USP8 mitigates cognitive dysfunction of mice with sepsis-associated encephalopathy.

BACKGROUND: Sepsis-associated encephalopathy (SAE) is a serious complication of sepsis which results from neuroinflammation and could lead to cognitive dysfunction. Ubiquitin-specific peptidase 8 (USP8) is involved in cognitive dysfunction. This study investigated the mechanism by which USP8 plays a role in cognitive dysfunction of SAE mice. METHODS: The SAE models were established by performing cecal ligation and puncture in the mice. Subsequently, a series of tests and procedures were conducted to assess the cognitive dysfunction and pathological impairment of mice, including the Morris water maze test, Y-maze test, open field test, tail suspension test, fear conditioning test, and haematoxylin-eosin staining. The levels of USP8 and Yin Yang 1 (YY1) in brain tissues of mice were detected. In order to determine the effects of USP8 or YY1 on cognitive function, SAE mice were injected with an adenovirus-packaged vector that had overexpressed levels of USP8 or YY1 short hairpin RNA. The binding of USP8 to YY1 and the ubiquitination level of YY1 were analyzed using immunoprecipitation and ubiquitination experiments. Lastly, chromatin immunoprecipitation was carried out to analyze enrichment of YY1 on the USP8 promoter. RESULTS: In SAE models, USP8 and YY1 were downregulated and cognitive functions were impaired. USP8 overexpression upregulated YY1 and attenuated the brain histopathological damage and cognitive dysfunction in SAE mice. USP8 upregulated YY1 protein level through deubiquitination, while YY1 was enriched on the USP8 promoter and activated USP8 transcription. The effects of USP8 overexpression on SAE mice was reversed secondary to YY1 silencing. CONCLUSION: USP8 upregulated YY1 protein level through deubiquitination and YY1 activated USP8 transcription, and USP8-YY1 feedback loop attenuated cognitive dysfunction in SAE mice, which could potentially serve as a novel theoretical foundation for the management of SAE.

Diffusion simulation and risk assessment model establishment of chlorine gas leakage based on terrain conditions.

This study researches the impact of terrain factors on chlorine gas diffusion processes based on SLAB model. Simulating the law of wind speed changing with altitude by calculating the real-time speed with vertical height combing actual terrain data, and integrating the influence of terrain on wind speed by using Reynolds Average Navier-Stokes (RANS) algorithm, K-turbulence model, and standard wall functions, then plotting the gas diffusion range in the map with terrain data according to the Gaussian-Cruger projection algorithm and dividing the hazardous areas according to the public exposure guidelines (PEG). The accidental chlorine gas releases near Lishan Mountain, Xi'an City, were simulated by the improved SLAB model. The results show that there are obvious differences analyzing contrastively the endpoint distance and area of chlorine gas dispersion under real terrain condition and ideal condition at different times; it can be found that the endpoint distance of the real terrain conditions is 1.34 km shorter than that of the ideal conditions at 300 s with terrain factors, and also the thermal area is 3,768,026m2 less than that of the ideal conditions. In addition, it can predict the specific number of casualties within different levels of harm at 2 min after chlorine gas dispersion, and casualties are constantly changing over time. The fusion of terrain factors can be used to optimize the SLAB model, which is expected to provide an important reference for effective rescue.

Irisin drives macrophage anti-inflammatory differentiation via JAK2-STAT6-dependent activation of PPARγ and Nrf2 signaling.

Irisin is an exercise-induced myokine that alleviates inflammation and obesity. The induction of anti-inflammatory (M2) macrophage is facilitated for treatment of sepsis and associated lung damage. However, whether irisin drives macrophage M2 polarization remains unclear. Here, we found that irisin induced-macrophage anti-inflammatory differentiation in vivo using an LPS-induced septic mice model and in vitro using RAW64.7 cells and bone marrow-derived macrophages (BMDMs). Irisin also promoted the expression, phosphorylation, and nuclear translocation of peroxisome proliferator-activated receptor gamma (PPAR-γ) and nuclear factor-erythroid 2-related factor 2 (Nrf2). Inhibition or knockdown of PPAR-γ and Nrf2 abolished irisin-induced accumulation of M2 macrophage markers, such as interleukin (IL)-10 and Arginase 1. Furthermore, dual-luciferase reporter and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assays confirmed that STAT6 boosts PPAR-γ and Nrf2 transcription by binding to their DNA promoters in irisin-stimulated macrophages. In contrast, STAT6 shRNA blocked the irisin-induced activation of Pparγ, Nrf2, and related downstream genes. Moreover, the interaction of irisin with its ligand integrin αVß5 remarkably promoted Janus kinase 2 (JAK2) phosphorylation, while inhibition or knockdown of integrin αVß5 and JAK2 attenuated the activation of STAT6, PPAR-γ, and Nrf2 signaling. Interestingly, co-immunoprecipitation (Co-IP) assay also revealed that the binding between JAK2 and integrin αVß5 is critical for irisin-induced macrophage anti-inflammatory differentiation by enhancing the activation of the JAK2-STAT6 pathway. In conclusion, irisin boosted M2 macrophage differentiation by inducing JAK2-STAT6-dependent transcriptional activation of the PPAR-γ-related anti-inflammatory system and Nrf2-related antioxidant genes. The findings of this study suggest that the administration of irisin is a novel and promising therapeutic strategy for infectious and inflammatory diseases.

Protective effects of pentoxifylline against chlorine-induced acute lung injury in rats.

OBJECTIVE: Chlorine is a chemical threat agent that can be harmful to humans. Inhalation of high levels of chlorine can lead to acute lung injury (ALI). Currently, there is no satisfactory treatment, and effective antidote is urgently needed. Pentoxifylline (PTX), a methylxanthine derivative and nonspecific phosphodiesterase inhibitor, is widely used for the treatment of vascular disorders. The present study was aimed to investigate the inhibitory effects of PTX on chlorine-induced ALI in rats. METHODS: Adult male Sprague-Dawley rats were exposed to 400 ppm Cl2 for 5 min. The histopathological examination was carried out and intracellular reactive oxygen species (ROS) levels were measured by the confocal laser scanning system. Subsequently, to evaluate the effect of PTX, a dose of 100 mg/kg was administered. The activities of superoxide dismutase (SOD) and the contents of malondialdehyde (MDA), glutathione (GSH), oxidized glutathione (GSSG) and lactate dehydrogenase (LDH) were determined by using commercial kits according to the manufacturer's instructions. Western blot assay was used to detect the protein expressions of SOD1, SOD2, catalase (CAT), hypoxia-inducible factor (HIF)-1α, vascular endothelial growth factor (VEGF), occludin, E-cadherin, bcl-xl, LC 3, Beclin 1, PTEN-induced putative kinase 1 (PINK 1) and Parkin. RESULTS: The histopathological examination demonstrated that chlorine could destroy the lung structure with hemorrhage, alveolar collapse, and inflammatory infiltration. ROS accumulation was significantly higher in the lungs of rats suffering from inhaling chlorine (P<0.05). PTX markedly reduced concentrations of MAD and GSSG, while increased GSH (P<0.05). The protein expression levels of SOD1 and CAT also decreased (P<0.05). Furthermore, the activity of LDH in rats treated with PTX was significantly decreased compared to those of non-treated group (P<0.05). Additionally, the results also showed that PTX exerted an inhibition effect on protein expressions of HIF-1α, VEGF and occludin, and increased the level of E-cadherin (P<0.05). While the up-regulation of Beclin 1, LC 3II/I, Bcl-xl, and Parkin both in the lung tissues and mitochondria, were found in PTX treated rats (P<0.05). The other protein levels were decreased when treated with PTX (P<0.05). CONCLUSION: PTX could ameliorate chlorine-induced lung injury via inhibition effects on oxidative stress, hypoxia and autophagy, thus suggesting that PTX could serve as a potential therapeutic approach for ALI.

Targeting PARK7 Improves Acetaminophen-Induced Acute Liver Injury by Orchestrating Mitochondrial Quality Control and Metabolic Reprogramming.

Mitochondrial dysfunction and oxidative stress are considered to be key events in acetaminophen (APAP)-induced acute liver injury. Mitochondrial quality control, including mitophagy and mitochondrial synthesis, can restore mitochondrial homeostasis and thus protect the liver. The role of PARK7, a mitochondrial stress protein, in regulating mitochondrial quality control in APAP-induced hepatotoxicity is unclear. In this study, L02 cells, AML12 cells and C57/BL6 mice were each used to establish models of APAP-induced acute liver injury. PARK7 was silenced in vitro by lentiviral transfection and knocked down in vivo by AAV adeno-associated virus. Changes in cell viability, apoptosis, reactive oxygen species (ROS) level, serum enzyme activity and pathological features were evaluated after APAP treatment. Western blotting, real-time PCR, immunofluorescence, electron microscopy and Seahorse assays were used to detect changes in key indicators of mitochondrial quality control. The results showed that APAP treatment decreased cell viability and increased the apoptosis rate, ROS levels, serum enzyme activity, pathological damage and PARK7 expression. PARK7 silencing or knockdown ameliorated APAP-induced damage to the cells and liver. Furthermore, PARK7 silencing enhanced mitophagy, increased mitochondrial synthesis, and led to a switch from oxidative phosphorylation to glycolysis. Taken together, these results suggest that PARK7 is involved in APAP-induced acute liver injury by regulating mitochondrial quality control and metabolic reprogramming. Therefore, PARK7 may be a promising therapeutic target for APAP-induced liver injury.

Heterogeneity of T cells and macrophages in chlorine-induced acute lung injury in mice using single-cell RNA sequencing.

OBJECTIVE: Chlorine (Cl2), as an asphyxiant toxicant, induced poisoning incidents and acute lung injury (ALI) occur frequently. The specific pathogenesis of Cl2-induced ALI remains unclear. Immune cells play an important role in the process of lung damage. We used single-cell RNA sequencing (scRNA-seq) technology to explore T cells and macrophages molecular mechanism. METHODS: Female BALB/c mice were exposed to 400 ppm Cl2 for 15 min. scRNA-seq technology was used to observe the heterogeneity of T cells and macrophages. Hematoxylin-eosin (H&E) staining was used to evaluate the degree of lung injury. Immunofluorescence was used to verify the highly expressed genes of our interest. RESULTS: A total of 5316 to 7742 cells were classified into eight different cell types. Several new highly expressed anti-inflammatory and pro-inflammatory genes were found in T cells and macrophages, which were further verified in vitro. Through the pseudotime analysis of macrophages, it was found that the expression of pro-inflammatory and anti-inflammatory genes showed opposite trends in the development of Cl2-induced ALI. This study also mapped T cells-macrophage communication and identified the development of several important receptor-ligand complexes in Cl2-induced ALI. CONCLUSIONS: These findings are worthy of further exploration and provide new resources and directions for the study of Cl2-induced ALI in mice, especially in immune and inflammation mechanisms.

Reactive Oxygen Species Bridge the Gap between Chronic Inflammation and Tumor Development.

According to numerous animal studies, adverse environmental stimuli, including physical, chemical, and biological factors, can cause low-grade chronic inflammation and subsequent tumor development. Human epidemiological evidence has confirmed the close relationship between chronic inflammation and tumorigenesis. However, the mechanisms driving the development of persistent inflammation toward tumorigenesis remain unclear. In this study, we assess the potential role of reactive oxygen species (ROS) and associated mechanisms in modulating inflammation-induced tumorigenesis. Recent reports have emphasized the cross-talk between oxidative stress and inflammation in many pathological processes. Exposure to carcinogenic environmental hazards may lead to oxidative damage, which further stimulates the infiltration of various types of inflammatory cells. In turn, increased cytokine and chemokine release from inflammatory cells promotes ROS production in chronic lesions, even in the absence of hazardous stimuli. Moreover, ROS not only cause DNA damage but also participate in cell proliferation, differentiation, and apoptosis by modulating several transcription factors and signaling pathways. We summarize how changes in the redox state can trigger the development of chronic inflammatory lesions into tumors. Generally, cancer cells require an appropriate inflammatory microenvironment to support their growth, spread, and metastasis, and ROS may provide the necessary catalyst for inflammation-driven cancer. In conclusion, ROS bridge the gap between chronic inflammation and tumor development; therefore, targeting ROS and inflammation represents a new avenue for the prevention and treatment of cancer.

Silent information regulator 1 suppresses epithelial-to-mesenchymal transition in lung cancer cells via its regulation of mitochondria status.

AIMS: Silent information regulator 1 (SIRT1) is a NAD+-dependent protein-modifying enzyme involved in regulating gene expression, DNA damage repair, cell metabolism, and mitochondrial functions. Given that it acts as both a tumor promoter and suppressor, the complex mechanisms underlying SIRT1 signaling in cancer remain controversial. Epithelial-to-mesenchymal transition (EMT) plays a key role in the progression of carcinogenesis and tumors metastasis. Studies have shown that mitochondrial defects are critical in EMT process, and SIRT1 is found to regulate the generation and energy metabolism of mitochondria. Here, we elucidate a novel mechanism by which SIRT1 affects EMT in lung cancer cells via its regulation on mitochondria. MAIN METHODS: SIRT1 signaling was detected in TGF-ß1-induced EMT and was found to regulate mitochondria status, including mitochondrial biogenesis-related protein levels as detected by western blotting, mitochondrial structure observed by transmission electron microscopy, and respiratory functions analyzed by a respiration capacity assay. The effects of modulating SIRT1 expression on EMT and migration of lung cancer cells or normal cells were evaluated by in vitro and in vivo models. KEY FINDINGS: We found that the regulation of SIRT1 signaling on the biogenesis or functions of mitochondria was critical to EMT. Overexpression of SIRT1 reduced EMT or metastasis potential of lung cancer cells by improving the quantity and quality of mitochondria, whereas silencing SIRT1 promote EMT in cancer cells, even in normal cells by disturbing mitochondria status. SIGNIFICANCE: Consequently, SIRT1 is an attractive therapeutic target for reversing EMT or tumor metastasis.

Electromagnetic Fields Ameliorate Insulin Resistance and Hepatic Steatosis by Modulating Redox Homeostasis and SREBP-1c Expression in db/db Mice.

PURPOSE: The prevalence of nonalcoholic fatty liver disease (NAFLD), which has recently become known as metabolic-associated fatty liver disease (MAFLD), has risen. However, pharmacotherapies for this disease have not been approved. Electromagnetic fields (EMFs) have excellent bioeffects on multiple diseases. However, the effects of EMFs on NAFLD are unknown. This study investigated the bioeffects of EMF exposure on insulin resistance, liver redox homeostasis and hepatic steatosis in db/db mice. METHODS: Animals were sacrificed after EMF exposure for 8 weeks. The fasting blood glucose and insulin levels in the serum were tested. The homeostatic model assessment of insulin resistance (HOMA-IR) was calculated by a formula. The levels of MDA, GSSG and GSH, biomarkers of redox, were assessed. The activities of CAT, SOD and GSH-Px were assessed. The body and liver weights were measured. Hepatic lipid accumulation was observed by Oil Red O staining. Hepatic CAT, GR, GSH-Px, SOD1, SOD2 and SREBP-1 expression was determined by Western blotting. RESULTS: EMF exposure ameliorated insulin resistance and oxidative stress in the liver by downregulating the MDA and GSSG levels, increasing the reduced GSH levels, and promoting the GSH-Px levels in db/db mice. In addition, liver weight and triglyceride (TG) levels were reduced by EMF exposure. Simultaneously, EMF exposure improved hepatic steatosis by downregulating the protein expression of SREBP-1c. CONCLUSION: The present findings suggest that EMF exposure has positive effects in the treatment of NAFLD.

Gestational bisphenol A exposure induces fatty liver development in male offspring mice through the inhibition of HNF1b and upregulation of PPARγ.

Bisphenol A (BPA) is an endocrine-disrupting chemical (EDC) associated with non-alcoholic fatty liver disease (NAFLD). The effects of gestational BPA exposure on hepatic lipid accumulation in offspring are not fully understood. Here, we investigate the sex-dependent effects of gestational BPA exposure on hepatic lipid and glucose metabolism in the offspring of mice to reveal the mechanisms underlying gestational BPA exposure-associated NAFLD. Pregnant mice were administered gavage with or without 1 µg kg-1 day-1 BPA at embryonic day 7.5 (E7.5)-E16.5. Hepatic glucose and lipid metabolism were evaluated in these models. Both male and female offspring mice exhibited hepatic fatty liver after BPA treatment. Lipid accumulation and dysfunction of glucose metabolism were observed in male offspring. We revealed abnormal expression of lipid regulators in the liver and that inhibition of peroxisome proliferator-activated receptor γ (PPARγ) repressed hepatic lipid accumulation induced by gestational BPA exposure. We also found a sex-dependent decrease of hepatocyte nuclear factor 1b (HNF1b) expression in male offspring. The transcriptional repression of PPARγ by HNF1b was confirmed in L02 cells. Downregulation of HNF1b, upregulation of PPARγ, and subsequent upregulation of hepatic lipid accumulation were essential for NAFLD development in male offspring gestationally exposed to BPA as well as BPA-exposed adult male mice. Dysregulation of the HNF1b/PPARγ pathway may be involved in gestational BPA exposure-induced NAFLD in male offspring. These data provide new insights into the mechanism of gestational BPA exposure-associated sex-dependent glucose and lipid metabolic dysfunction. Graphical abstract Schematic of the mechanism of gestational BPA exposure-induced glucose and lipid metabolic dysfunction.

Cubic Membranes Formation in Synchronized Human Hepatocellular Carcinoma Cells Reveals a Possible Role as a Structural Antioxidant Defense System in Cell Cycle Progression.

Cubic membranes (CMs) represent unique biological membrane structures with highly curved three-dimensional periodic minimal surfaces, which have been observed in a wide range of cell types and organelles under various stress conditions (e. g., starvation, virus-infection, and oxidation). However, there are few reports on the biological roles of CMs, especially their roles in cell cycle. Hence, we established a stable cell population of human hepatocellular carcinoma cells (HepG2) of 100% S phase by thymidine treatment, and determined certain parameters in G2 phase released from S phase. Then we found a close relationship between CMs formation and cell cycle, and an increase in reactive oxygen species (ROS) and mitochondrial function. After the synchronization of HepG2 cells were induced, CMs were observed through transmission electron microscope in G2 phase but not in G1, S and M phase. Moreover, the increased ATP production, mitochondrial and intracellular ROS levels were also present in G2 phase, which demonstrated a positive correlation with CMs formation by Pearson correlation analysis. This study suggests that CMs may act as an antioxidant structure in response to mitochondria-derived ROS during G2 phase and thus participate in cell cycle progression.

Stat2-Drp1 mediated mitochondrial mass increase is necessary for pro-inflammatory differentiation of macrophages.

Macrophage recruitment and pro-inflammatory differentiation are hallmarks of various diseases, including infection and sepsis. Although studies suggest that mitochondria may regulate macrophage immune responses, it remains unclear whether mitochondrial mass affects macrophage pro-inflammatory differentiation. Here, we found that lipopolysaccharide (LPS)-activated macrophages possess higher mitochondrial mass than resting cells. Therefore, this study aimed to explore the functional role and molecular mechanisms of increased mitochondrial mass in pro-inflammatory differentiated macrophages. Results show that an increase in the mitochondrial mass of macrophages positively correlates with inflammatory cytokine generation in response to LPS. RNA-seq analysis revealed that LPS promotes signal transducers and activators of transcription 2 (Stat2) and dynamin-related protein 1 (Drp1) expression, which are enriched in positive mitochondrial fission regulation. Meanwhile, knockdown or pharmacological inhibition of Drp1 blunts LPS-induced mitochondrial mass increase and pro-inflammatory differentiation. Moreover, Stat2 boosts Drp1 phosphorylation at serine 616, required for Drp1-mediated mitochondrial fission. LPS also causes Stat2-and Drp1-dependent biogenesis, which contributes to the generation of additional mitochondria. However, these mitochondria are profoundly remodeled, displaying fragmented morphology, loose cristae, reduced Δψm, and metabolic programming. Furthermore, these remodeled mitochondria shift their function from ATP synthesis to reactive oxygen species (ROS) production, which drives NFκB-dependent inflammatory cytokine transcription. Interestingly, an increase in mitochondrial mass with constitutively active phosphomimetic mutant of Drp1 (Drp1S616E) boosted pro-inflammatory response in macrophages without LPS stimulation. In vivo, we also demonstrated that Mdivi-1 administration inhibits LPS-induced macrophage pro-inflammatory differentiation. Importantly, we observed Stat2 phosphorylation and Drp1-dependent mitochondrial mass increase in macrophages isolated from LPS-challenged mice. In conclusion, we comprehensively demonstrate that a Stat2-Drp1 dependent mitochondrial mass increase is necessary for pro-inflammatory differentiation of macrophages. Therefore, targeting the Stat2-Drp1 axis may provide novel therapeutic approaches for treating infection and inflammatory diseases.

Chlorogenic acid prevents paraquat-induced apoptosis via Sirt1-mediated regulation of redox and mitochondrial function.

Paraquat (PQ) is a widely used agro-chemical in agriculture and highly toxic to humans. Although the mechanism of PQ poisoning is not clear, it has been well documented that reactive oxygen species (ROS) generation and apoptosis play pivotal roles. Alternatively, chlorogenic acid (CA) is a biologically active dietary polyphenol, playing several therapeutic roles. However, it is not known whether CA has protective effect on PQ-induced apoptosis. Here, we investigated the effect of CA in preventing PQ-induced apoptosis and explored the underlying mechanisms. A549 cells were pretreated with 100 µM CA for 24 h and then exposed to 160 µM PQ for 24 h. We found that CA was effective in preventing PQ-induced apoptotic features, including the release of cytochrome c from the mitochondria to cytoplasm, the cleavages of caspase 3 and caspase 9, and the increases in levels of Bcl-2-associated X protein (Bax) and intracellular calcium ions. CA alleviated ROS production and prevented the reduction of antioxidant capacity in cells exposed to PQ by increasing NF-E2-related factor 2 (Nrf2), superoxide dismutase 2 (SOD2) and glutathione levels. In addition, CA also attenuated PQ-induced alterations of mitochondrial structure and function (such as the decreases in membrane potential and adenosine triphosphate level), and the impaired autophagic flux was improved by CA. Down-regulation of sirtuin 1 (Sirt1) by short hairpin RNA reversed the protective effects of CA. Thus, CA may be viewed as a potential drug to treat PQ-induced lung epithelial cell apoptosis and other disorders with similar pathologic mechanisms.

TGF-ß1 induces epithelial-to-mesenchymal transition via inhibiting mitochondrial functions in A549 cells.

Epithelial-to-mesenchymal transition (EMT) is critical to the progression of several disease processes including carcinoma metastasis and organ fibrosis. Recent studies show that reactive oxygen species (ROS) and mitochondrial dysfunction have been associated with EMT. However, the role of mitochondria in the EMT process remains to be elucidated. Through the induction of EMT using TGF-ß1, we demonstrated that mitochondrial functions were abnormal by increasing ROS production and reducing mitochondrial membrane potential, ATP content and mitochondrial complex protein expression. Resveratrol, a mitochondria protective agent, was found to prevent EMT by preserving mitochondrial functions during the process. However, the inhibitory effects of resveratrol on EMT were abolished in mitochondrial DNA-depleted cells. These findings suggest a critical role for mitochondria in EMT and implicate the protection of mitochondria as a potential target to prevent EMT to treat tumour metastasis or tissue fibrosis, and other diseases involving with mitochondria.

Inhibition of hepatocyte nuclear factor 1b induces hepatic steatosis through DPP4/NOX1-mediated regulation of superoxide.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder that is closely associated with insulin resistance and type 2 diabetes. Previous studies have suggested that hepatocyte nuclear factor 1b (HNF1b) ameliorates insulin resistance. However, the role of HNF1b in the regulation of lipid metabolism and hepatic steatosis remains poorly understood. We found that HNF1b expression was decreased in steatotic livers. We injected mice with lentivirus (LV) expressing HNF1b shRNA to generate mice with hepatic knockdown of HNF1b. We also injected high fat (HF) diet-induced obese and db/db diabetic mice with LV expressing HNF1b to overexpress HNF1b. Knockdown of HNF1b increased hepatic lipid contents and induced insulin resistance in mice and in hepatocytes. Knockdown of HNF1b worsened HF diet-induced increases in hepatic lipid contents, liver injury and insulin resistance in mice and PA-induced lipid accumulation and impaired insulin signaling in hepatocytes. Moreover, overexpression of HNF1b alleviated HF diet-induced increases in hepatic lipid content and insulin resistance in mice. Knockdown of HNF1b increased expression of genes associated with lipogenensis and endoplasmic reticulum (ER) stress. DPP4 and NOX1 expression was increased by knockdown of HNF1b and HNF1b directly bound with the promoters of DPP4 and NOX1. Overexpression of DPP4 or NOX1 was associated with an increase in lipid droplets in hepatocytes and decreased expression of DPP4 or NOX1 suppressed the effects of knockdown of HNF1b knockdown on triglyceride (TG) formation and insulin signaling. Knockdown of HNF1b increased superoxide level and decreased glutathione content, which was inhibited by downregulation of DPP4 and NOX1. N-acetylcysteine (NAC) suppressed HNF1b knockdown-induced ER stress, TG formation and insulin resistance. Palmitic acid (PA) decreased HNF1b expression which was inhibited by NAC. Taken together, these studies demonstrate that HNF1b plays an essential role in controlling hepatic TG homeostasis and insulin sensitivity by regulating DPP4/NOX1mediated generation of superoxide.

HO-1 Is Essential for Tetrahydroxystilbene Glucoside Mediated Mitochondrial Biogenesis and Anti-Inflammation Process in LPS-Treated RAW264.7 Macrophages.

2,3,5,4'-Tetrahydroxystilbene-2-O-ß-D-glucoside (TSG), an important monomer extracted from Polygonum multiflorum, can prevent a number of inflammation associated chronic diseases. However, the mechanism involved in TSG inducing anti-inflammatory role remains unclear. As an inducible antioxidant enzyme, Heme oxygenase-1 (HO-1), is crucial for protecting the mammalian cells against adverse stimuli. Here, we found that the TSG treatment strongly induces the expression of HO-1 in an NRF2-depended manner. Meanwhile, TSG increased the mitochondrial mass through upregulation of the mitochondrial biogenesis activators (PGC-1α, NRF1, and TFAM) as well as the mitochondrial complex IV. Furthermore, TSG attenuated Lipopolysaccharide (LPS) mediated RAW264.7 cells activation and secretion of proinflammatory cytokines, including interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Zinc Protoporphyrin (ZnPP), a selective inhibitor of HO-1 activity, was able to attenuate TSG mediated mitochondrial biogenesis and anti-inflammatory process. Finally, we observed that LPS induced obvious mtDNA depletion and ATP deficiency, which indicated a severe damage of mitochondria. TSG restored the LPS induced mitochondrial dysfunction via activation of the mitochondrial biogenesis. ZnPP treatment markedly reversed the inhibitory effects of TSG on mitochondrial damage and oxidative stress in LPS stimulated macrophages. Taken together, these findings suggest that TSG enhances mitochondrial biogenesis and function mainly via activation the HO-1. TSG can be developed as a potential drug for treatment of inflammatory diseases.

Differential effect of Se on insulin resistance: regulation of adipogenesis and lipolysis.

Insulin resistance is the characteristic of type 2 diabetes mellitus and metabolic disorder. The biological effect of selenium (Se) on insulin sensitivity and metabolic function was contradictory. In this study, we designed two animal protocols to investigate the effect of physiological Se on high-fat (HF) diet-induced insulin resistance in mice and examined the influence of Se on adipocyte differentiation and lipolysis in isolated bone marrow stromal stem cells. The results showed that pre-treatment with Se, mimicking thiazolidinediones, increased adipocyte differentiation and fat deposit in adipose tissue and reduced ectopic lipid content and consequent ROS generation and mitochondrial dysfunction in livers, protecting against HF diet-induced insulin resistance. Post-treatment with Se promoted lipolysis in adipose tissue and ectopic lipid accumulation in livers and aggravated subsequent ROS generation and mitochondrial dysfunction, exacerbating insulin resistance induced by HF diet. Activation of GPx1 and Sepp1 was responsible for Se-exhibited bi-directional significance, which was at the crossroad of the biological effect of Se, leading to differential directions: one way is to accelerate mitotic clonal expansion and increase key regulators of adipocyte differentiation, such as PPARγ and C/EBPα/ß, leading to enhancement of adipogenic differentiation; the other way is to activate PKA/HSL pathway, reinforcing lipolysis. Further studies are needed to elucidate the mechanism underlying GPx1 and Sepp1-exerted differential effects under different conditions. Anyhow, these findings may partly explain the contradiction of the biological significance of Se and demonstrate a novel understanding of the mechanism of Se-exerted benefit or harmful effects in the context of high consumption of fat.

Cyclosporine A Suppressed Glucose Oxidase Induced P53 Mitochondrial Translocation and Hepatic Cell Apoptosis through Blocking Mitochondrial Permeability Transition.

P53 is known as a transcription factor to control apoptotic cell death through regulating a series of target genes in nucleus. There is accumulating evidences show that p53 can directly induce cell apoptosis through transcription independent way at mitochondria. However, the mechanism by which p53 translocation into mitochondria in response to oxidative stress remains unclear. Here, glucose oxidase (GOX) was used to induce ROS generation in HepG2 cells and liver tissues of mice. The results showed that p53 was stabilized and translocated to mitochondria in a time and dose dependent manner after GOX exposure. Interestingly, as an inhibitor of mitochondrial permeability transition, cyclosporine A (CsA) was able to effectively reduce GOX mediated mitochondrial p53 distribution without influencing on the expression of p53 target genes including Bcl-2 and Bax. These indicated that CsA could just block p53 entering into mitochondria, but not affect p53-dependent transcription. Meanwhile, CsA failed to inhibit the ROS generation induced by GOX, which indicated that CsA had no antioxidant function. Moreover, GOX induced typical apoptosis characteristics including, mitochondrial dysfunction, accumulation of Bax and release of cytochrome C in mitochondria, accompanied with activation of caspase-9 and caspase-3. These processions were suppressed after pretreatment with CsA and pifithrin-µ (PFT-µ, a specific inhibitor of p53 mitochondrial translocation). In vivo, CsA was able to attenuate p53 mitochondrial distribution and protect mice liver against from GOX mediated apoptotic cell death. Taken together, these suggested that CsA could suppress ROS-mediated p53 mitochondrial distribution and cell apoptosis depended on its inhibition effect to mitochondrial permeability transition. It might be used to rescue the hepatic cell apoptosis in the patients with acute liver injury.

Gastrodin alleviates cerebral ischemic damage in mice by improving anti-oxidant and anti-inflammation activities and inhibiting apoptosis pathway.

Gastrodin (GAS), an active constituent of the Chinese herbal medicine Tianma, has anti-oxidant and anti-inflammation activities but its protective effect to the prevention of neurotoxicity induced by ischemic stroke is unclear. In the present study, middle cerebral artery occlusion (MCAO) was used to establish a mice ischemic stroke model. Infarct volume ratio and neurobehavioral score were evaluated, Nissl staining was performed and the expression of cleaved Caspase 3, Bax and B cell lymphoma 2 (Bcl-2) were assessed at 24 h or 7 days after reperfusion. In addition, the total superoxide dismutase (SOD) activity and malondialdehyde (MDA) content, as well as the expression of Nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), SOD1, phospho-Akt and total Akt and TNF-α and IL-1ß in the ischemic hemispheres were also observed at 6 h after reperfusion to assess oxidative stress and inflammatory changes after GAS treatment. It was found that GAS, especially at high dose (100 mg/kg) reduced tested neuronal injury and neurobehavioral deficient in MCAO mice. Enhanced expression of cleaved Caspase 3 and Bax and decreased expression of Bcl-2 by MCAO were also reversed by GAS. Moreover, GAS treatment decreased the MDA content and the expression of TNF-α and IL-1ß, and increased amount of SOD activity and the expression of HO-1 and SOD1 in GAS-treated ischemic brain. Furthermore, GAS significantly increased Akt phosphorylation and Nrf2 expression. These results support the neuroprotective effects of GAS, and the activation of Akt/Nrf2 pathway may play a critical role in the pharmacological action of GAS.

RARγ-C-Fos-PPARγ2 signaling rather than ROS generation is critical for all-trans retinoic acid-inhibited adipocyte differentiation.

Obesity has become a worldwide public health problem, which is mainly determined by excess energy intake and adipose tissue expansion. Adipose tissue expansion can occur through hyperplasia (adipocyte differentiation) or hypertrophy. Retinoic acid was shown to inhibit adipocyte differentiation. However, the molecular mechanism is unclear. In the study, we found that all-trans-retinoic acid (ATRA) inhibited 3T3-L1 adipocyte differentiation. We did not observe significant apoptosis in differentiated adipocytes treated by ATRA. ATRA increased ROS generation and disturbed redox balance. However, antioxidant treatment did not ameliorate the reduction of lipid accumulation induced by ATRA, indicating that ROS generation was not involved in ATRA-inhibited adipocyte differentiation. ATRA reduced C/EBPα, PPARγ and its target gene expression. In the presence of ATRA, retinoic acid receptor (RAR) α/γ expression was increased. Inhibition of RARγ, but not RARα, blocked ATRA-induced reduction of PPARγ2 expression. ATRA induced a profound interaction between RARγ and C-Fos protein, reflected by Co-IP results. C-Fos was found to exhibit a differentiation-dependent DNA binding activity to PPARγ2 promoter. RARγ inhibitor significantly suppressed ATRA-inhibited DNA binding activity of C-Fos to PPARγ2 promoter, indicating that downregulation of C-Fos activity mediated activation of RARγ-exerted reduction of PPARγ2 expression and thus inhibition of adipocyte differentiation induced by ATRA. Taken together, these data demonstrates that RARγ-C-Fos-PPARγ2 signaling rather than ROS generation is critical for ATRA-inhibited adipocyte differentiation.