Pyrrolidine dithiocarbamate restores gastric damages and suppressive autophagy induced by hydrogen peroxide

Abstract

It is well known that gastric barrier is very important for protecting host from various insults. Simultaneously, autophagy serving as a prominent cytoprotective and survival pathway under oxidative stress conditions is being increasingly recognized. Thus, this study was conducted for investigating the effect of pyrrolidine dithiocarbamate (PDTC) on gastric barrier function and autophagy under oxidative stress induced by intragastric administration of hydrogen peroxide (H₂O₂). The gastric tight junction proteins [zonula occludens-1 (ZO1), occludin, and claudin1], autophagic proteins [microtubule-associated protein light chain 3I(LC3I), LC3II, and beclin1], and nuclear factor kappa B (NF-κB) signaling pathway (p65 and IκB kinase α/β) were determined by Western blot. The results showed that H₂O₂ exposure disturbed gastric barrier function with decreased expression of ZO1, occludin, and claudin1, and reduced gastric autophagy with decreased conversion of LC3I into LC3II in mice. However, treatment with PDTC restored these adverse effects evidenced by increased expression of ZO1 and claudin1 and increased conversion of LC3I into LC3II. Meanwhile, H₂O₂ exposure decreased normal human gastric epithelial mucosa cell line (GES-1) viability in a concentration-dependent way. However, after being exposed to H₂O₂, GES-1 exhibited autophagic response which was inconsistent with our in vivo results in mice, while PDTC failed to decrease autophagy in GES-1 induced by H₂O₂. Simultaneously, the beneficial effect of PDTC on gastric damage and autophagy in mice might be independent of inhibition of NF-κB. In conclusion, PDTC treatment restores gastric damages and reduced autophagy induced by H₂O₂. Therefore, PDTC may serve as a potential adjuvant therapy for gastric damages.     

Icariside II,a novel phosphodiesterase 5 inhibitor,protects against H2O2‐induced PC12 cells death by inhibiting mitochondria‐mediated autophagy

Oxidative stress is a major cause of cellular injury in a variety of human diseases including neurodegenerative disorders. Thus, removal of excessive reactive oxygen species (ROS) or suppression of ROS generation may be effective in preventing oxidative stress‐induced cell death. This study was designed to investigate the effect of icariside II (ICS II), a novel phosphodiesterase 5 inhibitor, on hydrogen peroxide (H₂O₂)‐induced death of highly differentiated rat neuronal PC12 cells, and to further examine the underlying mechanisms. We found that ICS II pre‐treatment significantly abrogated H₂O₂‐induced PC12 cell death as demonstrated by the increase of the number of metabolically active cells and decrease of intracellular lactate dehydrogenase (LDH) release. Furthermore, ICS II inhibited H₂O₂‐induced cell death through attenuating intracellular ROS production, mitochondrial impairment, and activating glycogen synthase kinase‐3β (GSK‐3β) as demonstrated by reduced intracellular and mitochondrial ROS levels, restored mitochondrial membrane potential (MMP), decreased p‐tyr216‐GSK‐3β level and increased p‐ser9‐GSK‐3β level respectively. The GSK‐3β inhibitor SB216763 abrogated H₂O₂‐induced cell death. Moreover, ICS II significantly inhibited H₂O₂‐induced autophagy by the reducing autophagosomes number and the LC3‐II/LC3‐I ratio, down‐regulating Beclin‐1 expression, and up‐regulating p62/SQSTM1 and HSP60 expression. The autophagy inhibitor 3‐methyl adenine (3‐MA) blocked H₂O₂‐induced cell death. Altogether, this study demonstrated that ICS II may alleviate oxidative stress‐induced autophagy in PC12 cells, and the underlying mechanisms are related to its antioxidant activity functioning via ROS/GSK‐3β/mitochondrial signalling pathways.     

The role of AKT1 and autophagy in the protective effect of hydrogen sulphide against hepatic ischemia/reperfusion injury in mice

Hydrogen sulphide (H₂S) exerts a protective effect in hepatic ischemia-reperfusion (I/R) injury. However, the exact mechanism of H₂S action remains largely unknown. This study was designed to investigate the role of the PtdIns3K-AKT1 pathways and autophagy in the protective effect of H₂S against hepatic I/R injury. Primary cultured mouse hepatocytes and livers with or without NaHS (a donor of H₂S) preconditioning were exposed to anoxia/reoxygenation (A/R) and I/R, respectively. In certain groups, they were also pretreated with LY294002 (AKT1-specific inhibitor), 3-methyladenine (3MA, autophagy inhibitor) or rapamycin (autophagy enhancer), alone or simultaneously. Cell viability, expression of P-AKT1, T-AKT1, LC3 and BECN1 were examined. The severity of liver injury was measured by the levels of serum aminotransferase and inflammatory cytokine, apoptosis and histological examination. GFP-LC3 redistribution and transmission electron microscopy were used to test the activity of autophagy. H₂S preconditioning activated PtdIns3K-AKT1 signaling in hepatocytes. LY294002 could abolish the AKT1 activation and attenuate the protective effect of H₂S on hepatocytes A/R and hepatic I/R injuries. H₂S suppressed hepatic autophagy in vitro and in vivo. Further reducing autophagy by 3MA also diminished the protective effect of H₂S, while rapamycin could reverse the autophagy inhibitory effect and enhance the protective effect of H₂S against hepatocytes A/R and hepatic I/R injuries, consequently. Taken together, H₂S protects against hepatocytic A/R and hepatic I/R injuries, at least in part, through AKT1 activation but not autophagy. An autophagy agonist could be applied to potentiate this hepatoprotective effect by reversing the autophagy inhibition of H₂S.     

Oxidative stress impairs autophagic flux in prion protein-deficient hippocampal cells

We previously reported that autophagy is upregulated in Prnp-deficient (Prnp^0/0) hippocampal neuronal cells in comparison to cellular prion protein (PrP^C)-expressing (Prnp^+/+) control cells under conditions of serum deprivation. In this study, we determined whether a protective mechanism of PrP^C is associated with autophagy using Prnp^0/0 hippocampal neuronal cells under hydrogen peroxide (H₂O₂)-induced oxidative stress. We found that Prnp^0/0 cells were more susceptible to oxidative stress than Prnp^+/+ cells in a dose- and time-dependent manner. In addition, we observed enhanced autophagy by immunoblotting, which detected the conversion of microtubule-associated protein 1 light chain 3 β (LC3B)-I to LC3B-II, and we observed increased punctate LC3B immunostaining in H₂O₂-treated Prnp^0/0 cells compared with H₂O₂-treated control cells. Interestingly, this enhanced autophagy was due to impaired autophagic flux in the H₂O₂-treated Prnp^0/0 cells, while the H₂O₂-treated Prnp^+/+ cells showed enhanced autophagic flux. Furthermore, caspase-dependent and independent apoptosis was observed when both cell lines were exposed to H₂O₂. Moreover, the inhibition of autophagosome formation by Atg7 siRNA revealed that increased autophagic flux in Prnp^+/+ cells contributes to the prosurvival effect of autophagy against H₂O₂ cytotoxicity. Taken together, our results provide the first experimental evidence that the deficiency of PrP^C may impair autophagic flux via H₂O₂-induced oxidative stress.     

Adiponectin Modulates Oxidative Stress-Induced Autophagy in Cardiomyocytes

Diastolic heart failure (HF) i.e., “HF with preserved ejection fraction” (HF-preserved EF) accounts for up to 50% of all HF presentations; however there have been no therapeutic advances. This stems in part from an incomplete understanding about HF-preserved EF. Hypertension is the major cause of HF-preserved EF whilst HF-preserved EF is also highly associated with obesity. Similarly, excessive reactive oxygen species (ROS), i.e., oxidative stress occurs in hypertension and obesity, sensitizing the heart to the renin-angiotensin-aldosterone system, inducing autophagic type-II programmed cell death and accelerating the propensity to adverse cardiac remodeling, diastolic dysfunction and HF. Adiponectin (APN), an adipokine, mediates cardioprotective actions but it is unknown if APN modulates cardiomyocyte autophagy. We tested the hypothesis that APN ameliorates oxidative stress-induced autophagy in cardiomyocytes. Isolated adult rat ventricular myocytes were pretreated with recombinant APN (30µg/mL) followed by 1mM hydrogen peroxide (H₂O₂) exposure. Wild type (WT) and APN-deficient (APN-KO) mice were infused with angiotensin (Ang)-II (3.2mg/kg/d) for 14 days to induced oxidative stress. Autophagy-related proteins, mTOR, AMPK and ERK expression were measured. H₂O₂ induced LC3I to LC3II conversion by a factor of 3.4±1.0 which was abrogated by pre-treatment with APN by 44.5±10%. However, neither H₂O₂ nor APN affected ATG5, ATG7, or Beclin-1 expression. H₂O₂ increased phospho-AMPK by 49±6.0%, whilst pretreatment with APN decreased phospho-AMPK by 26±4%. H₂O₂ decreased phospho-mTOR by 36±13%, which was restored by APN. ERK inhibition demonstrated that the ERK-mTOR pathway is involved in H₂O₂-induced autophagy. Chronic Ang-II infusion significantly increased myocardial LC3II/I protein expression ratio in APN-KO vs. WT mice. These data suggest that excessive ROS caused cardiomyocyte autophagy which was ameliorated by APN by inhibiting an H₂O₂-induced AMPK/mTOR/ERK-dependent mechanism. These findings demonstrate the anti-oxidant potential of APN in oxidative stress-associated cardiovascular diseases, such as hypertension-induced HF-preserved EF.     

Autophagy induction is a survival response against oxidative stress in bone marrow–derived mesenchymal stromal cells

Background aimsBone marrow–derived mesenchymal stromal cells (BMSCs) are being extensively investigated as cellular therapeutics for many diseases, including cardiovascular diseases. Although preclinical studies indicated that BMSC transplantation into infarcted hearts improved heart function, there are problems to be resolved, such as the low survival rate of BMSCs during the transplantation process and in the ischemic region with extreme oxidative stress. Autophagy plays pivotal roles in maintaining cellular homeostasis and defending against environmental stresses. However, the precise roles of autophagy in BMSCs under oxidative stress remain largely uncharacterized.MethodsBMSCs were treated with H₂O₂, and autophagic flux was examined by means of microtubule-associated protein 1A/1B-light chain 3 II/I ratio (LC3 II/I), autophagosome formation and p62 expression. Cytotoxicity and cell death assays were performed after co-treatment of BMSCs by autophagy inhibitor (3-methyladenine) or autophagy activator (rapamycin) together with H₂O₂.ResultsWe show that short exposure (1 h) of BMSCs to H₂O₂ dramatically elevates autophagic flux (2- to 4-fold), whereas 6-h prolonged oxidative treatment reduces autophagy but enhances caspase-3 and caspase-6–associated apoptosis. Furthermore, we show that pre- and co-treatment with rapamycin ameliorates H₂O₂-induced caspase-3 and caspase-6 activation and cell toxicity but that 3-methyladenine exacerbates H₂O₂-induced cell apoptotic cell death.ConclusionsOur results demonstrate that autophagy is critical for the survival of BMSCs under oxidative conditions. Importantly, we also suggest that the early induction of autophagic flux is possibly a self-defensive mechanism common in oxidant-tolerant cells.     

Induction of nuclear factor-κB signal-mediated apoptosis and autophagy by reactive oxygen species is associated with hydrogen peroxide-impaired growth performance of broilers

The oxidative study has always been particularly topical in poultry science. However, little information about the occurrence of cellular apoptosis and autophagy through the reactive oxygen species (ROS) generation in nuclear factor-κB (NF-κB) signal pathway was reported in the liver of broilers exposed to hydrogen peroxide (H₂O₂). So we investigated the change of growth performance of broilers exposed to H₂O₂ and further explored the occurrence of apoptosis and autophagy, as well as the expression of NF-κB in these signaling pathways in the liver. A total of 320 1-day-old Arbor Acres male broiler chickens were raised on a basal diet and randomly divided into five treatments which were arranged as non-injected treatment (Control), physiological saline (0.75%) injected treatment (Saline) and H₂O₂ treatments (H₂O_2(0.74), H₂O_2(1.48) and H₂O_2(2.96)) received an intraperitoneal injection of H₂O₂ with 0.74, 1.48 and 2.96 mM/kg BW. The results showed that compared to those in the control and saline treatments, 2.96 mM/kg BW H₂O₂-treated broilers exhibited significantly higher feed/gain ratio at 22 to 42 days and 1 to 42 days, ROS formation, the contents of oxidation products, the mRNA expressions of caspases (3, 6, 8), microtubule-associated protein 1 light chain 3 (LC3)-II/LC3-I, autophagy-related gene 6, Bcl-2 associated X and protein expressions of total caspase-3 and total LC3-II, and significantly lower BW gain at 22 to 42 days and 1 to 42 days, the activities of total superoxide dismutase and glutathione peroxidase, the expression of NF-κB in the liver. Meanwhile, significantly higher feed/gain ratio at 1 to 42 days, ROS formation, the contents of protein carbonyl and malondialdehyde, the mRNA expression of caspase-3 and the protein expressions of total caspase-3 and total LC3-II, as well as significantly lower BW gain at 22 to 42 days and 1 to 42 days were observed in broilers received 1.48 mM/kg BW H₂O₂ treatment than those in control and saline treatments. These results indicated that oxidative stress induced by H₂O₂ had a negative effect on histomorphology and redox status in the liver of broilers, which was associated with a decline in growth performance of broilers. This may attribute to apoptosis and autophagy processes triggered by excessive ROS that suppress the NF-κB signaling pathway.     

1,25-Dyhydroxyvitamin D3 attenuates rotenone-induced neurotoxicity in SH-SY5Y cells through induction of autophagy

Background and objectives

Dysregulation of the autophagy pathway has been suggested as an important mechanism in the pathogenesis of Parkinson’s disease (PD). Therefore, modulation of autophagy may be a novel strategy for the treatment of PD. Recently, an active form of vitamin D₃ has been reported to have neuroprotective properties. Therefore, we investigated the protective, autophagy-modulating effects of 1,25-dyhydroxyvitamin D₃ (calcitriol) in an in vitro model of Parkinson’s disease.

Methods

An in vitro model of Parkinson’s disease, the rotenone-induced neurotoxicity model in SH-SY5Y cells was adapted. We measured cell viability using an MTT assay, Annexin V/propidium iodide assay, and intracellular reactive oxygen species levels and analyzed autophagy-associated intracellular signaling proteins by Western blotting.

Results

Rotenone treatment of SH-SY5Y cells reduced their viability. This treatment also increased reactive oxygen species levels and decreased levels of intracellular signaling proteins associated with cell survival; simultaneous exposure to calcitriol significantly reversed these effects. Additionally, calcitriol increased levels of autophagy markers, including LC3, beclin-1, and AMPK. Rotenone inhibited autophagy, as indicated by decreased beclin-1 levels and increased mTOR levels, and this effect was reversed by calcitriol treatment.

Discussion

Calcitriol protects against rotenone-induced neurotoxicity in SH-SY5Y cells by enhancing autophagy signaling pathways such as those involving LC3 and beclin-1. These neuroprotective effects of calcitriol against rotenone-induced dopaminergic neurotoxicity provide an experimental basis for its clinical use in the treatment of PD.     

Are mitochondrial reactive oxygen species required for autophagy?

Reactive oxygen species (ROS) are said to participate in the autophagy signaling. Supporting evidence is obscured by interference of autophagy and apoptosis, whereby the latter heavily relies on ROS signaling. To dissect autophagy from apoptosis we knocked down expression of cytochrome c, the key component of mitochondria-dependent apoptosis, in HeLa cells using shRNA. In cytochrome c deficient HeLa1.2 cells, electron transport was compromised due to the lack of electron shuttle between mitochondrial respiratory complexes III and IV. A rapid and robust LC3-I/II conversion and mitochondria degradation were observed in HeLa1.2 cells treated with staurosporine (STS). Neither generation of superoxide nor accumulation of H₂O₂ was detected in STS-treated HeLa1.2 cells. A membrane permeable antioxidant, PEG-SOD, plus catalase exerted no effect on STS-induced LC3-I/II conversion and mitochondria degradation. Further, STS caused autophagy in mitochondria DNA-deficient ρ° HeLa1.2 cells in which both electron transport and ROS generation were completely disrupted. Counter to the widespread view, we conclude that mitochondrial ROS are not required for the induction of autophagy.     

Calcineurin suppresses AMPK-dependent cytoprotective autophagy in cardiomyocytes under oxidative stress

Calcineurin signalling plays a critical role in the pathogenesis of many cardiovascular diseases. Calcineurin has been proven to affect a series of signalling pathways and to exert a proapoptotic effect in cardiomyocytes. However, whether it is able to regulate autophagy remains largely unknown. Here, we report that prolonged oxidative stress-induced activation of calcineurin contributes to the attenuation of adaptive AMP-activated protein kinase (AMPK) signalling and inhibits autophagy in cardiomyocytes. Primary cardiomyocytes exhibited rapid formation of autophagosomes, microtubule-associated protein 1 light chain 3 (LC3) expression and phosphorylation of AMPK in response to hydrogen peroxide (H₂O₂) treatment. However, prolonged (12 h) H₂O₂ treatment attenuated these effects and was accompanied by a significant increase in calcineurin activity and apoptosis. Inhibition of calcineurin by FK506 restored AMPK function and LC3 expression, and decreased the extent of apoptosis caused by prolonged oxidative stress. In contrast, overexpression of the constitutively active form of calcineurin markedly attenuated the increase in LC3 induced by short-term (3 h) H₂O₂ treatment and sensitised cells to apoptosis. In addition, FK506 failed to induce autophagy and alleviate apoptosis in cardiomyocytes expressing a kinase-dead K45R AMPK mutant. Furthermore, inhibition of autophagy by 3-methylanine (3-MA) or by knockdown of the essential autophagy-related gene ATG7 abrogated the protective effect of FK506. These findings suggest a novel role of calcineurin in suppressing adaptive autophagy during oxidative stress by downregulating the AMPK signalling pathway. The results also provide insight into how altered calcineurin and autophagic signalling is integrated to control cell survival during oxidative stress and may guide strategies to prevent cardiac oxidative damage.     

Exogenous hydrogen sulfide protects fatty liver against ischemia–reperfusion injury by regulating endoplasmic reticulum stress‐induced autophagy in macrophage through mediating the class A scavenger receptor pathway in rats

Fatty liver disease is a disease manifested with excessive alcohol intake and obese. Importantly, hydrogen sulfide (H₂S) has been revealed to participate in the progression of fatty liver; however, the underlying mechanism has not been clearly elucidated yet. In this study, we aimed to investigate the effects of exogenous H₂S on fatty liver ischemia–reperfusion injury (IRI) through mediating class A scavenger receptor (SRA) pathway in rats. By determining endoplasmic reticulum stress (ERS)‐related factors, autophagy markers and apoptosis‐related factors in liver tissue and liver function, levels of oxidative stress, inflammatory factors, and hepatocyte apoptosis, the effects of H₂S on IRI‐induced autophagy, oxidative stress, and inflammation were all examined in rat model of fatty liver IRI. Results from obtained data showed that H₂S decreased the expression of SRA, Grp78, PERK, CHOP, and Caspase‐3, and increased that of LC3‐II/LC3‐I, in addition to alleviating the pathological changes of liver and reducing the levels of ALT, AST, LDH TBARS, and MDA. Moreover, H₂S decreased the levels of oxidative stress, the expression of pro‐inflammatory factors including tumor necrosis factor α, interleukin 1, and interleukin 6, and the apoptosis of hepatocytes. Our findings suggested exogenous H₂S could reduce ERS by mediating the SRA pathway and protect liver function by inducing autophagy, and protect against IRI by reducing oxidative stress and inflammation.     

Liraglutide alleviates H2O2-induced retinal ganglion cells injury by inhibiting autophagy through mitochondrial pathways

Retinal ganglion cells (RGCs), which exist in the inner retina, are the retinal neurons which can be damaged in the early stage of diabetic retinopathy (DR). Liraglutide, a glucagon-like peptide-1 (GLP-1) analog, exerts biological functions by binding the receptor (GLP-1R), the expression of which in RGC-5 cells was first shown by our team in 2012. It was reported that liraglutide prevented retinal neurodegeneration in diabetic subjects. However, the involvement of mechanisms such as autophagy and mitochondrial balance in liraglutide-induced retinal protection is unknown. Here, we aimed to investigate the protective effects of liraglutide and explore the potential mechanisms of liraglutide-induced retinal RGC protection. RGC-5 cells were treated with H₂O₂ and/or liraglutide. Cell viability was detected with the CCK-8 kit. The axon marker GAP43, autophagy and mitophagy indicators LC3A/B, Beclin-1, p62, Parkin, BCL2/Adenovirus E1B 19 kDa protein-interacting protein 3-like (BNIP3L) and the key regulator of mitochondrial biogenesis PGC-1α were examined via western blot analysis. Autophagy was also evaluated using the ImageXpress Micro XLS system and transmission electron microscopy (TEM). Reactive oxygen species (ROS), mitochondrial membrane potential and fluorescent staining for mitochondria were also measured using the ImageXpress Micro XLS system. Our results showed that pretreatment with liraglutide significantly prevented H₂O₂-induced cell viability decline, mitochondrial morphological deterioration and induction of autophagy, which appeared as increased expression of LC3 II/I and Beclin-1, along with p62 degradation. Moreover, liraglutide suppressed the H₂O₂-induced decline in GAP43 expression, thus protecting cells. However, rapamycin induced autophagy and blocked the protective process. Liraglutide also provided mitochondrial protection and appeared to alleviate H₂O₂-induced ROS overproduction and a decline in mitochondrial membrane potential, partially by promoting mitochondrial generation and attenuating mitophagy. In conclusion, liraglutide attenuates H₂O₂ induced RGC-5 cell injury by inhibiting autophagy through maintaining a balance between mitochondrial biogenesis and mitophagy.     

Effect of main ingredients of Danhong Injection against oxidative stress induced autophagy injury via miR-19a/SIRT1 pathway in endothelial cells

BackgroundAutophagy plays an important role in cellular homeostasis. Oxidative stress stimulated endothelial excessive autophagy has been proposed as a major risk factor for cardiovascular diseases (CVD). Danhong injection (DHI), the most prescribed traditional Chinese medicine for the treatment of CVD, has been shown to elicit vascular protective effects. However, its underlying mechanisms remain poorly defined. This study aimed to uncover the protective effects of DHI and its main bioactive components on autophagy injury of human umbilical vein endothelial cells (HUVECs) induced by H₂O₂ and reveal the possible mechanisms.MethodsHUVECs were treated with different concentrations of DHI or its components, after exposed to H₂O₂. The protective effects of DHI and its components in H₂O₂-induced HUVECs were examined via a cytotoxicity assay and western blot. Apoptosis was evaluated with flow cytometry. Autophagy flux was assessed by transmission electron microscopy and LC3 plasmid transfection. Besides, the role miR-19a and SIRT1 in DHI and components-mediated anti-autophagy responses were validated with inhibitors transfection.ResultsOur results showed that DHI and its components do have different effects on different aspects. In terms of HUVECs survival rate, Salvianolic acid B (Sal B) and danshensu (DSS) performed better than DHI, Hydroxysafflor yellow A (HSYA) and Tanshinone IIA (DST-IIA). As for the proliferation effect on HUVECs, only Sal B has the most obvious performance as same as 3MA. Besides, DHI and its components are sensitive and superior in regulating and balancing ROS concentration. Among the GSH/GSSG indicators, DSS and HSYA performed better. In terms of SOD content and apoptotic rate, the SOD level showed the opposite trend compared with H₂O₂ group. For the expression of LC3, Beclin-1 and P62, DHI and its components all had significant effects. When miR-19a or SIRT1 was inhibited, Sal B (0.5 μg/ml) can not decrease autophagy-related protein effectively.ConclusionDHI and its components all had anti-autophagy effects. And Sal B (0.5 μg/ml) inhibited HUVECs autophagy via miR-19a/SIRT1 pathway.     

Curcumin induces autophagy to protect vascular endothelial cell survival from oxidative stress damage

Our study first proposed that curcumin could protect human endothelial cells from the damage caused by oxidative stress via autophagy. Furthermore, our results revealed that curcumin causes some novel cellular mechanisms that promote autophagy as a protective effect. Pretreatment with curcumin remarkably improves the survival of human umbilical vein endothelial cells (HUVECs) from H₂O₂-induced viability loss, which specifically evokes an autophagic response. Exposed to H₂O₂, curcumin-treated HUVECs upregulate the level of microtubule-associated protein 1 light chain 3-II (LC3-II), the number of autophagosomes, and the degradation of p62. We show that this compound promotes BECN1 expression and inhibits the phosphatidylinositol 3-kinase (PtdIns3K)-AKT-mechanistic target of rapamycin (MTOR) signaling pathway. Curcumin can also reverse FOXO1 (a mediator of autophagy) nuclear localization along with causing an elevated level of cytoplasmic acetylation of FOXO1 and the interaction of acetylated FOXO1 and ATG7, under the circumstance of oxidative stress. Additionally, knockdown of FOXO1 by shRNA inhibits not only the protective effects that curcumin induced, but the autophagic process, from the quantity of LC3-II to the expression of RAB7. These results suggest that curcumin induces autophagy, indicating that curcumin has the potential for use as an autophagic-related antioxidant for prevention and treatment of oxidative stress. These data uncover a brand new protective mechanism involving FOXO1 as having a critical role in regulating autophagy in HUVECs, and suggest a novel role for curcumin in inducing a beneficial form of autophagy in HUVECs, which may be a potential multitargeted therapeutic avenue for the treatment of oxidative stress-related cardiovascular diseases.     

Trolox protection of myelin membrane in hydrogen peroxide-treated mature oligodendrocytes

Oligodendrocytes have the highest rate of metabolic activity in the brain and are highly vulnerable to oxidative stress. In this work we determined the protective effect of Trolox, a water-soluble analogue of vitamin E, and insulin, a peptide shown to be neuroprotective, in oligodendrocyte lesion induced by hydrogen peroxide (H₂O₂). Exposure of primary cultures of rat oligodendrocytes to H₂O₂ dose-dependently decreased their reducing capacity, as determined by the MTT assay. H₂O₂ (100 μM) had no effect on Bax levels, active-caspase-3, DNA fragmentation or lactate dehydrogenase (LDH) leakage. Nevertheless, under these conditions, H₂O₂ decreased the levels of myelin basic protein (MBP), used as a marker for oligodendrocyte myelin membrane. Treatment with insulin alone increased MBP levels, but no changes were observed in the presence of insulin plus H₂O₂. In contrast, incubation with Trolox completely prevented H₂O₂-induced decrease in MBP expression, suggesting that vitamin E analogues may prevent against oligodendrocyte oxidative damage.     

MiR‐19a Affects Hepatocyte Autophagy via Regulating lncRNA NBR2 and AMPK/PPARα in D‐GalN/Lipopolysaccharide‐Stimulated Hepatocytes

This study aims to evaluate the potential involvement and regulatory mechanism of miR‐19a in hepatocytes autophagy of acute liver failure (ALF). The in vitro hepatocytes injury model of primary hepatocyte and hepatocytes line HL‐7702 was established by D‐galactosamine (D‐GalN) and lipopolysaccharide (LPS) co‐treatment. Relative expression level of miR‐19a and NBR2 was determined by qRT‐PCR. Protein expression of AMPK/PPARα and autophagy‐related gene was determined by Western blot. In hepatic tissue of 20 ALF patients and D‐GalN/LPS‐stimulated hepatocytes, miR‐19a was upregulated and NBR2 was downregulated. D‐GalN/LPS stimulation caused the inactivation of AMPK/PPARα signaling and the decrease of autophagy‐related LC3‐II/LC3‐I ratio and beclin‐1 expression in hepatocytes. The expression of both AMPK/PPARα and NBR2 were negatively controlled by miR‐19a overexpression or knockdown. Moreover, both NBR2 and PPARα were targeted regulated by miR‐19a according to luciferase reporter assay. In D‐GalN/LPS‐stimulated hepatocytes, AMPK activation promoted PPARα expression. AMPK inactivation inhibited the pro‐autophagy effect of miR‐19a and caused the decrease of LC3‐II/LC3‐I ratio and beclin‐1 expression. PPARα activation abrogated the anti‐autophagy effect of miR‐19a mimic and caused the increase of LC3‐II/LC3‐I ratio and beclin‐1 expression. NBR2 knockdown reversed the anti‐autophagy impact of miR‐19a inhibitor and caused the decrease of LC3‐II/LC3‐I ratio and beclin‐1 expression. In summary, our data suggested that miR‐19a negatively controlled the autophagy of hepatocytes attenuated in D‐GalN/LPS‐stimulated hepatocytes via regulating NBR2 and AMPK/PPARα signaling. J. Cell. Biochem. 119: 358–365, 2018. © 2017 Wiley Periodicals, Inc.