MicroRNA-152 attenuates neuroinflammation in intracerebral hemorrhage by inhibiting thioredoxin interacting protein (TXNIP)-mediated NLRP3 inflammasome activation

Neuroinflammation significantly contributes to brain injury and neurological deterioration following intracerebral hemorrhage (ICH). MicroRNA-152(miR-152) was reported to be downregulated in ICH patients and to possess anti-inflammatory properties in other diseases. In this study, we aimed to explore the role of miR-152 in ICH, and the underlying mechanisms, using a collagenase-induced rat ICH model and hemin-exposure as a cell model. We first confirmed that miR-152 was consistently downregulated in both models. Overexpression of miR-152 in microglial BV2 cells reduced hemin-induced inflammatory response and reactive oxygen species (ROS) generation, thus protecting co-cultured neuronal HT22 cells. Moreover, overexpression of miR-152 by intracerebroventricular lentivirus injection in ICH rats significantly alleviated neurodecifits, brain edema, and hematoma. These changes were associated with a marked reduction in ICH-induced neuronal death, as detected by co-staining of NeuN and TUNEL, and ICH-induced neuroinflammation, as revealed by inflammatory cytokine levels as well as by the number of Iba1 positive-stained cells in the perihematomal region. Mechanistically, miR-152 significantly inhibited ICH-induced TXNIP expression, and its overexpression blocked the interaction between TXNIP and NOD-like receptor pyrin domain containing 3(NLRP3), thus inhibiting NLRP3-driven inflammasome activation to attenuate neuroinflammation in vivo and in vitro. Moreover, the results of si-TXNIP transfection further confirmed that TXNIP inhibition was involved in the reduction of NLRP3 inflammasome activation by the overexpression of miR-152. Collectively, the present study demonstrates that miR-152 confers protection against ICH-induced neuroinflammation and brain injury by inhibiting TXNIP-mediated NLRP3 inflammasome activation, indicating a potential strategy for ICH treatment.     

Carnosic Acid Prevents 6-Hydroxydopamine-Induced Cell Death in SH-SY5Y Cells via Mediation of Glutathione Synthesis

Understanding the neuroprotective effects of the rosemary phenolic diterpene carnosic acid (CA) has attracted increasing attention. We explored the mechanism by which CA modulates the neurotoxic effects of 6-hydroxydopamine (6-OHDA) in SH-SY5Y cells. Cells were pretreated with CA for 12 h followed by treatment with 100 μM 6-OHDA for 12 or 24 h. Cell viability determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolim bromide (MTT) assay indicated that 0.1 to 1 μM CA dose-dependently attenuated the cell death induced by 6-OHDA, whereas the effect of 3-5 μM CA was weaker. CA at 1 μM suppressed the 6-OHDA-induced nuclear condensation, reactive oxygen species generation, and cleavage of caspase 3 and PARP. Immunoblots showed that the phosphorylation of c-Jun NH(2)-terminal kinase (JNK) and p38 by 6-OHDA was reduced in the presence of CA. Incubation of cells with CA resulted in significant increases in the total glutathione (GSH) level and the protein expression of the γ-glutamylcysteine ligase catalytic subunit and modifier subunit. l-Buthionine-sulfoximine, an inhibitor of GSH synthesis, attenuated the effect of CA on cell death and apoptosis. Treatment with CA also led to an increase in nuclear factor erythroid-2 related factor 2 (Nrf2) activation, antioxidant response element (ARE)-luciferase reporter activity, and DNA binding to the ARE. Silencing of Nrf2 expression alleviated the reversal of p38 and JNK1/2 activation by CA. These results suggest that the attenuation of 6-OHDA-induced apoptosis by CA is associated with the Nrf2-driven synthesis of GSH, which in turn down-regulates the JNK and p38 signaling pathways. The CA compound may be a promising candidate for neuroprotection in Parkinson's disease.     

Activation of Nrf2 pathway and inhibition of NLRP3 inflammasome activation contribute to the protective effect of chlorogenic acid on acute liver injury

Chlorogenic acid (CGA), a kind of polyphenol found in coffee, fruits and vegetables, has potent anti-oxidant and anti-inflammatory properties. Our previous studies showed CGA could efficiently alleviate liver fibrosis in rats. However, whether CGA regulates nuclear factor erythroid-2-related factor 2 (Nrf2) anti-oxidant pathway and NLRP3 inflammasome activation and protects against carbon tetrachloride (CCl₄)-induced acute liver injury are unknown. We found that CGA could increase Nrf2 activation and expression of Nrf2-related anti-oxidant genes, including HO-1, NQO1 and GCLC. Pretreatment with CGA could reduce CCl₄-induced elevation of serum transaminases and alleviate liver pathological abnormalities. CGA also reversed CCl₄-induced increase in MDA level and decrease in the levels of GSH, SOD and CAT in liver tissues. Meanwhile, CGA inhibited NLRP3 inflammasome activation, as indicated by the reduced protein expression of NLRP3, Pro-Caspase-1, Caspase-1, Pro-IL-1β and IL-1β. Moreover, CGA reduced serum levels and liver mRNA expression of TNF-α, IL-6 and IL-1β. These results demonstrate that CGA protects against CCl₄-induced acute liver injury probably through enhancing Nrf2-mediated anti-oxidant pathway and inhibiting NLRP3 inflammasome activation.     

Cellular adaptive response to glutathione depletion modulates endothelial dysfunction triggered by TNF-α

Several interrelated cellular signaling molecules are involved in modulating adaptive compensatory changes elicited by low exposures to toxins and other stressors. The most prominent example of signaling pathway typically involved in this adaptive stress response, is represented by the activation of a redox-sensitive gene regulatory network mediated by the NF-E2-related factor-2 (Nrf2) which is intimately involved in mediating the Antioxidant Responsive Element (ARE)-driven response to oxidative stress and xenobiotics.We investigated if Nrf2 pathway activation following intracellular glutathione depletion through buthionine sulfoximine (BSO) exposure, might be able to alter the response to TNF-α, a proinflammatory cytokine, in cultured human umbilical vein endothelial cells. Herein, we revealed that such a change in the cellular redox status is able to reduce TNF-α induced endothelial activation (as shown by a decreased gene expression of adhesion molecules) by activating an adaptive response mediated by an increased Nrf2 nuclear translocation and overexpression of the ARE genes HO-1 and NQO-1. Furthermore, we have demonstrated the involvement of ERK1/2 kinases in Nrf2 nuclear translocation activated by BSO-induced glutathione depletion. The coordinate induction of endogenous cytoprotective proteins through adaptive activation of Nrf2 pathway is a field of great interest for potential application in prevention and therapy of inflammatory diseases such as atherosclerosis.     

Isorhamnetin protects against oxidative stress by activating Nrf2 and inducing the expression of its target genes

Isorhamentin is a 3′-O-methylated metabolite of quercetin, and has been reported to have anti-inflammatory and anti-proliferative effects. However, the effects of isorhamnetin on Nrf2 activation and on the expressions of its downstream genes in hepatocytes have not been elucidated. Here, we investigated whether isorhamnetin has the ability to activate Nrf2 and induce phase II antioxidant enzyme expression, and to determine the protective role of isorhamnetin on oxidative injury in hepatocytes. In HepG2 cells, isorhamnetin increased the nuclear translocation of Nrf2 in a dose- and time-dependent manner, and consistently, increased antioxidant response element (ARE) reporter gene activity and the protein levels of hemeoxygenase (HO-1) and of glutamate cysteine ligase (GCL), which resulted in intracellular GSH level increases. The specific role of Nrf2 in isorhamnetin-induced Nrf2 target gene expression was verified using an ARE-deletion mutant plasmid and Nrf2-knockout MEF cells. Deletion of the ARE in the promoter region of the sestrin2 gene, which is recently identified as the Nrf2 target gene by us, abolished the ability of isorhamnetin to increase luciferase activity. In addition, Nrf2 deficiency completely blocked the ability of isorhamnetin to induce HO-1 and GCL. Furthermore, isorhamnetin pretreatment blocked t-BHP-induced ROS production and reversed GSH depletion by t-BHP and consequently, due to reduced ROS levels, decreased t-BHP-induced cell death. In addition isorhamnetin increased ERK1/2, PKCδ and AMPK phosphorylation. Finally, we showed that Nrf2 deficiency blocked the ability of isorhamnetin to protect cells from injury induced by t-BHP. Taken together, our results demonstrate that isorhamnetin is efficacious in protecting hepatocytes against oxidative stress by Nrf2 activation and in inducing the expressions of its downstream genes.     

Antioxidative effects of quercetin-glycosides isolated from the flower buds of Tussilago farfara L.

A bioassay-guided fractionation of the ethylacetate soluble fraction from the flower buds of Tussilago farfara L. (Compositae) yielded two flavonoids, quercetin 3-O-beta-L-arabinopyranoside and quercetin 3-O-beta-D-glucopyranoside. These two sugar conjugates of quercetin exhibited higher antioxidative activity than their aglycone, quercetin by NBT superoxide scavenging assay. Moreover, treatment with quercetin 3-O-beta-L-arabinopyranoside significantly increased the total glutathione (GSH) contents and the protein level of gamma-glutamylcysteine ligase (gamma-GCL), a key enzyme required for glutathione (GSH) synthesis in a rat hepatocyte cell line. Subcellular fractionation and reporter gene analysis using antioxidant response element (ARE) construct revealed that quercetin 3-O-beta-L-arabinopyranoside increased the level of nuclear Nrf2 and reporter activity, and that these were associated with the induction of the gamma-GCL gene. After 24 h incubation of cells with quercetin 3-O-beta-L-arabinopyranoside, 23% of the glycoside was converted to its aglycone, quercetin, but gamma-GCL was not induced by 7 microM (23%) quercetin. These results suggest that the two quercetin-glycosides isolated from T. farfara L. have direct antioxidative properties, and that quercetin 3-O-beta-L-arabinopyranoside increases the cellular GSH level by inducing the gamma-GCL gene. These novel effects of quercetin-glycosides are suggestive to underlie the potential putative chemopreventive effects of T. farfara L.