioxidant enzyme activities, histopathological analysis, serum levels and hepatic gene expression of tumor necrosis factor-alpha (TNF-α), macrophage inflammatory protein-2 (MIP-2), and nitric oxide (NO) or inducible nitric oxide synthase (iNOS), and nuclear factor-kappa B (NF-κB) p65 content in GalN/LPS-treated ICR mice. RAW264 mouse macrophages were used to confirm the anti-inflammatory effects of WTQR and GD on the macrophages.Key findingsWTQR and GD increased the survival rate, suppressed the serum aminotransferase activities, serum levels and hepatic gene expression of TNF-α, MIP-2, and NO or iNOS, and nuclear NF-κB p65 content in GalN/LPS-treated mice; decreased the oxidized glutathione content, increased the superoxide dismutase activity, and decreased the histopathological grade values of the hepatocyte necrosis and lobular inflammation in GalN/LPS-injured liver; and suppressed the release levels and gene expression of TNF-α, MIP-2, and NO or iNOS, and nuclear NF-κB p65 content in LPS-stimulated RAW264 macrophages. WTQR and GD may improve the antioxidant defense system and inflammatory status in GalN/LPS-injured liver.SignificanceThese findings indicate that WTQR and GD have hepatoprotective effects on GalN/LPS-induced liver injury in ICR mice.     

Depletion of 14-3-3 protein exacerbates cardiac oxidative stress, inflammation and remodeling process via modulation of MAPK/NF-?B signaling pathways after streptozotocin-induced diabetes mellitus

Diabetic cardiomyopathy is associated with increased oxidative stress and inflammation. Mammalian 14-3-3 proteins are dimeric phosphoserine-binding proteins that participate in signal transduction and regulate several aspects of cellular biochemistry. The aim of the study presented here was to clarify the role of 14-3-3 protein in the mitogen activated protein kinase (MAPK) and nuclear factor-kB (NF-κB) signaling pathway after experimental diabetes by using transgenic mice with cardiac-specific expression of a dominant-negative 14-3-3 protein mutant (DN 14-3-3). Significant p-p38 MAPK activation in DN 14-3-3 mice compared to wild type mice (WT) after diabetes induction and with a corresponding up regulation of its downstream effectors, p-MAPK activated protein kinase 2 (MAPKAPK-2). Marked increases in cardiac hypertrophy, fibrosis and inflammation were observed with a corresponding up-regulation of atrial natriuretic peptide, osteopontin, connective tissue growth factor, tumor necrosis factor α, interleukin (IL)-1β, IL-6 and cellular adhesion molecules. Moreover, reactive oxygen species, left ventricular expression of NADPH oxidase subunits, p22 phox, p67 phox, and Nox4, and lipid peroxidation levels were significantly increased in diabetic DN 14-3-3mice compared to diabetic WT mice. Furthermore, myocardial NF-κB activation, inhibitor of kappa B-α degradation and mRNA expression of proinflammatory cytokines were significantly increased in DN 14-3-3 mice compared to WT mice after diabetes induction. In conclusion, our data suggests that depletion of 14-3-3 protein induces cardiac oxidative stress, inflammation and remodeling after experimental diabetes induction mediated through p38 MAPK, MAPKAPK-2 and NF-κB signaling.     

Fisetin,a dietary flavonoid,ameliorates experimental colitis in mice: Relevance of NF-κB signaling

Fisetin, a dietary flavonoid, is commonly found in many fruits and vegetables. Although studies indicate that fisetin has an anti-inflammatory property, little is known about its effects on intestinal inflammation. The present study investigated the effects of the fisetin on dextran sulphate sodium (DSS)-induced murine colitis, an animal model that resembles human inflammatory bowel disease. Fisetin treatment to DSS-exposed mice significantly reduced the severity of colitis and alleviated the macroscopic and microscopic signs of the disease. Moreover, fisetin reduced the levels of myeloperoxidase activity, the production of proinflammatory cytokines, tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) and the expressions of COX-2 and iNOS in the colon tissues. Further studies revealed that fisetin suppressed the activation of NF-κB (p65) by inhibiting IκBα phosphorylation and NF-κB (p65)–DNA binding activity and attenuated the phosphorylation of Akt and the p38, but not ERK and JNK MAPKs in the colon tissues of DSS-exposed mice. In addition, DSS-induced decline in reduced glutathione (GSH) and the increase in malondialdehyde (MDA) levels were significantly restored by oral fisetin. Furthermore, the results from in vitro studies showed that fisetin significantly reduced the pro-inflammatory cytokine and mediator release and suppressed the degradation and phosphorylation of IκBα with subsequent nuclear translocation of NF-κB (p65) in lipopolysaccharide (LPS)-stimulated mouse primary peritoneal macrophages. These results suggest that fisetin exerts anti-inflammatory activity via inhibition of Akt, p38 MAPK and NF-κB signaling in the colon tissues of DSS-exposed mice. Thus, fisetin may be a promising candidate as pharmaceuticals or nutraceuticals in the treatment of inflammatory bowel disease.     

Avicularin alleviates acute liver failure by regulation of the TLR4/MyD88/NF-κB and Nrf2/HO-1/GPX4 pathways to reduce inflammation and ferroptosis

Acute liver failure (ALF) is an inflammation-mediated hepatocyte death process associated with ferroptosis. Avicularin (AL), a Chinese herbal medicine, exerts anti-inflammatory and antioxidative effects. However, the protective effect of AL and the mechanism on ALF have not been reported. Our in vivo results suggest that AL significantly alleviated lipopolysaccharide (LPS)/D-galactosamine (D-GalN)-induced hepatic pathological injury, liver enzymes, inflammatory cytokines, reactive oxygen species and iron levels and increased the antioxidant enzyme activities (malondialdehyde and glutathione). Our further in vitro experiments demonstrated that AL suppressed inflammatory response in LPS-stimulated RAW 264.7 cells via blocking the toll-like receptor 4 (TLR4)/myeloid differentiation protein-88 (MyD88)/nuclear factor kappa B (NF-κB) pathway. Moreover, AL attenuated ferroptosis in D-GalN-induced HepG2 cells by activating the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1)/glutathione peroxidase 4 (GPX4) pathway. Therefore, AL can alleviate inflammatory response and ferroptosis in LPS/D-GalN-induced ALF, and its protective effects are associated with blocking TLR4/MyD88/NF-κB pathway and activating Nrf2/HO-1/GPX4 pathway. Moreover, AL is a promising therapeutic option for ALF and should be clinically explored.     

TP-58, a novel thienopyridine derivative, protects mice from concanavalinA-induced hepatitis by suppressing inflammation

Hepatitis represents a ubiquitous human health problem but effective therapies with limited side effects are still lacking. In this study, we investigated the effect and mechanism of TP-58, a novel thienopyridine derivative, on a murine fulminant hepatitis model induced by concanavalin A (ConA). We found TP-58 markedly alleviated ConA-caused liver injury and increased survival ratio of mice injected with a lethal dose of ConA. Oral administration of TP-58 significantly alleviated ConA-caused liver injury in mice by the reduction of serum aminotransferases and liver necrosis.The analysis of proinflammatory cytokines showed that TP-58 decreased both hepatic mRNA expressions and serum protein levels of TNF-α and IL-6. And the result from LPS-stimulated RAW 264.7 cells showed TP-58 suppressed the production of TNF-α, IL-6, and Nitro Oxide (NO) in the supernatant of LPS-stimulated RAW 264.7 cells. The study of activation of nuclear factor-κB (NF-κB) by electrophoretic mobility shift assay (EMSA) showed that TP-58 inhibited the activation of NF-κB both in vivo and in vitro. The inhibitory effect was also accompanied by a parallel reduction of IκB phosphorylation. These results indicate that TP-58 protects against liver injury by inhibition of the NF-κB-mediated inflammation and suggest a potential role of TP-58 against acute liver injury and other inflammatory diseases.     

Stercurensin inhibits nuclear factor-κB-dependent inflammatory signals through attenuation of TAK1-TAB1 complex formation

We identified a chalcone, 2',4'-dihydroxy-6'-methoxy-3'-methylchalcone (stercurensin), as an active compound isolated from the leaves of Syzygium samarangense. In the present study, the anti-inflammatory effects and underlying mechanisms of stercurensin were examined using lipopolysaccharide (LPS)-stimulated RAW264.7 cells and mice. To determine the effects of stercurensin in vitro, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression were analyzed by RT-PCR and immunoblotting. Nuclear factor-κB (NF-κB) activation and its upstream signaling cascades were also investigated using a dual-luciferase reporter assay, electrophoretic mobility shift assay, immunoblotting, immunofluorescence, and immunoprecipitation. To verify the effects of stercurensin in vivo, the mRNA expression levels of iNOS and COX-2 were evaluated in isolated mouse peritoneal macrophages by quantitative real-time PCR, and the production of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β were assessed in serum samples from mice using a Luminex system. Pretreatment with stercurensin reduced LPS-induced iNOS and COX-2 expression, thereby inhibiting nitric oxide (NO) and prostaglandin E(2) production, respectively. In addition, an inhibitory effect of stercurensin on NF-κB activation was shown by the recovery of LPS-induced inhibitor of κB (I-κB) degradation after blocking the transforming growth factor-β-activated kinase 1 (TAK1)/I-κB kinase signaling pathway. In mouse models, stercurensin negatively regulated NF-κB-dependent pro-inflammatory mediators and cytokines. These results demonstrate that stercurensin modulates NF-κB-dependent inflammatory pathways through the attenuation of TAK1-TAB1 complex formation. Our findings demonstrating the anti-inflammatory effects of stercurensin in vitro and in vivo will aid in understanding the pharmacology and mode of action of stercurensin.