Comparison of the inhibition effects of naringenin and its glycosides on LPS-induced inflammation in RAW 264.7 macrophages.

BACKGROUND: Inflammation is intricately linked to the development of various diseases, such as diabetes, cardiovascular diseases, and cancer. Flavonoids, commonly found in plants, are known for their diverse health benefits, including antioxidant and anti-inflammatory properties. These compounds are categorized into different classes based on their chemical structure. structures. However, limited research has compared the effects of flavonoid aglycones and flavonoid glycosides. This study aims to assess the anti-inflammatory effects of naringenin and its glycosides (naringin and narirutin) in RAW264.7 macrophages. METHODS AND RESULTS: RAW264.7 cells were treated with naringenin, naringin, and narirutin, followed by stimulation with lipopolysaccharide. The levels of inflammatory mediators, including tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß), nitric oxide (NO), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2), were assessed. Additionally, the study examined nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) activation using western blot analysis. Among the compounds tested, narirutin exhibited the most potent anti-inflammatory effect against TNF-α, NO, and iNOS. Naringin and narirutin showed comparable inhibitory effects on IL-1ß and COX-2. Both naringin and narirutin suppressed the expression of pro-inflammatory mediators by targeting different levels of the NF-κB and MAPK pathways. Naringenin demonstrated the weakest anti-inflammatory effect, primarily inhibiting NF-κB and reducing the phosphorylation levels of p38. CONCLUSIONS: This study suggests that the presence of glycosides on naringenin and the varied binding forms of sugars in naringenin glycosides significantly influence the anti-inflammatory effects compared with naringenin in RAW 264.7 macrophages.

Protein Kinases in Obesity, and the Kinase-Targeted Therapy.

The action of protein kinases and protein phosphatases is essential for multiple physiological responses. Each protein kinase displays its own unique substrate specificity and a regulatory mechanism that may be modulated by association with other proteins. Protein kinases are classified as dual-specificity kinases and dual-specificity phosphatases. Dual-specificity phosphatases are important signal transduction enzymes that regulate various cellular processes in coordination with protein kinases and play an important role in obesity. Impairment of insulin signaling in obesity is largely mediated by the activation of the inhibitor of kappa B-kinase beta and the c-Jun N-terminal kinase (JNK). Oxidative stress and endoplasmic reticulum (ER) stress activate the JNK pathway which suppresses insulin biosynthesis. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) are important for proper regulation of glucose metabolism in mammals at both the hormonal and cellular levels. Additionally, obesity-activated calcium/calmodulin dependent-protein kinase II/p38 suppresses insulin-induced protein kinase B phosphorylation by activating the ER stress effector, activating transcription factor-4. To alleviate lipotoxicity and insulin resistance, promising targets are pharmacologically inhibited. Nifedipine, calcium channel blocker, stimulates lipogenesis and adipogenesis by downregulating AMPK and upregulating mTOR, which thereby enhances lipid storage. Contrary to the nifedipine, metformin activates AMPK, increases fatty acid oxidation, suppresses fatty acid synthesis and deposition, and thus alleviates lipotoxicity. Obese adults with vascular endothelial dysfunction have greater endothelial cells activation of unfolded protein response stress sensors, RNA-dependent protein kinase-like ER eukaryotic initiation factor-2 alpha kinase (PERK), and activating transcription factor-6. The transcriptional regulation of adipogenesis in obesity is influenced by AGC (protein kinase A (PKA), PKG, PKC) family signaling kinases. Obesity may induce systemic oxidative stress and increase reactive oxygen species in adipocytes. An increase in intracellular oxidative stress can promote PKC-ß activation. Activated PKC-ß induces growth factor adapter Shc phosphorylation. Shc-generated peroxides reduce mitochondrial oxygen consumption and enhance triglyceride accumulation and lipotoxicity. Liraglutide attenuates mitochondrial dysfunction and reactive oxygen species generation. Co-treatment of antiobesity and antidiabetic herbal compound, berberine with antipsychotic drug olanzapine decreases the accumulation of triglyceride. While low-dose rapamycin, metformin, amlexanox, thiazolidinediones, and saroglitazar protect against insulin resistance, glucagon-like peptide-1 analog liraglutide inhibits palmitate-induced inflammation by suppressing mTOR complex 1 (mTORC1) activity and protects against lipotoxicity.

DUSP7 regulates the activity of ERK2 to promote proper chromosome alignment during cell division.

Human cell division is a highly regulated process that relies on the accurate capture and movement of chromosomes to the metaphase plate. Errors in the fidelity of chromosome congression and alignment can lead to improper chromosome segregation, which is correlated with aneuploidy and tumorigenesis. These processes are known to be regulated by extracellular signal-regulated kinase 2 (ERK2) in other species, but the role of ERK2 in mitosis in mammals remains unclear. Here, we have identified the dual-specificity phosphatase 7 (DUSP7), known to display selectivity for ERK2, as important in regulating chromosome alignment. During mitosis, DUSP7 bound to ERK2 and regulated the abundance of active phospho-ERK2 through its phosphatase activity. Overexpression of DUSP7, but not catalytically inactive mutants, led to a decrease in the levels of phospho-ERK2 and mitotic chromosome misalignment, while knockdown of DUSP7 also led to defective chromosome congression that resulted in a prolonged mitosis. Consistently, knockdown or chemical inhibition of ERK2 or chemical inhibition of the MEK kinase that phosphorylates ERK2 led to chromosome alignment defects. Our results support a model wherein MEK-mediated phosphorylation and DUSP7-mediated dephosphorylation regulate the levels of active phospho-ERK2 to promote proper cell division.

Potentiation of B2 receptor signaling by AltB2R, a newly identified alternative protein encoded in the human bradykinin B2 receptor gene.

Recent functional and proteomic studies in eukaryotes (www.openprot.org) predict the translation of alternative open reading frames (AltORFs) in mature G-protein-coupled receptor (GPCR) mRNAs, including that of bradykinin B2 receptor (B2R). Our main objective was to determine the implication of a newly discovered AltORF resulting protein, termed AltB2R, in the known signaling properties of B2R using complementary methodological approaches. When ectopically expressed in HeLa cells, AltB2R presented predominant punctate cytoplasmic/perinuclear distribution and apparent cointeraction with B2R at plasma and endosomal/vesicular membranes. The presence of AltB2R increases intracellular [Ca2+] and ERK1/2-MAPK activation (via phosphorylation) following B2R stimulation. Moreover, HEK293A cells expressing mutant B2R lacking concomitant expression of AltB2R displayed significantly decreased maximal responses in agonist-stimulated Gαq-Gαi2/3-protein coupling, IP3 generation, and ERK1/2-MAPK activation as compared with wild-type controls. Conversely, there was no difference in cell-surface density as well as ligand-binding properties of B2R and in efficiencies of cognate agonists at promoting B2R internalization and ß-arrestin 2 recruitment. Importantly, both AltB2R and B2R proteins were overexpressed in prostate and breast cancers, compared with their normal counterparts suggesting new associative roles of AltB2R in these diseases. Our study shows that BDKRB2 is a dual-coding gene and identifies AltB2R as a novel positive modulator of some B2R signaling pathways. More broadly, it also supports a new, unexpected alternative proteome for GPCRs, which opens new frontiers in fields of GPCR biology, diseases, and drug discovery.

DUSP3/VHR: A Druggable Dual Phosphatase for Human Diseases.

Protein tyrosine kinases (PTK), discovered in the 1970s, have been considered master regulators of biological processes with high clinical significance as targets for human diseases. Their actions are countered by protein tyrosine phosphatases (PTP), enzymes yet underrepresented as drug targets because of the high homology of their catalytic domains and high charge of their catalytic pocket. This scenario is still worse for some PTP subclasses, for example, for the atypical dual-specificity phosphatases (ADUSPs), whose biological functions are not even completely known. In this sense, the present work focuses on the dual-specificity phosphatase 3 (DUSP3), also known as VH1-related phosphatase (VHR), an uncommon regulator of mitogen-activated protein kinase (MAPK) phosphorylation. DUSP3 expression and activities are suggestive of a tumor suppressor or tumor-promoting enzyme in different types of human cancers. Furthermore, DUSP3 has other biological functions involving immune response mediation, thrombosis, hemostasis, angiogenesis, and genomic stability that occur through either MAPK-dependent or MAPK-independent mechanisms. This broad spectrum of actions is likely due to the large substrate diversity and molecular mechanisms that are still under scrutiny. The growing advances in characterizing new DUSP3 substrates will allow the development of pharmacological inhibitors relevant for possible future clinical trials. This review covers all aspects of DUSP3, since its gene cloning and crystallographic structure resolution, in addition to its classical and novel substrates and the biological processes involved, followed by an update of what is currently known about the DUSP3/VHR-inhibiting compounds that might be considered potential drugs to treat human diseases.

Sustained Incompatibility between MAPK Signaling and Pathogen Effectors.

In plants, Mitogen-Activated Protein Kinases (MAPKs) are important signaling components involved in developemental processes as well as in responses to biotic and abiotic stresses. In this review, we focus on the roles of MAPKs in Effector-Triggered Immunity (ETI), a specific layer of plant defense responses dependent on the recognition of pathogen effector proteins. Having inspected the literature, we synthesize the current state of knowledge concerning this topic. First, we describe how pathogen effectors can manipulate MAPK signaling to promote virulence, and how in parallel plants have developed mechanisms to protect themselves against these interferences. Then, we discuss the striking finding that the recognition of pathogen effectors can provoke a sustained activation of the MAPKs MPK3/6, extensively analyzing its implications in terms of regulation and functions. In line with this, we also address the question of how a durable activation of MAPKs might affect the scope of their substrates, and thereby mediate the emergence of possibly new ETI-specific responses. By highlighting the sometimes conflicting or missing data, our intention is to spur further research in order to both consolidate and expand our understanding of MAPK signaling in immunity.

Downregulation of microRNA-143-5p is required for the promotion of odontoblasts differentiation of human dental pulp stem cells through the activation of the mitogen-activated protein kinases 14-dependent p38 mitogen-activated protein kinases signaling pathway.

MicroRNAs (miRNAs) play critical roles in various biological processes including cell differentiation. Some researchers suggested that the p38 mitogen-activated protein kinases (MAPK) signaling pathway had an effect on regulating the odontoblastic differentiation of human dental pulp stem cells (hDPSCs). This study focuses on the effects of miR-143-5p on hDPSCs by regulating the p38 MAPK signaling pathway. The targeting relationship of MAPK14 and miR-143-5p targets were verified by TargetScan and dual-luciferase reporter gene assay. Through overexpression of miR-143-5p or silencing of miR-143-5p, expressions of miR-143-5p, MAPK14, Ras, MAPK kinase (MKK) 3/6, dentin sialophosphoprotein (DSPP), alkaline phosphatase (ALP), and osteocalcin (OCN) were detected by reverse transcription quantitative polymerase chain reaction. Protein expressions of MAPK14, Ras, and MKK3/6 were determined by western blot analysis. ALP and alizarin red S staining were used to detect mineralization. Initially, MAPK14 was found to be negatively regulated by miR-143-5p. Meanwhile, the upregulated miR-143-5p decreased the p38 MAPK signaling pathway related genes (MAPK14, Ras, and MKK3/6) and odontoblastic differentiation markers (ALP, DSPP, and OCN) expression. On the contrary, the downregulated miR-143-5p increased the p38 MAPK signaling pathway related genes (MAPK14, Ras, and MKK3/6) and odontoblastic differentiation markers (ALP, DSPP, and OCN) expression. Furthermore, ALP activity and mineralized nodules increased after downregulation of miR-143-5p, and after its upregulation, ALP activity and mineralized nodules decreased. Our data suggest that poor expression of miR-143-5p promotes hDPSCs odontoblastic differentiation through the activation of the p38 MAPK signaling pathway by upregulating MAPK14.

Immunostimulatory Effects of Chitooligosaccharides on RAW 264.7 Mouse Macrophages via Regulation of the MAPK and PI3K/Akt Signaling Pathways.

Chitooligosaccharides (COS), the hydrolyzed products of chitin and chitosan, can be obtained by various methods. In this study, water-soluble COS were prepared from α- and ß-chitosan by microwave-assisted degradation and their immunostimulatory effects were investigated in RAW 264.7 macrophages. The results indicated that α-COS were more active than ß-COS in promoting the production of nitric oxide (NO) and cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6). Quantitative real-time reverse transcription polymerase chain reaction and Western blotting indicated that COS also enhanced the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and TNF-α. Further analyses demonstrated that COS induced the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38, p85 and Akt, and the nuclear translocation of p65, indicating that they are able to activate the mitogen-activated protein kinases (MAPKs) and phosphoinositide 3-kinases (PI3K)/Akt signaling pathways dependent on nuclear factor (NF)-κB activation. In conclusion, COS activate RAW 264.7 cells via the MAPK and PI3K/Akt signaling pathways and are potential novel immune potentiators.

Astaxanthin Modulation of Signaling Pathways That Regulate Autophagy.

Autophagy is a lysosomal pathway that degrades and recycles unused or dysfunctional cell components as well as toxic cytosolic materials. Basal autophagy favors cell survival. However, the aberrant regulation of autophagy can promote pathological conditions. The autophagy pathway is regulated by several cell-stress and cell-survival signaling pathways that can be targeted for the purpose of disease control. In experimental models of disease, the carotenoid astaxanthin has been shown to modulate autophagy by regulating signaling pathways, including the AMP-activated protein kinase (AMPK), cellular homolog of murine thymoma virus akt8 oncogene (Akt), and mitogen-activated protein kinase (MAPK), such as c-Jun N-terminal kinase (JNK) and p38. Astaxanthin is a promising therapeutic agent for the treatment of a wide variety of diseases by regulating autophagy.

Atractylodis Rhizoma Alba Attenuates Neuroinflammation in BV2 Microglia upon LPS Stimulation by Inducing HO-1 Activity and Inhibiting NF-κB and MAPK.

Microglial activation and the resulting neuroinflammation are associated with a variety of brain diseases, such as Alzheimer's disease and Parkinson's disease. Thus, the control of microglial activation is an important factor in the development of drugs that can treat or prevent inflammation-related neurodegenerative disorders. Atractylodis Rhizoma Alba (ARA) has been reported to exhibit antioxidant, gastroprotective, and anti-inflammatory effects. However, the effects of ARA ethanolic extract (ARAE) on microglia-mediated neuroinflammation have not been fully elucidated. In this work, we explored the anti-neuroinflammatory properties and underlying molecular mechanisms of ARAE in lipopolysaccharide (LPS)-stimulated microglial BV2 cells. Our results showed that ARAE significantly attenuates the production of nitric oxide (NO) and inflammatory cytokines induced by LPS. ARAE treatment also inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 without causing cytotoxicity. ARAE markedly attenuated the transcriptional activities of nuclear factor (NF)-κB and mitogen-activated protein kinases (MAPK) phosphorylation, and induced heme oxygenase (HO)-1 expression. High-performance liquid chromatography (HPLC) analysis showed that ARAE contains three main components-atractylenolide I, atractylenolide III, and atractylodin-all compounds that significantly inhibit the production of inflammatory factors. These findings indicate that ARAE may be a potential therapeutic agent for the treatment of inflammation-related neurodegenerative diseases.

Recent Advances in Basic Research for Brain Arteriovenous Malformation.

Arteriovenous malformations (AVMs) are abnormal connections of vessels that shunt blood directly from arteries into veins. Rupture of brain AVMs (bAVMs) can cause life-threatening intracranial bleeding. Even though the majority of bAVM cases are sporadic without a family history, some cases are familial. Most of the familial cases of bAVMs are associated with a genetic disorder called hereditary hemorrhagic telangiectasia (HHT). The mechanism of bAVM formation is not fully understood. The most important advances in bAVM basic science research is the identification of somatic mutations of genes in RAS-MAPK pathways. However, the mechanisms by which mutations of these genes lead to AVM formation are largely unknown. In this review, we summarized the latest advance in bAVM studies and discussed some pathways that play important roles in bAVM pathogenesis. We also discussed the therapeutic implications of these pathways.

Human Protein Kinases and Obesity.

The action of protein kinases and protein phosphatases is essential for multiple physiological responses. Each protein kinase displays its own unique substrate specificity, and a regulatory mechanism that may be modulated by association with other proteins. Protein kinases are classified by the target amino acid in their substrates. Some protein kinases can phosphorylate both serine/threonine, as well as tyrosine residues. This group of kinases has been known as dual specificity kinases. Unlike the dual specificity kinases, a heterogeneous group of protein phosphatases are known as dual-specificity phosphatases. These phosphatases remove phosphate groups from tyrosine and serine/threonine residues on their substrate. Dual-specificity phosphatases are important signal transduction enzymes that regulate various cellular processes in coordination with protein kinases. The protein kinase-phosphoproteins interactions play an important role in obesity . In obesity, the pro- and anti-inflammatory effects of adipokines and cytokines through intracellular signaling pathways mainly involve the nuclear factor kappa B (NF-kappaB) and the c-Jun N-terminal kinase (JNK) systems as well as the inhibitor of kappaB-kinase beta (IKK beta). Impairment of insulin signaling in obesity is largely mediated by the activation of the IKKbeta and the JNK. Furthermore, oxidative stress and endoplasmic reticulum (ER) stress activate the JNK pathway which suppresses insulin biosynthesis. Additionally, obesity-activated calcium/calmodulin dependent-protein kinase II/p38 suppresses insulin-induced protein kinase B phosphorylation by activating the ER stress effector, activating transcription factor-4. Obese adults with vascular endothelial dysfunction have greater endothelial cells activation of unfolded protein response stress sensors, RNA-dependent protein kinase-like ER eukaryotic initiation factor-2alpha kinase (PERK) and activating transcription factor-6. The transcriptional regulation of adipogenesis in obesity is influenced by AGC (protein kinase A (PKA), PKG, PKC) family signaling kinases. Obesity may induce systemic oxidative stress and increase reactive oxygen species in adipocytes. Increase in intracellular oxidative stress can promote PKC-beta activation. Activated PKC-beta induces growth factor adapter Shc phosphorylation. Shc-generated peroxides reduce mitochondrial oxygen consumption and enhances triglyceride accumulation. Obesity is fundamentally caused by cellular energy imbalance and dysregulation. Like adenosine monophosphate (AMP)-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR), N-terminal Per-ARNT-Sim (PAS) kinase are nutrient responsive protein kinases and important for proper regulation of glucose metabolism in mammals at both the hormonal and cellular level. Defective responses of AMPK to leptin may contribute to resistance to leptin action on food intake and energy expenditure in obese states.

Exenatide Regulates Substrate Preferences Through the p38γ MAPK Pathway After Ischaemia/Reperfusion Injury in a Rat Heart.

BACKGROUND: It is reported that glucagon-like peptide-1 (GLP-1) has direct cardioprotective effects. We hypothesise that Exenatide, a long half-life analog of GLP-1, might protect the heart against ischaemia/reperfusion (I/R) injury. In this study, the role and mechanism of Exenatide in I/R was investigated. METHODS: Two p38 mitogen-activated protein kinase (MAPK) isoforms p38α or p38γ, were knocked down by recombinant adeno-associated virus (rAAV) in male Sprague-Dawley rats. Then, rats were randomly treated with Exenatide or phosphate buffered saline (PBS) before I/R. Left ventricular function was measured. The translocation of glucose transporter 4 (GLUT4), GLUT1 and fatty acid transporter (FAT)/CD36 was assessed. RESULTS: Exenatide treatment increased the p38γ expression, but not p38α, in I/R rats. Exenatide significantly improved post-ischaemic cardiac function of I/R rats. The administration of Exenatide stimulated the translocation of GLUT4 and GLUT1, while it also increased the GLUT1 expression in the cytoplasm. Meanwhile, it reduced the translocation of FAT/CD36 (p<0.05). However, cardiac down-regulation of p38γ mediated by rAAV abolished not only the Exenatide-induced cardioprotective effects but also the GLUT4, GLUT1 and FAT/CD36 translocation. CONCLUSIONS: These results demonstrated that Exenatide improved cardiac function, increased translocation of GLUTs, and suppressed translocation of FAT/CD36 after myocardial I/R injury. This protective effect was mediated, at least in part, through modulation of the cardiac p38γ MAPK.

cAMP-Induced Histones H3 Dephosphorylation Is Independent of PKA and MAP Kinase Activations and Correlates With mTOR Inactivation.

cAMP is a second messenger well documented to be involved in the phosphorylation of PKA, MAP kinase, and histone H3 (H3). Early, we reported that cAMP also induced H3 dephosphorylation in a variety of proliferating cell lines. Herein, it is shown that cAMP elicits a biphasic H3 dephosphorylation independent of PKA activation in cycling cells. H89, a potent inhibitor of PKA catalytic sub-unite, could not abolish this effect. Additionally, H89 induces a rapid and biphasic H3 serine 10 dephosphorylation, while a decline in the basal phosphorylation of CREB/ATF-1 is observed. Rp-cAMPS, an analog of cAMP and specific inhibitor of PKA, is unable to suppress cAMP-mediated H3 dephosphorylation, whereas Rp-cAMPS effectively blocks CREB/ATF-1 hyper-phosphorylation by cAMP and its inducers. Interestingly, cAMP exerts a rapid and profound H3 dephosphorylation at much lower concentration (50-fold lower, 0.125 mM) than the concentration required for maximal CREB/ATF-1 phosphorylation (5 mM). Much higher cAMP concentration is required to fully induce CREB/ATF-1 gain in phosphate (5 mM), which correlates with the inhibition of H3 dephosphorylation. Also, the dephosphorylation of H3 does not overlap at onset of MAP kinase phosphorylation pathways, p38 and ERK. Surprisingly, rapamycin (an mTOR inhibitor), cAMP, and its natural inducer isoproterenol, elicit identical dephosphorylation kinetics on both S6K1 ribosomal kinase (a downstream mTOR target) and H3. Finally, cAMP-induced H3 dephosphorylation is PP1/2-dependent. The results suggest that a pathway, requiring much lower cAMP concentration to that required for CREB/ATF-1 hyper-phosphorylation, is responsible for histone H3 dephosphorylation and may be linked to mTOR down regulation.

RAS/MAPK pathway hyperactivation determines poor prognosis in undifferentiated pleomorphic sarcomas.

BACKGROUND: Undifferentiated pleomorphic sarcoma (UPS) constitutes the most common subtype of soft tissue sarcoma. However, UPS is clinically and molecularly poorly understood, in great extent due to its intrinsic phenotypic and cytogenetic complexity, which in turn results in the absence of specific prognostic or predictive biomarkers. The RAS/mitogen-activated protein kinases (MAPK) and phosphoinositide 3-kinase inhibitor (PI3K)/mammalian target of rapamycin (mTOR) pathways are considered to be 2 major mechanisms for sarcoma proliferation and survival and to the authors' knowledge their role in UPS remains unclear. The objective of the current study was to investigate whether the RAS/MAPK and PI3K/mTOR pathways are activated in UPS, and whether pathway activation is associated with outcome. METHODS: Records for patients diagnosed and treated for UPS in the study institution between 2000 and 2009 were reviewed. Phosphorylation status of 4E-binding protein (4E-BP1), eukaryotic translation initiation factor 4E (eIF-4E), S6-RP, and ERK 1/2, together with total forms of 4E-BP1 and eIF-4E, were assessed using immunohistochemistry in paraffin-embedded tumor tissue. Mutational analysis for KRAS; NRAS; BRAF; and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) oncogenic mutations was performed as well. RESULTS: Critical lymph nodes within the RAS/MAPK and PI3K/mTOR pathways were found to be activated in >80% of UPS cases. Hyperactivation of the RAS/MAPK pathway, as assessed by expression of phosphorylated ERK 1/2, was found to independently predict a higher risk of disease recurrence and impaired overall survival. Only a KRAS A146V mutation was detected in 1 tumor. CONCLUSIONS: The RAS/MAPK and PI3K/mTOR pathways are activated in the majority of cases of UPS. The RAS/MAPK pathway distinguishes a subgroup of patients with localized UPS with a worse outcome.

Lactobacillus plantarum L67 glycoprotein protects against cadmium chloride toxicity in RAW 264.7 cells.

The food and water we consume may be contaminated with a range of chemicals and heavy metals, such as lead, cadmium, arsenic, chromium, and mercury by accumulation through the food chain. Cadmium is known to be one of the major components in cigarette smoke and can cause lesions in many organs. Some lactobacilli can bind and remove heavy metals such as cadmium, lead, and copper. However, the mechanisms of cadmium toxicity and inhibition by probiotics are not clear. In this study, we demonstrated that glycoprotein (18 kDa) isolated from Lactobacillus plantarum L67 protected RAW 264.7 cells from expression of inflammation-related factors stimulated by cadmium chloride (100 µM). Furthermore, we evaluated the cytotoxicity of cadmium using the MTT assay and intracellular Ca(2+) using fluorescence, and assessed activities of activator protein kinase C (PKC-α), inducible nitric oxide synthase, activator protein (AP)-1, and mitogen-activated protein kinases using immunoblot. Our results indicated that glycoprotein isolated from L. plantarum L67 inhibited intracellular Ca(2+) mobilization. It also significantly suppressed inflammatory factors such as AP-1 (c-Jun and c-Fos), mitogen-activated protein kinases (ERK, JNK, and p38), and inducible nitric oxide synthase. Our findings suggest that the 24-kDa glycoprotein isolated from L. plantarum L67 might be used as a food component for protection of inflammation caused by cadmium ion.

Achillolide A Protects Astrocytes against Oxidative Stress by Reducing Intracellular Reactive Oxygen Species and Interfering with Cell Signaling.

Achillolide A is a natural sesquiterpene lactone that we have previously shown can inhibit microglial activation. In this study we present evidence for its beneficial effects on astrocytes under oxidative stress, a situation relevant to neurodegenerative diseases and brain injuries. Viability of brain astrocytes (primary cultures) was determined by lactate dehydrogenase (LDH) activity, intracellular ROS levels were detected using 2',7'-dichlorofluorescein diacetate, in vitro antioxidant activity was measured by differential pulse voltammetry, and protein phosphorylation was determined using specific ELISA kits. We have found that achillolide A prevented the H2O2-induced death of astrocytes, and attenuated the induced intracellular accumulation of reactive oxygen species (ROS). These activities could be attributed to the inhibition of the H2O2-induced phosphorylation of MAP/ERK kinase 1 (MEK1) and p44/42 mitogen-activated protein kinases (MAPK), and to the antioxidant activity of achillolide A, but not to H2O2 scavenging. This is the first study that demonstrates its protective effects on brain astrocytes, and its ability to interfere with MAPK activation. We propose that achillolide A deserves further evaluation for its potential to be developed as a drug for the prevention/treatment of neurodegenerative diseases and brain injuries where oxidative stress is part of the pathophysiology.

Chitosan oligosaccharides prevented retinal ischemia and reperfusion injury via reduced oxidative stress and inflammation in rats.

The purpose of the present study was to investigate the protective effect and mechanism of chitosan oligonucleotides (COS) on retinal ischemia and reperfusion (I/R) injury. Rats pretreated with PBS, low-dose COS (5 mg/kg), or high-dose COS (10 mg/kg) were subjected to retinal ischemia by increasing their intraocular pressure to 130 mm Hg for 60 min. The protective effect of COS was evaluated by determining the electroretinograms (ERGs), morphology of the retina, and survival of retinal ganglion cells (RGCs). The oxidative damage was determined by imuunohistochemistry and ELISA, respectively. The expressions of inflammatory mediators (TNF-α, IL-1ß, MCP-1, iNOS, ICAM-1) and apoptotic-related proteins (p53, Bax, Bcl-2) were quantified by PCR and Western blots. The detection of NF-κB p65 in the retina was performed by immunofluorescence. The protein levels of IκB and phosphorylated mitogen-activated protein kinases [MAPK; viz. extracellular signal-regulated protein kinases (ERK), c-Jun N-terminal kinases (JNK) and p38] and the NF-κB/DNA binding ability were assessed by Western blot analysis and EMSA. We found that pretreatment with COS, especially a high dosage, effectively ameliorated the I/R-induced reduction of the b-wave ratio in ERGs and the retinal thickness and the survival of RGCs at 24 h. COS decreased the expression of inflammatory mediators, p53 and Bax, increasing Bcl-2 expression and thereby reducing retinal oxidative damage and the number of apoptotic cells. More importantly, COS attenuated IκB degradation and p65 presence in the retina, thus decreasing NF-κB/DNA binding activity after I/R. In addition, COS decreased the phosphorylation levels of JNK and ERK but increased the phosphorylation level of p38. Pretreatment with p38 inhibitor (SB203580) abolished the protective effect of COS on retinal oxidative damage, as indicated by increased retinal 8-OHdG stains, and significantly increased the expression of inflammatory mediators (TNF-α, MCP-1, iNOS, ICAM-1) in I/R-injured rats. In conclusion, COS prevented retinal I/R injury through its inhibition of oxidative stress and inflammation. These effects were achieved by blocking the activation of NF-κB, JNK, and ERK but promoting the activation of p38 activation.

Dihydroartemisinin, a potential PTGS1 inhibitor, potentiated cisplatin-induced cell death in non-small cell lung cancer through activating ROS-mediated multiple signaling pathways.

Dihydroartemisinin (DHA) exerts an anti-tumor effect in multiple cancers, however, the molecular mechanism of DHA and whether DHA facilitates the anti-tumor efficacy of cisplatin in non-small cell lung cancer (NSCLC) are unclear. Here, we found that DHA potentiated the anti-tumor effects of cisplatin in NSCLC cells by stimulating reactive oxygen species (ROS)-mediated endoplasmic reticulum (ER) stress, C-Jun-amino-terminal kinase (JNK) and p38 MAPK signaling pathways both in vitro and in vivo. Of note, we demonstrated for the first time that DHA inhibits prostaglandin G/H synthase 1 (PTGS1) expression, resulting in enhanced ROS production. Importantly, silencing PTGS1 sensitized DHA-induced cell death by increasing ROS production and activating ER-stress, JNK and p38 MAPK signaling pathways. In summary, our findings provided new experimental basis and therapeutic prospect for the combined therapy with DHA and cisplatin in some NSCLC patients.

Matrix metalloproteinase-7 induces E-cadherin cleavage in acid-exposed primary human pharyngeal epithelial cells via the ROS/ERK/c-Jun pathway.

Laryngopharyngeal reflux disease (LPRD) is caused by pharyngeal mucosal damage due to the reflux of gastric contents, including acid, pepsin, and bile juice. Our previous study has demonstrated that LPRD is associated with the cleavage of E-cadherin, which is facilitated by the acid-activated matrix metalloproteinase-7 (MMP-7); however, the mechanism by which the acid activates MMP-7 remains unclear. The purpose of this study was to investigate the mechanism by which MMP-7 is activated in the pharyngeal epithelial cells that are exposed to acid. The levels of reactive oxygen species (ROS) were measured in the epithelial cells exposed to acid. To investigate the signaling mechanism of ROS in the expression of MMP-7, the mechanism of action of the mitogen-activated protein kinase was examined. The expression of various signaling factors was determined, according to the presence or absence of each inhibitor in the acid-exposed pharyngeal epithelial cells. To identify changes in the cleavage of E-cadherin, the integrity of the mucosal membrane was assessed using a transepithelial permeability test. We found that acid exposure increased the levels of ROS, phosphorylated-extracellular signal-regulated kinase (p-ERK) 1/2, and phosphorylated-c-Jun (p-c-Jun) in pharyngeal epithelial cells. The ROS inhibitor reduced the expression of p-ERK and MMP-7, while the ERK inhibitor reduced the expression of p-c-Jun and MMP-7. Moreover, the c-Jun inhibitor reduced the expression of MMP-7 and blocked the degradation of E-cadherin. In addition, decrease in the levels of immunostained E-cadherin and increase in transepithelial permeability after acid exposure were collectively alleviated by the inhibitors of ROS, ERK, and c-Jun. The degradation of E-cadherin that occurs after human mucosal cells are exposed to acid appears to be caused by an increase in the expression of MMP-7 via the ROS/ERK/c-Jun pathway, which is thought to be an important mechanism associated with the development of LPRD. KEY MESSAGES: • ROS is triggered when reflux occurs. • ROS regulates the transcription factor c-Jun via the ERK pathway. • The increase in MMP-7 that induces LPRD is induced via the ROS/ERK/c-Jun pathway. • This study revealed for the first time the expression mechanism of MMP-7, which is one of the causes of LPRD.