Kellerin alleviates cerebral ischemic injury by inhibiting ferroptosis via targeting Akt-mediated transcriptional activation of Nrf2.

BACKGROUND: Ischemic stroke (IS) is characterized as a detrimental cerebrovascular disease with high mortality and disability. Ferroptosis is a novel mechanism involved in neuronal death. There is a close connection between IS and ferroptosis, and inhibiting ferroptosis may provide an effective strategy for treating IS. Our previous investigations have discovered that kellerin, the active compound of Ferula sinkiangensis K. M. Shen, possesses the capability to shield against cerebral ischemia injury. PURPOSE: Our objective is to clarify the relationship between the neuroprotective properties of kellerin against IS and its ability to modulate ferroptosis, and investigate the underlying regulatory pathway. STUDY DESIGN: We investigated the impact and mechanism of kellerin in C57BL/6 mice underwent middle cerebral artery occlusion/reperfusion (MCAO/R) as well as SH-SY5Y cells exposed to oxygen-glucose deprivation/ re-oxygenation (OGD/R). METHODS: The roles of kellerin on neurological severity, cerebral infarction and edema were investigated in vivo. The regulatory impacts of kellerin on ferroptosis, mitochondrial damage and Akt/Nrf2 pathway were explored. Molecular docking combined with drug affinity responsive target stability assay (DARTS) and cellular thermal shift assay (CETSA) were performed to analyze the potential target proteins for kellerin. RESULTS: Kellerin protected against IS and inhibited ferroptosis in vivo. Meanwhile, kellerin improved the neuronal damage caused by OGD/R and suppressed ferroptosis by inhibiting the production of mitochondrial ROS in vitro. Further we found that kellerin directly interacted with Akt and enhanced its phosphorylation, leading to the increase of Nrf2 nuclear translocation and its downstream antioxidant genes expression. Moreover, kellerin's inhibitory effect on ferroptosis and mitochondrial ROS release was eliminated by inhibiting Akt/Nrf2 pathway. CONCLUSIONS: Our study firstly demonstrates that the neuroprotective properties of kellerin against IS are related to suppressing ferroptosis through inhibiting the production of mitochondrial ROS, in which its modulation on Akt-mediated transcriptional activation of Nrf2 plays an important role. This finding shed light on the potential mechanism that kellerin exerts therapeutic effects in IS.

Emodin ameliorates antioxidant capacity and exerts neuroprotective effect via PKM2-mediated Nrf2 transactivation.

Pyruvate kinase M2 (PKM2) is overexpressed in neuronal cells. However, there are few studies on the involvement of PKM2 modulators in neurodegenerative diseases. Emodin, a dominating anthraquinone derivative extracting from the rhizome of rhubarb, has received expanding consideration due to its pharmacological properties. Our data reveal that emodin could resist hydrogen peroxide- or 6-hydroxydopamine-mediated mitochondrial fission and apoptosis in PC12 cells (a neuron-like rat pheochromocytoma cell line). Notably, emodin at nontoxic concentrations significantly inhibits PKM2 activity and promotes dissociation of tetrameric PKM2 into dimers in cells. The PKM2 dimerization enhances the interaction of PKM2 and NFE2-related factor 2 (Nrf2), which further triggers the activation of the Nrf2/ARE pathway to upregulate a panel of cytoprotective genes. Modulating the PKM2/Nrf2/ARE axis by emodin unveils a novel mechanism for understanding the pharmacological functions of emodin. Our findings indicate that emodin is a potential candidate for the treatment of oxidative stress-related neurodegenerative disorders.

Transcriptional Profiling of the Candida auris Response to Exogenous Farnesol Exposure.

The antifungal resistance threat posed by Candida auris necessitates bold and innovative therapeutic options. Farnesol is a quorum-sensing molecule with a potential antifungal and/or adjuvant effect; it may be a promising candidate in alternative treatment regimens. To gain further insights into the farnesol-related effect on C. auris, genome-wide gene transcription analysis was performed using transcriptome sequencing (RNA-Seq). Farnesol exposure resulted in 1,766 differentially expressed genes. Of these genes, 447 and 304 genes with at least 1.5-fold increase or decrease in transcription, respectively, were selected for further investigation. Genes involved in morphogenesis, biofilm events (maturation and dispersion), gluconeogenesis, iron metabolism, and regulation of RNA biosynthesis showed downregulation, whereas those related to antioxidative defense, transmembrane transport, glyoxylate cycle, fatty acid ß-oxidation, and peroxisome processes were upregulated. In addition, farnesol treatment increased the transcription of certain efflux pump genes, including MDR1, CDR1, and CDR2. Growth, measured by the change in the number of CFU, was significantly inhibited within 2 h of the addition of farnesol (5.8 × 107 ± 1.1 × 107 and 1.1 × 107 ± 0.3 × 107 CFU/ml for untreated control and farnesol-exposed cells, respectively) (P < 0.001). In addition, farnesol treatment caused a significant reduction in intracellular iron (152.2 ± 21.1 versus 116.0 ± 10.0 mg/kg), manganese (67.9 ± 5.1 versus 18.6 ± 1.8 mg/kg), and zinc (787.8 ± 22.2 versus 245.8 ± 34.4 mg/kg) (P < 0.05 to 0.001) compared to untreated control cells, whereas the level of cooper was significantly increased (274.6 ± 15.7 versus 828.8 ± 106.4 mg/kg) (P < 0.001). Our data demonstrate that farnesol significantly influences the growth, intracellular metal ion contents, and gene transcription related to fatty acid metabolism, which could open new directions in developing alternative therapies against C. auris. IMPORTANCE Candida auris is a dangerous fungal pathogen that causes outbreaks in health care facilities, with infections associated with a high mortality rate. As conventional antifungal drugs have limited effects against the majority of clinical isolates, new and innovative therapies are urgently needed. Farnesol is a key regulator molecule of fungal morphogenesis, inducing phenotypic adaptations and influencing biofilm formation as well as virulence. Alongside these physiological modulations, it has a potent antifungal effect alone or in combination with traditional antifungals, especially at supraphysiological concentrations. However, our knowledge about the mechanisms underlying this antifungal effect against C. auris is limited. This study has demonstrated that farnesol enhances the oxidative stress and reduces the fungal survival strategies. Furthermore, it inhibits manganese, zinc transport, and iron metabolism as well as increases fungal intracellular copper content. In addition, metabolism was modulated toward ß-oxidation. These results provide definitive explanations for the observed antifungal effects.

Hemistepsin a Induces Apoptosis of Hepatocellular Carcinoma Cells by Downregulating STAT3.

Hemistepta lyrata (Bunge) Bunge is a biennial medicinal plant possessing beneficial effects including anti-inflammation, and hemistepsin A (HsA) isolated from H. lyrata has been known as a hepatoprotective sesquiterpene lactone. In this report, we explored the cytotoxic effects of H. lyrata on hepatocellular carcinoma (HCC) cells and investigated the associated bioactive compounds and their relevant mechanisms. From the viability results of HCC cells treated with various H. lyrata extracts, HsA was identified as the major compound contributing to the H. lyrata-mediated cytotoxicity. HsA increased expression of cleaved PARP and cells with Sub-G1 phase, Annexin V binding, and TUNEL staining, which imply HsA induces apoptosis. In addition, HsA provoked oxidative stress by decreasing the reduced glutathione/oxidized glutathione ratio and accumulating reactive oxygen species and glutathione-protein adducts. Moreover, HsA inhibited the transactivation of signal transducer and activator of transcription 3 (STAT3) by its dephosphorylation at Y705 and glutathione conjugation. Stable expression of a constitutive active mutant of STAT3 prevented the reduction of cell viability by HsA. Finally, HsA enhanced the sensitivity of sorafenib-mediated cytotoxicity by exaggerating oxidative stress and Y705 dephosphorylation of STAT3. Therefore, HsA will be a promising candidate to induce apoptosis of HCC cells via downregulating STAT3 and sensitizing conventional chemotherapeutic agents.

Characterisation and validation of an in vitro transactivation assay based on the 22Rv1/MMTV_GR-KO cell line to detect human androgen receptor agonists and antagonists.

We describe the characterisation and validation of an androgen receptor (AR) transactivation assay for detection of AR agonists and antagonists using a stably transfected human prostate cancer cell line. This 22Rv1/mouse mammary tumour virus glucocorticoid knock-out cell line based AR transactivation assay was validated by criteria in Organisation for Economic Cooperation and Development Guidance Document 34 to determine if the assay performed equally well to the AR EcoScreen Assay included in Test Guideline for AR Transactivation (OECD TG 458). There was no Glucocorticoid Receptor (GR) crosstalk, and no changes in the AR DNA sequence in cells after the successful knock out of GR. Subsequently, the concordance of classifications of the 22 test chemicals was 100% in all laboratories. The AR agonistic and antagonistic inter-laboratory coefficients of variation based on log[10% effect for 10 nM DHT, PC10] and log[inhibitory response of 800 pM DHT by at 30%, IC30] from comprehensive tests were 2.75% and 2.44%, respectively. The AR agonist/antagonist test chemical classifications were consistent across AR EcoScreen ARTA assay data for 82/89%, and the balanced accuracy, sensitivity, and specificity were 83/90%, 88/100% and 78/80%, respectively. This assay was successfully validated and was approved for inclusion in TG 458 in 2020.

Diverse diterpenoids and sesquiterpenoids from Siegesbeckia pubescens and their activity against RANKL-induced osteoclastogenesis.

Phytochemical investigation of the aerial parts of Siegesbeckia pubescens led to seventeen diterpenoids (1-17) and twelve sesquiterpenoids (18-29). Their structures were varied including twelve ent-pimarane (1-12), three ent-kaurane (13-15), two acyclic diterpenoids (16-17), ten germacrene (18-27), one guaiane (28), and one caryolane (29) sesquiterpenoids. Eight of twenty-nine were new ones (1, 3, 4, 16-18, 23, and 28). Their structures were elucidated by extensive spectroscopic analysis. The absolute configurations of compounds 1 and 2 were identified using X-ray diffraction analysis, and of compounds 18, 23, and 28 were elucidated by the experimental and calculated electronic circular dichroism (ECD) spectra. All the isolated compounds (1-29) were assayed for their inhibition of RANKL-induced osteoclastogenesis in bone marrow macrophages (BMMs). Four sesquiterpenoids 18, 25, 26, and 27 exhibited potent inhibition of osteoclastogenesis with IC50 value of 0.51, 0.80, 0.50, and 0.83 µM, respectively. Here we demonstrated that S. pubescens may be a resource for discovery of anti-osteoporosis agents.

A G-quadruplex nanoswitch in the SGK1 promoter regulates isoform expression by K+/Na+ balance and resveratrol binding.

BACKGROUND: High sodium intake can up-regulate the level of renal serum- and glucocorticoid-inducible kinase-1 (SGK1), which plays a pivotal role in controlling blood pressure via activation of the epithelial sodium channel (ENaC), which can lead to salt-sensitive hypertension. Increased potassium intake, or a vegetarian diet, counteracts salt-sensitive hypertension, but the underlying mechanisms are not fully understood. METHODS: Bioinformatics and molecular modeling were used to identify G-quadruplex (G4) and their conformations in the SGK1 promoter. CD spectra and UV melting dynamics were measured to study the stability of G4 as influenced by potassium/sodium balance and resveratrol. RT-PCR and Western blot were employed to study the effects of potassium and resveratrol on the SGK1 isoform expression. RESULTS: The SGK1 gene encodes a G4 structure in the proximal upstream of promoter-2; the G4 structure is stabilized by potassium or resveratrol, but destabilized by sodium. Super-physiological levels of sodium stimulate the transcription of all SGK1 isoforms, whereas resveratrol or potassium supplementation inhibits the transcription of iso-2 and iso-3, but not iso-1. CONCLUSIONS: Stabilizing the G4 by potassium or resveratrol induces alternative promoter usage and/or pre-mRNA splicing in the transcription of SGK1. GENERAL SIGNIFICANCE: Potassium/sodium ion balance or resveratrol binding can act to regulate G4 molecular switches for controlling SGK1 gene expression, thereby presenting a new avenue for drug development.

Transcription factor Sp1 is upregulated by PKCι to drive the expression of YAP1 during pancreatic carcinogenesis.

Recently, we identified that the atypical protein kinase C isoform ι (PKCι) enhances the expression of Yes-associated protein 1 (YAP1) to promote the tumorigenesis of pancreatic adenocarcinoma harboring mutant KRAS (mu-KRAS). To advance our understanding about underlying mechanisms, we analyze the transcription of YAP1 in pancreatic cancer cells and reveal that transcription factor specificity protein 1 (Sp1) is upregulated by PKCι and subsequently binds to multiple sites in YAP1 promoter to drive the transactivation of YAP1 in pancreatic cancer cells carrying mu-KRAS. The bioinformatics analysis further substantiates that the expression of PKCι, Sp1 and YAP1 is correlated and associated with the stages and prognosis of pancreatic tumors. Moreover, our apoptotic detection data demonstrate that combination of PKCι and Sp1 inhibitors at subtoxic doses displays synergistic effects on inducing apoptosis and reversing the immunosuppression of pancreatic cancer cells, establishing the combination of PKCι and Sp1 inhibitors as a promising novel therapeutic approach, or an adjuvant strategy to potentiate the antitumor effects of other immunotherapeutic agents in pancreatic cancer treatment.

Artesunate inhibits intestinal tumorigenesis through inhibiting wnt signaling.

Artesunate (ART) is a clinically approved antimalarial drug and was revealed as a candidate of colorectal cancer chemopreventive agents in our drug screening system. Here, we aimed to understand the suppressive effects of ART on intestinal tumorigenesis. In vitro, ART reduced T-cell factor/lymphoid enhancer factor (TCF/LEF) promoter transcriptional activity. In vivo, ART inhibited intestinal polyp development. We found that ART reduces TCF1/TCF7 nuclear translocation by binding the Ras-related nuclear protein (RAN), suggesting that ART inhibits TCF/LEF transcriptional factor nuclear translocation by binding to RAN, thereby inhibiting Wnt signaling. Our results provide a novel mechanism through which artesunate inhibits intestinal tumorigenesis.

Application of Humanized Zebrafish Model in the Suppression of SARS-CoV-2 Spike Protein Induced Pathology by Tri-Herbal Medicine Coronil via Cytokine Modulation.

Zebrafish has been a reliable model system for studying human viral pathologies. SARS-CoV-2 viral infection has become a global chaos, affecting millions of people. There is an urgent need to contain the pandemic and develop reliable therapies. We report the use of a humanized zebrafish model, xeno-transplanted with human lung epithelial cells, A549, for studying the protective effects of a tri-herbal medicine Coronil. At human relevant doses of 12 and 58 µg/kg, Coronil inhibited SARS-CoV-2 spike protein, induced humanized zebrafish mortality, and rescued from behavioral fever. Morphological and cellular abnormalities along with granulocyte and macrophage accumulation in the swim bladder were restored to normal. Skin hemorrhage, renal cell degeneration, and necrosis were also significantly attenuated by Coronil treatment. Ultra-high-performance liquid chromatography (UHPLC) analysis identified ursolic acid, betulinic acid, withanone, withaferine A, withanoside IV-V, cordifolioside A, magnoflorine, rosmarinic acid, and palmatine as phyto-metabolites present in Coronil. In A549 cells, Coronil attenuated the IL-1ß induced IL-6 and TNF-α cytokine secretions, and decreased TNF-α induced NF-κB/AP-1 transcriptional activity. Taken together, we show the disease modifying immunomodulatory properties of Coronil, at human equivalent doses, in rescuing the pathological features induced by the SARS-CoV-2 spike protein, suggesting its potential use in SARS-CoV-2 infectivity.

Dexmedetomidine and levobupivacaine co-loaded, transcriptional transactivator peptide modified nanostructured lipid carriers or lipid-polymer hybrid nanoparticles, which performed better for local anesthetic therapy?

Local anesthetics (LAs) have been widely applied in clinic for regional anesthesia, postoperative analgesia, and management of acute and chronic pain. Nanostructured lipid carriers (NLCs) and lipid-polymer hybrid nanoparticles (LPNs) are reported as good choices for LA therapy. Transactivated transcriptional activator (TAT) was reported as a modifier for the topical delivery of drugs. In the present study, TAT modified, levobupivacaine (LEV) and dexmedetomidine (DEX) co-delivered NLCs (TAT-LEV&DEX-NLCs, T-L&D-N) and LPNs (TAT-LEV&DEX-LPNs, T-L&D-L) were designed and compared for the LA therapy. T-L&D-L exhibited better efficiency in improving the skin permeation, analgesic time, and pain control intensity than T-L&D-N both in vitro and in vivo. On the other side, T-L&D-N also improved the therapeutic effect of drugs to a large extent. These two systems both exhibited superiority in some respects. TAT modified LPNs are more promising platform for the long-term local anesthesia.

The tea catechin epigallocatechin gallate inhibits NF-κB-mediated transcriptional activation by covalent modification.

Potential health benefits of consuming tea are thought to include anti-inflammatory actions of its constituent flavonoids including catechins, which are well-recognized antioxidants. We analyzed and discovered a novel mechanism by which epigallocatechin gallate (EGCG), the most abundant polyphenol in tea and a putative health-promoting constituent, inhibits activation of the nuclear transcription factor NF-κB, which mediates inflammatory responses to cytokines and other agents. We found that EGCG inhibits NF-κB-p65 transcriptional activity, by preventing NF-κB-p65 binding to κBs in normal human bronchial epithelial cells. We also analyzed the chemical mechanism by which EGCG binds directly to NF-κB-p65, and found that it involves covalent reaction via enones within EGCG ring structures, as the oxidizer diamide, which prevents 1, 4-addition reactions, blocked adduct-forming reaction of biotinylated EGCG with NF-κB-p65. Such blockade was inhibited by competing unlabeled EGCG. Furthermore, such covalent binding reflected irreversible reaction of EGCG with sulfhydryls of NF-κB-p65, as it was inhibited by glutathione but not reversible by it. We identified the reactive sulfhydryl moiety as that of cysteine, as S-carboxymethylation to block cysteine sulfhydryls prevented NF-κB-p65-Cys-alkylation reaction with EGCG. We also tested if EGCG can inhibit NF-κB-p65 binding to DNA within the nucleus, after its phosphorylation and translocation (activation). EGCG did not alter intranuclear phosphorylation levels of NF-κB-p65, but strongly repressed DNA-binding ability of activated NF-κB-p65, indicating that EGCG inhibits NF-κB-p65 DNA binding activity even without altering NF-κB-p65 phosphorylation or expression. These findings thus reveal a novel mechanism by which EGCG inhibits transcriptional activity of NF-κB-p65, that may potentially contribute to anti-inflammatory and health-promoting effects of EGCG and consumption of tea.

Spirulina maxima derived marine pectin promotes the in vitro and in vivo regeneration and wound healing in zebrafish.

Purified bioactive components of marine algae have shown great pharmaceutical and biomedical potential, including wound healing activity. However, the activity of Spirulina maxima is the least documented with regard to wound healing potential. In the present study, we investigated the regenerative and wound healing activities of a Spirulina (Arthrospira) maxima based pectin (SmP) using in vitro human dermal fibroblasts (HDFs) and in vivo zebrafish model. SmP treated (12.5-50 µg/mL) HDFs showed increased cell proliferation by 20-40% compared to the untreated HDFs. Moreover, in vitro wound healing results in HDFs demonstrated that SmP decreased the open wound area % in concentration-dependent manner at 12.5 (32%) and 25 µg/mL (12%) compared to the control (44%). Further, zebrafish larvae displayed a greater fin regenerated area in the SmP exposed group at 25 (0.48 mm2) and 50 µg/mL (0.51 mm2), whereas the untreated group had the lowest regenerated area (0.40 mm2) at 3 days post amputation. However, fin regeneration was significantly (P < 0.001) higher only in the SmP treated group at 50 µg/mL. Furthermore, the open skin wound healing % in adult zebrafish was significantly higher (P < 0.05) after topical application (600 µg/fish) of SmP (46%) compared to the control (38%). Upregulation of genes such as tgfß1, timp2b, mmp9, tnf-α, and il-1ß, and chemokines such as cxcl18b, ccl34a.4, and ccl34b.4, in the muscle and kidney tissues of SmP treated fish compared to the respective control group was demonstrated using qRT-PCR. Histological analysis results further supported the rapid epidermal growth and tissue remodeling in SmP treated fish, suggesting that SmP exerts positive effects associated with wound healing. Therefore, SmP can be considered a potential regenerative and wound healing agent.

MK615 Suppresses Hypoxia Tolerance by Up-regulation of E-cadherin in Colorectal Cancer Cells With Mutant KRAS.

BACKGROUND/AIM: The Japanese apricot "Prunus mume" is a traditional Japanese medicine. MK615, a compound extract from Prunus mume has been reported to have anti-tumor effects. Herein, we used 3D floating (3DF) culture to evaluate the anticancer effects of MK615 against human colorectal cancer (CRC) cells that contain mutant (mt) KRAS. MATERIALS AND METHODS: HKe3 cells exogenously expressing mtKRAS (HKe3-mtKRAS) were treated with MK615 in 3DF cultures. The protein levels of hypoxia-inducible factor 1 (HIF-1) and E-cadherin were quantified by western blotting. RESULTS: MtKRAS enhanced hypoxia tolerance via up-regulation of HIF-1. The expression of HIF-1 protein was suppressed by constitutive overexpression of E-cadherin in CRC HCT116 spheroids. MK615 increased the expression of E-cadherin and decreased the expression of HIF-1 in HKe3-mtKRAS. These results suggest that MK615 suppresses hypoxia tolerance by up-regulation of E-cadherin in CRC cells with mtKRAS. CONCLUSION: MK615 exhibits properties useful for the potential treatment of CRC patients with mtKRAS.

Food for Bone: Evidence for a Role for Delta-Tocotrienol in the Physiological Control of Osteoblast Migration.

Bone remodeling and repair require osteogenic cells to reach the sites that need to be rebuilt, indicating that stimulation of osteoblast migration could be a promising osteoanabolic strategy. We showed that purified δ-tocotrienol (δ-TT, 10 µg/mL), isolated from commercial palm oil (Elaeis guineensis) fraction, stimulates the migration of both MC3T3-E1 osteoblast-like cells and primary human bone marrow mesenchymal stem cells (BMSC) as detected by wound healing assay or Boyden chamber assay respectively. The ability of δ-TT to promote MC3T3-E1 cells migration is dependent on Akt phosphorylation detected by Western blotting and involves Wnt/ß-catenin signalling pathway activation. In fact, δ-TT increased ß-catenin transcriptional activity, measured using a Nano luciferase assay and pretreatment with procaine (2 µM), an inhibitor of the Wnt/ß-catenin signalling pathway, reducing the wound healing activity of δ-TT on MC3T3-E1 cells. Moreover, δ-TT treatment increased the expression of ß-catenin specific target genes, such as Osteocalcin and Bone Morphogenetic Protein-2, involved in osteoblast differentiation and migration, and increased alkaline phosphatase and collagen content, osteoblast differentiation markers. The ability of δ-TT to enhance the recruitment of BMSC, and to promote MC3T3-E1 differentiation and migratory behavior, indicates that δ-TT could be considered a promising natural anabolic compound.

Limonin ameliorates acetaminophen-induced hepatotoxicity by activating Nrf2 antioxidative pathway and inhibiting NF-κB inflammatory response via upregulating Sirt1.

BACKGROUND: Limonin, a bioactive compound from citrus plants, exerts antioxidant activities, however its therapeutic potential in acetaminophen (APAP)-induced hepatotoxicity remains unclear. PURPOSE: Our study aims to investigate the protective effect of limonin on APAP-induced hepatotoxicity and illuminate the underlying mechanisms. STUDY: design In vitro, we chose L-02 cells to establish in vitro APAP-induced liver injury model. L-02 cells were treated with APAP (7.5 mM) for 24 h after pre-incubation with limonin (10, 25, 50 µM) or NAC (250 µM) for 2 h. In vivo, we used C57BL/6 mice as an in vivo APAP-induced liver injury model. C57BL/6 mice with pre-treatment of limonin (40, 80 mg/kg) or NAC (150 mg/kg) for 1 h, were given with a single dose of APAP (300 mg/kg). METHODS: After pre-incubation with limonin (10, 25, 50 µM) for 2 h, L-02 cells were treated with APAP (7.5 mM) for 24 h.The experiments in vitro included MTT assay, Annexin V/PI staining, measurement of reactive oxygen species (ROS), quantitative real-time PCR analysis, Western blot analysis, immunofluorescence microscopy and analysis of LDH activity. Transfection of Nrf2 or Sirt1 siRNA was also conducted in vitro. In vivo, C57BL/6 mice with pre-treatment of limonin (40, 80 mg/kg) or NAC (150 mg/kg) for 1 h, were given with a single dose of APAP (300 mg/kg). Mice were sacrificed at 4, 12 h after APAP poisoning, and analysis of ALT and AST in serum, GSH level in liver tissues, liver histological observation and immunohistochemistry were performed. RESULTS: Limonin increased the cell viability and alleviated APAP-induced apoptosis in hepatocytes. Limonin also inhibited APAP-induced mitochondrial-mediated apoptosis by decreasing the ratio of Bax/Bcl-2, recovery of mitochondrial membrane potential (MMP), inhibiting ROS production and cleavage of caspase-3 in L-02 cells. Moreover, limonin induced activation of Nrf2 and increased protein expression and mRNA levels of its downstream targets, including HO-1, NQO1 and GCLC/GCLM. The inhibition of limonin on apoptosis and promotion on Nrf2 antioxidative pathway were lessened after the application of Nrf2 siRNA. In addition, limonin inhibited NF-κB transcriptional activation, NF-κB-regulated genes and protein expression of inflammatory related proteins iNOS and COX2. Furthermore, limonin increased the protein expression of Sirt1. Sirt1 siRNA transfection confirmed that limonin activated Nrf2 antioxidative pathway and inhibited NF-κB inflammatory response by upregulating Sirt1. Finally, we established APAP-induced liver injury in vivo and demonstrated that limonin alleviated APAP-induced hepatotoxicity by activating Nrf2 antioxidative signals and inhibiting NF-κB inflammatory response via upregulating Sirt1. CONCLUSION: In summary, this study documented that limonin mitigated APAP-induced hepatotoxicity by activating Nrf2 antioxidative pathway and inhibiting NF-κB inflammatory response via upregulating Sirt1, and demonstrated that limonin had therapeutic promise in APAP-induced liver injury.

Up-regulation of miR-297 mediates aluminum oxide nanoparticle-induced lung inflammation through activation of Notch pathway.

Exposure to Aluminum oxide nanoparticles (Al2O3 NPs) has been associated with pulmonary inflammation in recent years; however, the underlying mechanism that causes adverse effects remains unclear. In the present study, we characterized microRNA (miRNA) expression profiling in human bronchial epithelial (HBE) cells exposed to Al2O3 NPs by miRNA microarray. Among the differentially expressed miRNAs, miR-297, a homologous miRNA in Homo sapiens and Mus musculus, was significantly up-regulated following exposure to Al2O3 NPs, compared with that in control. On combined bioinformatic analysis, proteomics analysis, and mRNA microarray, NF-κB-activating protein (NKAP) was found to be a target gene of miR-297 and it was significantly down-regulated in Al2O3 NPs-exposed HBE cells and murine lungs, compared with that in control. Meanwhile, inflammatory cytokines, including IL-1ß and TNF-α, were significantly increased in bronchoalveolar lavage fluid (BALF) from mice exposed to Al2O3 NPs. Then we set up a mouse model with intranasal instillation of antagomiR-297 to further confirm that inhibition of miR-297 expression can rescue pulmonary inflammation via Notch pathway suppression. Collectively, our findings suggested that up-regulation of miR-297 expression was an upstream driver of Notch pathway activation, which might be the underlying mechanism involved in lung inflammation induced by exposure to Al2O3 NPs.

Baicalin relieves TNF-α-evoked injury in human aortic endothelial cells by up-regulation of miR-145.

Hypertension is recognized to be associated with low-grade inflammation. Baicalin (BAI) is reported to possess various pharmacological including anti-inflammatory activities. This research explored the molecular mechanism by which BAI functions in human aortic endothelial cells (HAECs). HAECs were pretreated with BAI. Cell viability, apoptosis, and expressions of crucial proteins were respectively evaluated using cell counting kit-8 assay, flow cytometry, and western blot. Productions of cytokines were respectively assessed employing quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Cell transfection was utilized to alter miR-145 expression. The expressions of proteins participated in JNK and p38MAPK pathways were analyzed utilizing western blot. TNF-α inducement successfully evoked inflammatory injury in HAECs, exhibiting as prominently suppressed viability, while facilitated apoptosis and productions of cytokines. However, BAI pretreatment significantly ameliorated TNF-α-triggered inflammatory injuries. Besides, miR-145 expression was markedly inhibited by TNF-α inducement, while notably elevated by BAI pretreatment. Although miR-145 overexpression had no significant influence on apoptosis, miR-145 silence observably reversed BAI pretreatment-evoked protective influences on TNF-α-induced HAECs, as well as the inhibited impacts on the levels of key proteins involved in JNK and p38MAPK pathways. This investigation illustrated that BAI relieved TNF-α-triggered injuries through upregulating miR-145 via suppressing JNK and p38MAPK pathways.

Targeting NRF2 for the Treatment of Friedreich's Ataxia: A Comparison among Drugs.

NRF2 (Nuclear factor Erythroid 2-related Factor 2) signaling is impaired in Friedreich's Ataxia (FRDA), an autosomal recessive disease characterized by progressive nervous system damage and degeneration of nerve fibers in the spinal cord and peripheral nerves. The loss of frataxin in patients results in iron sulfur cluster deficiency and iron accumulation in the mitochondria, making FRDA a fatal and debilitating condition. There are no currently approved therapies for the treatment of FRDA and molecules able to activate NRF2 have the potential to induce clinical benefits in patients. In this study, we compared the efficacy of six redox-active drugs, some already adopted in clinical trials, targeting NRF2 activation and frataxin expression in fibroblasts obtained from skin biopsies of FRDA patients. All of these drugs consistently increased NRF2 expression, but differential profiles of NRF2 downstream genes were activated. The Sulforaphane and N-acetylcysteine were particularly effective on genes involved in preventing inflammation and maintaining glutathione homeostasis, the dimethyl fumarate, omaxevolone, and EPI-743 in counteracting toxic products accumulation, the idebenone in mitochondrial protection. This study may contribute to develop synergic therapies, based on a combination of treatment molecules.

The serum amyloid A3 promoter-driven luciferase reporter mice is a valuable tool to image early renal fibrosis development and shows the therapeutic effect of glucosyl-hesperidin treatment.

Tubulointerstitial fibrosis is a progressive process affecting the kidneys, causing renal failure that can be life-threatening. Thus, renal fibrosis has become a serious concern in the ageing population; however, fibrotic development cannot be diagnosed early and assessed noninvasively in both patients and experimental animal models. Here, we found that serum amyloid A3 (Saa3) expression is a potent indicator of early renal fibrosis; we also established in vivo Saa3/C/EBPß-promoter bioluminescence imaging as a sensitive and specific tool for early detection and visualization of tubulointerstitial fibrosis. Saa3 promoter activity is specifically upregulated in parallel with tumor necrosis factor α (TNF-α) and fibrotic marker collagen I in injured kidneys. C/EBPß, upregulated in injured kidneys and expressed in tubular epithelial cells, is essential for the increased Saa3 promoter activity in response to TNF-α, suggesting that C/EBPß plays a crucial role in renal fibrosis development. Our model successfully enabled visualization of the suppressive effects of a citrus flavonoid derivative, glucosyl-hesperidin, on inflammation and fibrosis in kidney disease, indicating that this model could be widely used in exploring therapeutic agents for fibrotic diseases.