Nrf1 Reduces COX-2 Expression and Maintains Cellular Homeostasis After Cerebral Ischemia/Reperfusion By Targeting IL-6/TNF-α Protein Production.

Neuroinflammation has been considered involved in the process of cerebral ischemia-reperfusion injury (CIRI). Transcription factors play a crucial role in regulating gene transcription and the expressions of specific proteins during the progression of various neurological diseases. Evidence showed that transcription factor nuclear factor erythroid 2-related factor 1 (NFE2L1, also known as Nrf1) possessed strong biological activities including antioxidant, anti-inflammatory and neuroprotective properties. However, its role and potential molecular mechanisms in CIRI remain unclear. In our study, we observed a significant elevation of Nrf1 in the cerebral cortex following cerebral ischemia-reperfusion in rats. The Nrf1 downregulation markedly raised COX-2, TNF-α, IL-1ß, and IL-6 protein levels during middle cerebral artery occlusion/reperfusion in rats, which led to worsened neurological deficits, higher cerebral infarct volume, and intensified cortical histopathological damage. In subsequent in vitro studies, the expression of Nrf1 protein increased following oxygen-glucose deprivation/reperfusion treatment on neurons. Subsequently, Nrf1 knockdown resulted in a significant upregulation of inflammatory factors, leading to a substantial increase in the cell death rate. Through analyzing the alterations in the expression of inflammatory factors under diverse interventions, it is indicated that Nrf1 possesses the capacity to discern variations in inflammatory factors via specific structural domains. Our findings demonstrate the translocation of the Nrf1 protein from the cytoplasm to the nucleus, thereby modulating the protein expression of IL-6/TNF-α and subsequently reducing the expression of multiple inflammatory factors. This study signifies, for the first time, that during cerebral ischemia-reperfusion, Nrf1 translocases to the nucleus to regulate the protein expression of IL-6/TNF-α, consequently suppressing COX-2 expression and governing cellular inflammation, ultimately upholding cellular homeostasis.

Nuclear respiratory factor 1 protects H9C2 cells against hypoxia-induced apoptosis via the death receptor pathway and mitochondrial pathway.

Hypoxia-induced cardiomyocyte apoptosis is one of the leading causes of heart failure. Nuclear respiratory factor 1 (NRF-1) was suggested as a protector against cell apoptosis; However, the mechanism is not clear. Therefore, the aim of this study was to elucidate the role of NRF-1 in hypoxia-induced H9C2 cardiomyocyte apoptosis and to explore its effect on regulating the death receptor pathway and mitochondrial pathway. NRF-1 was overexpressed or knocked down in H9C2 cells, which were then exposed to a hypoxia condition for 0, 3, 6, 12, and 24 h. Changes in cell proliferation, cell viability, reactive oxygen species (ROS) generation, and mitochondrial membrane potential (MMP) were investigated. The activities of caspase-3, -8, and -9, apoptosis rate, and the gene and protein expression levels of the death receptor pathway and mitochondrial pathway were analyzed. Under hypoxia exposure, NRF-1 overexpression improved the proliferation and viability of H9C2 cells and decreased ROS generation, MMP loss, caspase activities, and the apoptosis rate. However, the NRF-1 knockdown group showed the opposite results. Additionally, NRF-1 upregulated the expression of antiapoptotic molecules involved in the death receptor and mitochondrial pathways, such as CASP8 and FADD-like apoptosis regulator, B-cell lymphoma 2, B-cell lymphoma-extra-large, and cytochrome C. Conversely, the expression of proapoptotic molecules, such as caspase-8, BH3-interacting domain death agonist, Bcl-2-associated X protein, caspase-9, and caspase-3 was downregulated by NRF-1 overexpression in hypoxia-induced H9C2 cells. These results suggest that NRF-1 functions as an antiapoptotic factor in the death receptor and mitochondrial pathways to mitigate hypoxia-induced apoptosis in H9C2 cardiomyocytes.

Long-isoform NRF1 protects against arsenic cytotoxicity in mouse bone marrow-derived mesenchymal stem cells by suppressing mitochondrial ROS and facilitating arsenic efflux.

Acute exposure to arsenic is known to cause bone marrow depression and result in anemia, in which the dusfunction of cells in the bone marrow niche such as mesenchymal stem cells (MSCs) is vital. However, the mechanism underlying response of MSCs to arsenic challange is not fully understood. In the present study, we investigated the role of nuclear factor erythroid 2-related factor (NRF) 1 (NRF1), a sister member of the well-known master regulator in antioxidative response NRF2, in arsenite-induced cytotoxicity in mouse bone marrow-derived MSCs (mBM-MSCs). We found that arsenite exposure induced significant increase in the protein level of long-isoform NRF1 (L-NRF1). Though short-isoform NRF1 (S-NRF1) was induced by arsenite at mRNA level, its protein level was not obviously altered. Silencing L-Nrf1 sensitized the cells to arsenite-induced cytotoxicity. L-Nrf1-silenced mBM-MSCs showed decreased arsenic efflux with reduced expression of arsenic transporter ATP-binding cassette subfamily C member 4 (ABCC4), as well as compromised NRF2-mediated antioxidative defense with elevated level of mitochondrial reactive oxygen species (mtROS) under arsenite-exposed conditions. A specific mtROS scavenger (Mito-quinone) alleviated cell apoptosis induced by arsenite in L-Nrf1-silenced mBM-MSCs. Taken together, these findings suggest that L-NRF1 protects mBM-MSCs from arsenite-induced cytotoxicity via suppressing mtROS in addition to facilitating cellular arsenic efflux.

"Cold training" affects rat liver responses to continuous cold exposure.

Continuous exposure of homeothermic animals to low environmental temperatures elicits physiological adaptations necessary for animal survival, which are associated to higher generation of pro-oxidants in thermogenic tissues. It is not known whether intermittent cold exposure (cold training) is able to affect tissue responses to continuous cold exposure. Therefore, we investigated whether rat liver responses to continuous cold exposure of 2 days are modified by cold training (1h daily for 5 days per week for 3 consecutive weeks). Continuous cold increased liver oxidative metabolism by increasing tissue content of mitochondrial proteins and mitochondrial aerobic capacity. Cold training did not affect such parameters, but attenuated or prevented the changes elicited by continuous cold exposure. Two-day cold exposure increased lipid hydroperoxide and protein-bound carbonyl levels in homogenates and mitochondria, whereas cold training decreased such effects although it decreased only homogenate protein damage in control rats. The activities of the antioxidant enzymes GPX and GR and H2O2 production were increased by continuous cold exposure. Despite the increase in GPX and GR activities, livers from cold-exposed rats showed increased susceptibility to in vitro oxidative challenge. Such cold effects were decreased by cold training, which in control rats reduced only H2O2 production and susceptibility to stress. The changes of PGC-1, NRF-1, and NRF-2 expression levels were consistent with those induced by cold exposure and cold training in mitochondrial protein content and antioxidant enzyme activities. However, the mechanisms by which cold training attenuates the effects of the continuous cold exposure remain to be elucidated.

Activation of AMP-activated protein kinase by metformin protects against global cerebral ischemia in male rats: interference of AMPK/PGC-1α pathway.

Here, we have investigated the effect of metformin pretreatment in the rat models of global cerebral ischemia. Cerebral ischemia which leads to brain dysfunction is one of the main causes of neurodegeneration and death worldwide. Metformin is used in clinical drug therapy protocols of diabetes. It is suggested that metformin protects cells under hypoxia and ischemia in non-neuronal contexts. Protective effects of metformin may be modulated via activating the AMP activated protein kinase (AMPK). Our results showed that induction of 30 min global cerebral I/R injury using 4-vesseles occlusion model led to significant cell death in the rat brain. Metformin pretreatment (200 mg kg/once/day, p.o., 2 weeks) attenuated apoptotic cell death and induced mitochondrial biogenesis proteins in the ischemic rats, analyzed using histological and Western blot assays. Besides, inhibition of AMPK by compound c showed that metformin resulted in apoptosis attenuation via AMPK activation. Interestingly, AMPK activation was also involved in the induction of mitochondrial biogenesis proteins using metformin, inhibition of AMPK by compound c reversed such effect, further supporting the role of AMPK upstream of mitochondrial biogenesis proteins. In summary, Metformin pretreatment is able to modulate mitochondrial biogenesis and apoptotic cell death pathways through AMPK activation in the context of global cerebral ischemia, conducting the outcome towards neuroprotection.

Nrf1 is time-dependently expressed and distributed in the distinct cell types after trauma to skeletal muscles in rats.

Our goal was to elucidate the dynamic expression and distribution of the nuclear factor erythroid-derived factor 2-related factor 1 (Nrf1) by immunohistochemistry, Western blotting, and real-time PCR during wound healing of contused skeletal muscle in rats. An animal model of skeletal muscle contusion was established in 40 Sprague-Dawley male healthy rats. Samples were taken at 6 h, 12 h, 1 day, 3 days, 5 days, 7 days, 10 days, and 14 days post-injury, respectively (5 rats in each posttraumatic interval). 5 rats were employed as control. A weak immunoreactivity of Nrf1 was observed in the sarcoplasm and nuclei of normal myofibers in control rats. Prominent immunostaining for Nrf1 was seen in a large number of polymorphonuclear cells, round-shaped mononuclear cells and spindle-shaped fibroblastic cells, and regenerated multinucleated myotubes in the injured tissue. Subsequently, neutrophils, macrophages and myofibroblasts were identified as expressing Nrf1 by double immunofluorescent procedures. By real-time PCR analysis, Nrf1 expression was up-regulated and peaked at inflammatory phase. The expression tendency was also confirmed by Western blot. In conclusion, Nrf1 is time-dependently expressed in certain cell types, such as neutrophils, macrophages, myofibroblasts and regenerated multinucleated myotubes, suggesting that Nrf1 may modulate oxidative stress response and regeneration after trauma to skeletal muscles.

ERK and p38 inhibitors attenuate memory deficits and increase CREB phosphorylation and PGC-1α levels in Aß-injected rats.

In this study, we investigated the effect of intracerebroventricular administration of ERK and p38 specific inhibitors, U0126 and PD169316, respectively, on learning and memory deficits induced by amyloid beta (Aß) in rats. To investigate the effects of these compounds on learning and memory, we performed Morris water maze (MWM) test. U0126 and/or PD169316 improved spatial learning in MWM in Aß-injected rats, 20 days after Aß-injection. To determine the mechanisms of action of U0126 and PD169316, we studies their effect on some intracellular signaling pathways such as Ca(+)/cAMP-response element binding protein (CREB), c-fos, and transcription factors that regulate mitochondrial biogenesis. Based on our data, CREB and c-fos levels decreased 7 days after Aß-injection, while U0126 and/or PD169316 pretreatments significantly increased these levels. Moreover, U0126 and PD169316 activated peroxisome proliferator-activated receptor gamma coactivator-1a, nuclear respiratory factor 1, and mitochondrial transcription factor A, 7 days after Aß-injection. Surprisingly, these factors were returned to vehicle level, 20 days after Aß-injection. Our findings reinforce the potential neuroprotective effect of these inhibitors against the Aß toxicity.

Mitochondrial biogenesis fails in secondary biliary cirrhosis in rats leading to mitochondrial DNA depletion and deletions.

Chronic cholestasis is characterized by mitochondrial dysfunction, associated with loss of mitochondrial membrane potential, decreased activities of respiratory chain complexes, and ATP production. Our aim was to determine the molecular mechanisms that link long-term cholestasis to mitochondrial dysfunction. We studied a model of chronic cholestasis induced by bile duct ligation in rats. Key sensors and regulators of the energetic state and mitochondrial biogenesis, mitochondrial DNA (mtDNA)-to-nuclear DNA (nDNA) ratio (mtDNA/nDNA) relative copy number, mtDNA deletions, and indexes of apoptosis (BAX, BCL-2, and cleaved caspase 3) and cell proliferation (PCNA) were evaluated. Our results show that long-term cholestasis is associated with absence of activation of key sensors of the energetic state, evidenced by decreased SIRT1 and pyruvate dehydrogenase kinase levels and lack of AMPK activation. Key mitochondrial biogenesis regulators (PGC-1α and GABP-α) decreased and NRF-1 was not transcriptionally active. Mitochondrial transcription factor A (TFAM) protein levels increased transiently in liver mitochondria at 2 wk after bile duct ligation, but they dramatically decreased at 4 wk. Reduced TFAM levels at this stage were mirrored by a marked decrease (65%) in mtDNA/nDNA relative copy number. The blockade of mitochondrial biogenesis should not be ascribed to activation of apoptosis or inhibition of cell proliferation. Impaired mitochondrial turnover and loss of the DNA stabilizing effect of TFAM are likely the causative event involved in the genetic instability evidenced by accumulation of mtDNA deletions. In conclusion, the lack of stimulation of mitochondrial biogenesis leads to mtDNA severe depletion and deletions in long-term cholestasis. Hence, long-term cholestasis should be considered a secondary mitochondrial hepatopathy.

Hydroxytyrosol protects retinal pigment epithelial cells from acrolein-induced oxidative stress and mitochondrial dysfunction.

Hydroxytyrosol (HTS) is a natural polyphenol abundant in olive oil. Increasing evidence indicates HTS has beneficial effect on human health for preventing various diseases. In the present study, we investigated the protective effects of HTS on acrolein-induced toxicity in human retinal pigment epithelial cell line, ARPE-19, a cellular model of smoking- and age-related macular degeneration. Acrolein, a major component of the gas phase cigarette smoke and also a product of lipid peroxidation in vivo, at 75 µmol/L for 24 h caused significant loss of cell viability, oxidative damage (increase in oxidant generation and oxidative damage to proteins and DNA, decrease in antioxidants and antioxidant enzymes, and also inactivation of the Keap1/Nrf2 pathway), and mitochondrial dysfunction (decrease in membrane potential, activities of mitochondrial complexes, viable mitochondria, oxygen consumption, and factors for mitochondrial biogenesis, and increase in calcium). Pre-treatment with HTS dose dependently and also time dependently protected the ARPE-19 cells from acrolein-induced oxidative damage and mitochondrial dysfunction. A short-term pre-treatment with HTS (48 h) required > 75 µmol/L for showing protection while a long-term pre-treatment (7 days) showed protective effect from 5 µmol/L on. The protective effect of HTS in this model was as potent as that of established mitochondria-targeting antioxidant nutrients. These results suggest that HTS is also a mitochondrial-targeting antioxidant nutrient and that dietary administration of HTS may be an effective measure in reducing and or preventing cigarette smoke-induced or age-related retinal pigment epithelial degeneration, such as age-associated macular degeneration.