Aqueous and Alcoholic Extracts of Triphala and Their Active Compounds Chebulagic Acid and Chebulinic Acid Prevented Epithelial to Mesenchymal Transition in Retinal Pigment Epithelial Cells,by Inhibiting SMAD-3 Phosphorylation

Epithelial to Mesenchymal Transition (EMT) of the retinal pigment epithelium is involved in the pathogenesis of proliferative vitreoretinopathy (PVR) that often leads to retinal detachment. In this study, Triphala, an ayurvedic formulation and two of its active ingredients, namely chebulagic acid and chebulinic acid were evaluated for anti-EMT properties based on in vitro experiments in human retinal pigment epithelial cell line (ARPE-19) under TGFβ1 induced conditions. ARPE-19 cells were treated with TGFβ1 alone or co-treated with various concentrations of aqueous extract (AqE) (30 - 300 μg/ml); alcoholic extract (AlE) (50 - 500 μg/ml) of triphala and the active principles chebulagic acid (CA) and chebulinic acid (CI) (CA,CI: 50 - 200 μM). The expression of EMT markers namely MMP-2, αSMA, vimentin and the tight junction protein ZO-1 were evaluated by qPCR, western blot and immunofluorescence. The functional implications of EMT, namely migration and proliferation of cells were assessed by proliferation assay, scratch assay and transwell migration assay. AqE, AlE, CA and CI reduced the expression and activity of MMP-2 at an ED50 value of 100 μg/ml, 50 μg/ml, 100 μM and 100 μM, respectively. At these concentrations, a significant down-regulation of the expression of αSMA, vimentin and up-regulation of the expression of ZO-1 altered by TGFβ1 were observed. These concentrations also inhibited proliferation and migration of ARPE-19 cells induced by TGFβ1. EMT was found to be induced in ARPE-19 cells, through SMAD-3 phosphorylation and it was inhibited by AqE, AlE, CA and CI. Further studies in experimental animals are required to attribute therapeutic potential of these extracts and their active compounds, as an adjuvant therapy in the disease management of PVR.     

Vascular smooth muscle cells from small human omental arteries express P2X1 and P2X4 receptor subunits

Stimulation of P2X receptors by ATP in vascular smooth muscle cells (VSMCs) is proposed to mediate vascular tone. However, understanding of P2X receptor-mediated actions in human blood vessels is limited, and therefore, the current work investigates the role of P2X receptors in freshly isolated small human gastro-omental arteries (HGOAs). Expression of P2X1 and P2X4 receptor subunit messenger RNA (mRNA) and protein was identified in individual HGOA VSMCs using RT-PCR and immunofluorescent analysis and using Western blot in multi-cellular preparations. ATP of 10 μmol/l and αβ-meATP of 10 μmol/l, a selective P2X receptor agonist, evoked robust increases in [Ca²⁺]_i in fluo-3-loaded HGOA VSMCs. Pre-incubation with 1 μmol/l NF279, a selective P2X receptor antagonist, reduced the amplitude of αβ-meATP-induced increase in [Ca²⁺]_i by about 70 %. ATP of 10 μmol/l and αβ-meATP of 10 μmol/l produced similar contractile responses in segments of HGOA, and these contractions were greatly reduced by 2 μmol/l NF449, a selective P2X receptor inhibitor. These data suggest that VSMCs from HGOA express P2X1 and P2X4 receptor subunits with homomeric P2X1 receptors likely serving as the predominant target for extracellular ATP.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-014-9415-6) contains supplementary material, which is available to authorized users.     

PA28α overexpressing female mice maintain exploratory behavior and capacity to prevent protein aggregation in hippocampus as they age

With age, protein damage accumulates and increases the risk of age‐related diseases. The proteasome activator PA28αβ is involved in protein damage clearance during early embryogenesis and has demonstrated protective effects against proteinopathy. We have recently discovered that adult female mice overexpressing PA28α (PA28αOE) have enhanced learning and memory, and protein extracts from their hippocampi prevent aggregation more efficiently than wild type. In this study, we investigated the effect of overexpressing PA28α on aging using C57BL/6N×BALB/c F2 hybrid mice. We found that the hippocampal anti‐aggregation effect was maintained in young adult (7 months) to middle‐aged (15 months) and old (22 months) PA28αOE females. While the PA28αOE influence on learning and memory gradually decreased with aging, old PA28αOE females did not display the typical drop in explorative behavior—a behavioral hallmark of aging—but were as explorative as young mice. PA28αOE lowered PA28‐dependent proteasome capacity in both heart and hippocampus, and there was no indication of lower protein damage load in PA28αOE. The life span of PA28αOE was also similar to wild type. In both wild type and PA28αOE, PA28‐dependent proteasome capacity increased with aging in the heart, while 26S and 20S proteasome capacities were unchanged in the timepoints analyzed. Thus, PA28αOE females exhibit improved hippocampal ability to prevent aggregation throughout life and enhanced cognitive capabilities with different behavioral outcomes dependent on age; improved memory at early age and a youth‐like exploration at old age. The cognitive effects of PA28αβ combined with its anti‐aggregation molecular effect highlight the therapeutical potential of PA28αβ in combating proteinopathies.     

Cytoplasmic and Nuclear Anti-Apoptotic Roles of αB-Crystallin in Retinal Pigment Epithelial Cells

In addition to its well-characterized role in the lens, αB-crystallin performs other functions. Methylglyoxal (MGO) can alter the function of the basement membrane of retinal pigment epithelial (RPE) cells. Thus, if MGO is not efficiently detoxified, it can induce adverse reactions in RPE cells. In this study, we examined the mechanisms underlying the anti-apoptotic activity of αB-crystallin in the human retinal pigment epithelial cell line ARPE-19 following MGO treatment using various assays, including nuclear staining, flow cytometry, DNA electrophoresis, pulse field gel electrophoresis, western blot analysis, confocal microscopy and co-immunoprecipitation assays. To directly assess the role of phosphorylation of αB-crystallin, we used site-directed mutagenesis to convert relevant serine residues to alanine residues. Using these techniques, we demonstrated that MGO induces apoptosis in ARPE-19 cells. Silencing αB-crystallin sensitized ARPE-19 cells to MGO-induced apoptosis, indicating that αB-crystallin protects ARPE-19 cells from MGO-induced apoptosis. Furthermore, we found that αB-crystallin interacts with the caspase subtypes, caspase-2L, -2S, -3, -4, -7, -8, -9 and -12 in untreated control ARPE-19 cells and that MGO treatment caused the dissociation of these caspase subtypes from αB-crystallin; transfection of S19A, S45A or S59A mutants caused the depletion of αB-crystallin from the nuclei of untreated control RPE cells leading to the release of caspase subtypes. Additionally, transfection of these mutants enhanced MGO-induced apoptosis in ARPE-19 cells, indicating that phosphorylation of nuclear αB-crystallin on serine residues 19, 45 and 59 plays a pivotal role in preventing apoptosis in ARPE-19 cells. Taken together, these results suggest that αB-crystallin prevents caspase activation by physically interacting with caspase subtypes in the cytoplasm and nucleus, thereby protecting RPE cells from MGO-induced apoptosis.     

18β-Glycyrrhetinic acid exerts protective effects against cyclophosphamide-induced hepatotoxicity: potential role of PPARγ and Nrf2 upregulation

18β-Glycyrrhetinic acid (18β-GA) has been proposed as a promising hepatoprotective agent. The current study aimed to investigate the protective action and the possible mechanisms of 18β-GA against cyclophosphamide (CP)-induced liver injury in rats, focusing on the role of peroxisome proliferator-activated receptor gamma (PPARγ) and NF-E2-related factor-2 (Nrf2). Rats were administered 18β-GA at doses 25 and 50 mg/kg 2 weeks prior to CP injection. Five days after CP administration, animals were sacrificed and samples were collected. CP induced hepatic damage evidenced by the histopathological changes and significant increase in serum pro-inflammatory cytokines, liver marker enzymes, and liver lipid peroxidation and nitric oxide (NO) levels. 18β-GA counteracted CP-induced oxidative stress and inflammation as assessed by restoration of the antioxidant defenses and diminishing of pro-inflammatory cytokines, lipid peroxidation, and NO production. These hepatoprotective effects appear to depend on activation of Nrf2 and PPARγ, and subsequent suppression of nuclear factor-kappa B. In conclusion, the present study provides evidence that 18β-GA exerts hepatoprotective effects against CP through induction of antioxidant defenses and suppression of inflammatory response. This report also confers new information that 18β-GA protects liver against the toxic effect of chemotherapeutic alkylating agents via activation of Nrf2 and PPARγ.     

Anticholinesterse and antioxidant investigations of crude extracts,subsequent fractions,saponins and flavonoids of atriplex laciniata L.: potential effectiveness in Alzheimer’s and other neurological disorders

Background

Atriplex laciniata L. was investigated for phenolic, flavonoid contents, antioxidant, anticholinesterase activities, in an attempt to explore its effectiveness in Alzheimer’s and other neurological disorders. Plant crude methanolic extract (Al.MeF), subsequent fractions; n-hexane (Al.HxF), chloroform (Al.CfF), ethyl acetate (Al.EaF), aqueous (Al.WtF), Saponins (Al.SPF) and Flavonoids (Al.FLVF) were investigated for DPPH, ABTS and H₂O₂ free radical scavenging activities. Further these extracts were subjected to acetylcholinesterase (AChE) & butyrylcholinesterase (BChE) inhibitory activities using Ellman’s assay. Phenolic and Flavonoid contents were determined and expressed in mg Gallic acid GAE/g and Rutin RTE/g of samples respectively.

Results

In DPPH free radicals scavenging assay, Al.FLVF, Al.SPF and Al.MeF showed highest activity causing 89.41 ± 0.55, 83.37 ± 0.34 and 83.37 ± 0.34% inhibition of free radicals respectively at 1 mg/mL concentration. IC₅₀ for these fractions were 33, 83 and 82 μg/mL respectively. Similarly, plant extracts showed high ABTS scavenging potential, i.e. Al.FLVF (90.34 ± 0.55), Al.CfF (83.42 ± 0.57), Al.MeF (81.49 ± 0.60) with IC₅₀ of 30, 190 and 70 μg/ml respectively. further, H₂O₂ percent scavenging was highly appraised in Al.FLVF (91.29 ± 0.53, IC₅₀ 75), Al.SPF (85.35 ± 0.61, IC₅₀ 70) and Al.EaF (83.48 ± 0.67, IC₅₀ 270 μg/mL). All fractions exhibited concentration dependent AChE inhibitory activity as; Al.FLVF, 88.31 ± 0.57 (IC₅₀ 70 μg/mL), Al.SPF, 84.36 ± 0.64 (IC₅₀ 90 μg/mL), Al.MeF, 78.65 ± 0.70 (IC₅₀ 280 μg/mL), Al.EaF, 77.45 ± 0.46 (IC₅₀ 270 μg/mL) and Al.WtF 72.44 ± 0.58 (IC₅₀ 263 μg/mL) at 1 mg/mL. Likewise the percent BChE inhibitory activity was most obvious in Al.FLVF 85.46 ± 0.62 (IC₅₀ 100 μg/mL), Al.CfF 83.49 ± 0.46 (IC₅₀ 160 μg/mL), Al.MeF 82.68 ± 0.60 (IC₅₀ 220 μg/mL) and Al.SPF 80.37 ± 0.54 (IC₅₀ 120 μg/mL).

Conclusions

These results stipulate that A. laciniata is enriched with phenolic and flavonoid contents that possess significant antioxidant and anticholinestrase effects. This provide pharmacological basis for the presence of compounds that may be effective in Alzheimer’s and other neurological disorders.     

Activation of BV2 microglia by lipopolysaccharide triggers an inflammatory reaction in PC12 cell apoptosis through a toll-like receptor 4-dependent pathway

Microglia play an important role in neuronal protection and damage. However, the molecular and cellular relationship between microglia and neurons is unclear. We carried out a prospective study to detect that activation of BV2 microglia induced PC12 cell apoptosis in vitro through the TLR4/adapter protein myeloid differentiation factor 88 (MyD88)/nuclear factor-κB (NF-κB) signaling pathway. BV2 microglia were treated with different concentrations of LPS for 24 h. Western blot was utilized to detect the expression of TLR4 and the downstream signaling pathway. The level of inflammatory mediator was quantified using a specific ELISA kit. The supernatant of 10 μg/ml LPS-treated BV2 cells was used as conditioned medium (CM). PC12 cells were co-culture with CM for 24 h. Cell viability was determined by MTT assay and cell apoptosis was tested by flow cytometry. BV2 microglia were treated with 10, 20, or 30 μg/ml LPS for 24 h. The expression of TLR4, MyD88, and NF-κB significantly increased. When PC12 cells were co-cultured with CM for 24 h, cell viability decreased. CM up-regulated the Bax level and down-regulated the Bcl-2 protein level in PC12 cells. PC12 cells pretreated with interleukin-1 receptor antagonist (IL-1RA) for 30 min, significantly alleviated CM-induced PC12 cell apoptosis. These results suggest that BV2 microglia activated by LPS triggered TLR4/MyD88/NF-κB signaling pathway that induced the release of IL-1β and could participate in the PC12 cells injury.     

Peripheral inflammation increases seizure susceptibility via the induction of neuroinflammation and oxidative stress in the hippocampus

Background

Neuroinflammation with activation of microglia and production of proinflammatory cytokines in the brain plays an active role in epileptic disorders. Brain oxidative stress has also been implicated in the pathogenesis of epilepsy. Damage in the hippocampus is associated with temporal lobe epilepsy, a common form of epilepsy in human. Peripheral inflammation may exacerbate neuroinflammation and brain oxidative stress. This study examined the impact of peripheral inflammation on seizure susceptibility and the involvement of neuroinflammation and oxidative stress in the hippocampus.

Results

In male, adult Sprague-Dawley rats, peripheral inflammation was induced by the infusion of Escherichia coli lipopolysaccharide (LPS, 2.5 mg/kg/day) into the peritoneal cavity for 7 days via an osmotic minipump. Pharmacological agents were delivered via intracerebroventricular (i.c.v.) infusion with an osmotic minipump. The level of cytokine in plasma or hippocampus was analyzed by ELISA. Redox-related protein expression in hippocampus was evaluated by Western blot. Seizure susceptibility was tested by intraperitoneal (i.p.)  injection of kainic acid (KA, 10 mg/kg). We found that i.p. infusion of LPS for 7 days induced peripheral inflammation characterized by the increases in plasma levels of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). This is associated with a significant increase in number of the activated microglia (Iba-1⁺ cells), enhanced production of proinflammatory cytokines (including IL-1β, IL-6 and TNF-α), and tissue oxidative stress (upregulations of the NADPH oxidase subunits) in the hippocampus. These cellular and molecular responses to peripheral inflammation were notably blunted by i.c.v. infusion of a cycloxygenase-2 inhibitor, NS398 (5 μg/μl/h). The i.c.v. infusion of tempol (2.5 μg/μl/h), a reactive oxygen species scavenger, protected the hippocampus from oxidative damage with no apparent effect on microglia activation or cytokine production after peripheral inflammation. In the KA-induced seizure model, i.c.v. infusion of both NS398 and tempol ameliorated the increase in seizure susceptibility in animals succumbed to the LPS-induced peripheral inflammation.

Conclusions

Together these results indicated that LPS-induced peripheral inflammation evoked neuroinflammation and the subsequent oxidative stress in the hippocampus, resulting in the increase in KA-induced seizure susceptibility. Moreover, protection from neuroinflammation and oxidative stress in the hippocampus exerted beneficial effect on seizure susceptibility following peripheral inflammation.     

β-Glucan from Saccharomyces cerevisiae alleviates oxidative stress in LPS-stimulated RAW264.7 cells via Dectin-1/Nrf2/HO-1 signaling pathway

β-Glucan from Saccharomyces cerevisiae has been described to be effective antioxidants, but the specific antioxidation mechanism of β-glucan is unclear. The objectives of this research were to determine whether the β-glucan from Saccharomyces cerevisiae could regulate oxidative stress through the Dectin-1/Nrf2/HO-1 signaling pathway in lipopolysaccharides (LPS)-stimulated RAW264.7 cells. In this study, we examined the effects of β-glucan on the enzyme activity or production of oxidative stress indicators in LPS-stimulated RAW264.7 cells by biochemical analysis and the protein expression of key factors of Dectin-1/Nrf2/HO-1 signaling pathway by immunofluorescence and western blot. The biochemical analysis results showed that β-glucan increased the LPS-induced downregulation of enzyme activity of intracellular heme oxygenase (HO), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) while decreasing the production of reactive oxygen species (ROS) and malondialdehyde (MDA). Furthermore, immunofluorescence results showed that β-glucan can activate the nuclear factor erythroid 2-related factor 2 (Nrf2). The antioxidant mechanism study indicated that β-glucan activated dendritic-cell-associated C-type lectin 1 (Dectin-1) receptors mediated Nrf2/HO-1 signaling pathway, thereby downregulating the production of ROS and thus produced the antioxidant effects in LPS-stimulated RAW 264.7 cells. In conclusion, these results indicate that β-glucan potently alleviated oxidative stress via Dectin-1/Nrf2/HO-1 in LPS-stimulated RAW 264.7 cells.     

Asprin‐loaded strontium‐containing α‐calcium sulphate hemihydrate/nano‐hydroxyapatite composite promotes regeneration of critical bone defects

Our laboratory originally synthesized strontium(Sr)‐containing α‐calcium sulphate hemihydrate/nano‐hydroxyapatite composite (Sr‐α‐CSH/n‐HA) and demonstrated its ability to repair critical bone defects. This study attempted to incorporate aspirin into it to produce a better bone graft material for critical bone defects. After 5% Sr‐α‐CSH was prepared by coprecipitation and hydrothermal methods, it was mixed with aspirin solution of different concentrations (50 μg/ml, 200 μg/ml, 800 μg/ml and 3200 μg/ml) at a fixed liquid‐solid ratio (0.54 v/w) to obtain aspirin‐loaded Sr‐α‐CSH/n‐HA composite. In vitro experiments were performed on the composite extracts. The tibial defects (3 mm*5 mm) in SD rat model were filled with the composite for 4 weeks and 12 weeks to evaluate its osteogenic capacity in vivo. Our results showed its capability of proliferation, migration and osteogenesis of BMSCs in vitro got improved. In vivo treatment with 800 μg/ml aspirin–loaded Sr‐α‐CSH/n‐HA composite led to significantly more new bone formation in the defects compared with Sr‐α‐CSH/n‐HA composite and significantly promoted the expression of osteogenic‐related genes and inhibited osteoclast activity. In general, our research suggests that aspirin‐loaded Sr‐α‐CSH/n‐HA composite may have a greater capacity of repairing tibial defects in SD rats than simple Sr‐α‐CSH/n‐HA composite.     

Collaboration of geldanamycin-activated P70S6K and Hsp70 against beta-amyloid-induced hippocampal apoptosis: an approach to long-term memory and learning

One of the neuropathological hallmarks of Alzheimer’s disease (AD) is the accumulation of beta-amyloid peptides (Aβ) in senile plaques. Aβ-induced oxidative stress is believed to be responsible for degeneration and apoptosis of neurons and consequent cognitive and memory deficits. Here, we investigated the possible neuroprotective effect of the heat shock protein 90 (Hsp90) inhibitor geldanamycin (GA) against amyloid pathogenesis in adult male Wistar rats. GA or vehicle was injected into the lateral cerebral ventricles of rats 24 h before injection of Aβ (1–42) in CA1 area of hippocampus. The learning and memory of the rats were assessed 7 days after injection of Aβ using passive avoidance (PA) task. As potential contributing factors in Aβ-induced memory decline, we evaluated apoptotic markers and also used terminal-transferase UTP nick end labeling (TUNEL) technique to detect apoptosis in the hippocampus of Aβ-injected rats. Our behavioral data suggest that GA pretreatment can significantly suppress memory deficits in Aβ-injected rats. There was also not only a marked increase in Hsp70 level but also upregulated 70 kDa ribosomal protein S6 kinase (p70S6K) in the hippocampus of GA-treated groups with a reduction in apoptotic factors including caspase-3, poly (ADP-ribose) polymerase, Bax/Bcl-2 ratio, and TUNEL-positive cells as well. Thus, we conclude that GA exerts its protective effects against Aβ (1–42) toxicity and memory deficits, at least in part, by upregulating of Hsp70 and P70S6K.     

Synthesis and biological evaluation of thieno[3,2-c]pyrazol-3-amine derivatives as potent glycogen synthase kinase 3β inhibitors for Alzheimer’s disease

Glycogen synthase kinase 3β (GSK-3β) catalyses the hyperphosphorylation of tau protein in the Alzheimer’s disease (AD) pathology. A series of novel thieno[3,2-c]pyrazol-3-amine derivatives were designed and synthesised and evaluated as potential GSK-3β inhibitors by structure-guided drug rational design approach. The thieno[3,2-c]pyrazol-3-amine derivative 16b was identified as a potent GSK-3β inhibitor with an IC₅₀ of 3.1 nM in vitro and showed accepted kinase selectivity. In cell levels, 16b showed no toxicity on the viability of SH-SY5Y cells at the concentration up to 50 μM and targeted GSK-3β with the increased phosphorylated GSK-3β at Ser9. Western blot analysis indicated that 16b decreased the phosphorylated tau at Ser396 in a dose-dependent way. Moreover, 16b effectively increased expressions of β-catenin as well as the GAP43, N-myc, and MAP-2, and promoted the differentiated neuronal neurite outgrowth. Therefore, the thieno[3,2-c]pyrazol-3-amine derivative 16b could serve as a promising GSK-3β inhibitor for the treatment of AD.     

Randomised,double-blind,placebo-controlled crossover study to investigate different dosing regimens of olodaterol delivered via Respimat? in patients with moderate to severe persistent asthma

Background

A Phase II, multicentre, randomised, double-blind, placebo-controlled, crossover trial comparing the 24-h forced expiratory volume in 1 s (FEV₁) time profile after 3 weeks’ treatment with once-daily (QD) or twice-daily (BID) olodaterol (at the same total daily dose) versus placebo delivered via Respimat® in patients with moderate to severe asthma.

Methods

Patients were randomised to different sequences of olodaterol with 2-week washout, either as a total daily dose of 5 μg (5 μg QD [AM] or 2.5 μg BID) or placebo, or 10 μg (10 μg QD [AM] or 5 μg BID) or placebo. Primary end point was FEV₁ area under the curve from 0 to 24 h (AUC_0–24) response (defined as change from study baseline FEV₁) after 3 weeks. Key secondary end points were FEV₁ AUC_0–12 and AUC_12–24 responses.

Results

Two hundred and six patients received treatment. All olodaterol treatments demonstrated statistically significant improvements in FEV₁ AUC_0–24 response at 3 weeks versus placebo (p < 0.0001); adjusted mean treatment difference versus placebo was 0.191 L for olodaterol 2.5 μg BID (95 % confidence interval [CI] 0.152, 0.229), 0.150 L for 5 μg QD (95 % CI 0.111, 0.189), 0.228 L for 5 μg BID (95 % CI 0.190, 0.266) and 0.209 L for 10 μg QD (95 % CI 0.170, 0.247). These results were supported by the key secondary end points. Olodaterol 5 μg QD provided numerically lower mean values for 24-h bronchodilation than olodaterol 2.5 μg BID (p = 0.0465), with no statistically significant difference between treatment with olodaterol 10 μg QD and 5 μg BID. No relevant differences in morning and evening peak expiratory flow or Asthma Control Questionnaire scores at 3 weeks were observed between different doses and regimens. Adverse events were generally mild to moderate and comparable between groups.

Conclusions

All doses and dose frequencies provided adequate 24-h bronchodilation superior to placebo. Based on the results of this study, it would be reasonable to include both posologies of 5 μg olodaterol daily (5 μg QD or 2.5 μg BID, both delivered in two puffs per dose from the Respimat® inhaler) in subsequent studies. Further studies are necessary to confirm the optimum dosing regimen in asthma. No safety concerns were identified.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01311661","term_id":"NCT01311661"}}NCT01311661

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0243-1) contains supplementary material, which is available to authorized users.     

Hydrolyzed camel whey protein alleviated heat stress-induced hepatocyte damage by activated Nrf2/HO-1 signaling pathway and inhibited NF-κB/NLRP3 axis

Liver damage is the most severe complication of heat stress (HS). Hydrolyzed camel whey protein (CWP) possesses bioactive peptides with obviously antioxidant and anti-inflammatory activities. The current study aims to investigate whether CWP that is hydrolyzed by a simulated gastrointestinal digestion process, named S-CWP, protects BRL-3A hepatocytes from HS-induced damage via antioxidant and anti-inflammatory mechanisms. BRL-3A cells were pretreated with S-CWP before being treated at 43 °C for 1 h, and the levels of the cellular oxidative stress, inflammation, apoptosis, biomarkers for liver function, the activities of several antioxidant enzymes, and the cell viability were analyzed. The expression level of pivotal proteins in correlative signaling pathways was evaluated by western blotting. We confirmed that S-CWP alleviated HS-induced hepatocytes oxidative stress by decreased reactive oxygen species (ROS), nitric oxide (NO), 8-Hydroxy-2′-deoxyguanosine (8-OHdG), lipid peroxidation (LPO), protein carbonylation (PCO), and the activities of NADPH oxidase while enhanced superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), heme oxygenase-1 (HO-1) activities, and GSH content. S-CWP suppressed HS-induced inflammatory response by reducing the phosphorylation of NF-κB p65, the expression of NLRP3, and caspase-1 and finally alleviated caspase-3-mediated apoptosis. S-CWP also alleviated HS-induced hepatocyte injury by reducing alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) levels and restoring Heat Shock Protein 70 (HSP70) expression. Furthermore, S-CWP treatment significantly enhanced the expression of NF-E2-related nuclear factor erythroid-2 (Nrf2) and HO-1. The antioxidant and anti-inflammatory effects of S-CWP were weakened by ML385, a specific Nrf2 inhibitor. Additionally, zinc protoporphyrin (ZnPP), a specific HO-1 inhibitor, significantly reversed S-CWP-induced reduction in the phosphorylation of NF-κB p65. Thus, our results revealed that S-CWP protected against HS-induced hepatocytes damage via activating the Nrf2/HO-1 signaling pathway and inhibiting NF-κB/NLRP3 axis.     

Necrosis-Induced Sterile Inflammation Mediated by Interleukin-1α in Retinal Pigment Epithelial Cells

Endogenous danger signals released from necrotic cells contribute to retinal inflammation. We have now investigated the effects of necrotic cell extracts prepared from ARPE-19 human retinal pigment epithelial cells (ANCE) on the release of proinflammatory cytokines and chemokines by healthy ARPE-19 cells. ANCE were prepared by subjection of ARPE-19 cells to freeze-thaw cycles. The release of various cytokines and chemokines from ARPE-19 cells was measured with a multiplex assay system or enzyme-linked immunosorbent assays. The expression of interleukin (IL)–1α and the phosphorylation and degradation of the endogenous nuclear factor–κB (NF-κB) inhibitor IκB-α were examined by immunoblot analysis. Among the various cytokines and chemokines examined, we found that ANCE markedly stimulated the release of the proinflammatory cytokine IL-6 and the chemokines IL-8 and monocyte chemoattractant protein (MCP)–1 by ARPE-19 cells. ANCE-induced IL-6, IL-8, and MCP-1 release was inhibited by IL-1 receptor antagonist and by an IKK2 inhibitor (a blocker of NF-κB signaling) in a concentration-dependent manner, but was not affected by a pan-caspase inhibitor (Z-VAD-FMK). Recombinant IL-1α also induced the secretion of IL-6, IL-8, and MCP-1 from ARPE-19 cells, and IL-1α was detected in ANCE. Furthermore, ANCE induced the phosphorylation and degradation of IκB-α in ARPE-19 cells. Our findings thus suggest that IL-1α is an important danger signal that is released from necrotic retinal pigment epithelial cells and triggers proinflammatory cytokine and chemokine secretion from intact cells in a manner dependent on NF-κB signaling. IL-1α is therefore a potential therapeutic target for amelioration of sterile inflammation in the retina.