A new alkaloid and two organic acids from Portulaca oleracea L. and their bioactivities.

A new alkaloid, identified as 1-benzyl-2-nitroso-1,2,3,4-tetrahydroisoquinoline-6,7-diol, named oleraisoquinoline (1), and five organic acids and two esters, identified as 5-(hydroxymethyl)furan-2-carboxylic acid (2), 1H-pyrrole-2,5-dicarboxylic acid (3), (7E,10E)-octadeca-7,10-dienoic acid (4), (10E,13E)-octadeca-10,13-dienoic acid (5), (7E,10E)-hexadeca-7,10-dienoic acid (6), methyl tridecanoate (7) and methyl (9E,12E)-octadeca-9,12-dienoate (8), were isolated from Portulaca oleracea L., among which compounds 2 and 4‒7 were isolated for the first time. Moreover, the anti-inflammatory activities of compounds 1‒3 were studied, especially, compound 1 presented good inhibitory effects on the production of inflammatory factors IL-1ß and TNF-α.

Co-exposure to low-dose lead, cadmium, and mercury promotes memory deficits in rats: Insights from the dynamics of dendritic spine pruning in brain development.

Lead (Pb), cadmium (Cd), and mercury (Hg) are environmentally toxic heavy metals that can be simultaneously detected at low levels in the blood of the general population. Although our previous studies have demonstrated neurodevelopmental toxicity upon co-exposure to these heavy metals at these low levels, the precise mechanisms remain largely unknown. Dendritic spines are the structural foundation of memory and undergo significant dynamic changes during development. This study focused on the dynamics of dendritic spines during brain development following Pb, Cd, and Hg co-exposure-induced memory impairment. First, the dynamic characteristics of dendritic spines in the prefrontal cortex were observed throughout the life cycle of normal rats. We observed that dendritic spines increased rapidly from birth to their peak value at weaning, followed by significant pruning and a decrease during adolescence. Dendritic spines tended to be stable until their loss in old age. Subsequently, a rat model of low-dose Pb, Cd, and Hg co-exposure from embryo to adolescence was established. The results showed that exposure to low doses of heavy metals equivalent to those detected in the blood of the general population impaired spatial memory and altered the dynamics of dendritic spine pruning from weaning to adolescence. Proteomic analysis of brain and blood samples suggested that differentially expressed proteins upon heavy metal exposure were enriched in dendritic spine-related cytoskeletal regulation and axon guidance signaling pathways and that cofilin was enriched in both of these pathways. Further experiments confirmed that heavy metal exposure altered actin cytoskeleton dynamics and disturbed the dendritic spine pruning-related LIM domain kinase 1-cofilin pathway in the rat prefrontal cortex. Our findings demonstrate that low-dose Pb, Cd, and Hg co-exposure may promote memory impairment by perturbing dendritic spine dynamics through dendritic spine pruning-related signaling pathways.

A new alkaloid from Portulaca oleracea L. with its anti-inflammatory activity.

A new isoindole alkaloid, 6-hydroxy-2-(4'''-hydroxy-3'''-methoxyphenethyl)-4-(4'-hydroxy-3'-methoxyphenyl)-7-methoxy-1H-benzo[f]isoindole-1,3(2H)-dione, named oleraisoindole B was isolated from Portulaca oleracea L., its structure was elucidated using NMR and UHPLC-ESI-Q-TOF/MS spectroscopic methods, and presented anti-inflammatory activity at 5 µM.

Insight into the effect of a heavy metal mixture on neurological damage in rats through combined serum metabolomic and brain proteomic analyses.

The heavy metals lead (Pb), cadmium (Cd), and mercury (Hg) that cause neurocognitive impairment have been extensively studied. These elements typically do not exist alone in the environment; they are often found with other heavy metals and can enter the body through various routes, thereby impacting health. Our previous research showed that low Pb, Cd, and Hg levels cause neurobehavioral impairments in weaning and adult rats. However, little is known about the biomarkers and mechanisms underlying Pb, Cd, and Hg mixture-induced neurological impairments. A combined analysis of metabolomic and proteomic data may reveal heavy metal-induced alterations in metabolic and protein profiles, thereby improving our understanding of the molecular mechanisms underlying heavy metal-induced neurological impairments. Therefore, brain tissue and serum samples were collected from rats exposed to a Pb, Cd, and Hg mixture for proteomic and metabolomic analyses, respectively. The analysis revealed 363 differential proteins in the brain and 206 metabolites in serum uniquely altered in the Pb, Cd, and Hg mixture exposure group, compared to those of the control group. The main metabolic impacted pathways were unsaturated fatty acids biosynthesis, linoleic acid metabolism, phenylalanine metabolism, and tryptophan metabolism. We further identified that the levels of arachidonic acid (C20:4 n-3) and, adrenic acid (C22:4 n-3) were elevated and that kynurenic acid (KA) and quinolinic acid (QA) levels and the KA/QA ratio, were decreased in the group exposed to the Pb, Cd, and Hg mixture. A joint analysis of the proteome and metabolome showed that significantly altered proteins such as LPCAT3, SLC7A11, ASCL4, and KYAT1 may participate in the neurological impairments induced by the heavy metal mixture. Overall, we hypothesize that the dysregulation of ferroptosis and kynurenine pathways is associated with neurological damage due to chronic exposure to a heavy metal mixture.

[Determination of 39 fatty acids in liver of rats by gas chromatography-mass spectrometry].

Fatty acids not only form phospholipids that contribute to the formation of cell membranes but also participate in many metabolic activities, such as energy storage and cell signal transduction. The liver plays a key role in the synthesis and metabolism of fatty acids. The composition and contents of fatty acids in the liver are closely related to body health. Most fatty acid-detection methods require a large sample size and can detect only a small number of fatty acids. Therefore, a sensitive and efficient method to determine fatty acids in the liver is urgently required. Herein, a method based on gas chromatography-mass spectrometry (GC-MS) was established for the simultaneous determination of 39 fatty acids in 1.1 mg of liver tissue. Different extraction methods and derivatization conditions were compared to develop an optimal sample-treatment method. The performance of two different columns in separating the target fatty acids were also compared. A total of 10 mg of liver was added to 450 µL of normal saline and ground at -35 ℃ to obtain a homogenate. Next, 50 µL of the homogenate (equivalent to 1.1 mg of liver) was added with 750 µL of chloroform-methanol (1∶2, v/v) to extract total fatty acids. The fatty acid extracts were dried under nitrogen, and then derivatized at 100 ℃ for 90 min after being added with methanol containing 5% sulfuric acid. The fatty acid methyl esters were extracted with hexane and then separated on an SP-2560 capillary column (100 m×0.25 mm×0.2 µm; Supelco, USA) via GC-MS. The results revealed that all 39 fatty acid methyl esters detected had good linearities in the certain mass concentration ranges with correlation coefficients (R2) greater than 0.9940. The limits of detection (LOD) and quantification (LOQ) of these methyl esters in the liver were 2-272 ng/mg and 7-906 ng/mg, respectively. The accuracy and precision of the method were evaluated by spiking the liver homogenate with tridecylic acid and eicosanoic acid at low (0.09 µg/mg), moderate (0.90 µg/mg), and high (5.40 µg/mg) concentration levels. The recoveries ranged from 82.4% to 101.0% with an intraday relative standard deviations (RSDs) (n=5) of 3.2%-12.0% and interday RSDs (n=3) of 5.4%-13.4%. The method was successfully applied to detect fatty acids in the livers of four healthy male Sprague-Dawley (SD) rats and four male SD rats with abnormal liver function induced by perfluorooctane sulfonate (PFOS). PFOS is a persistent organic pollutant. Twenty-six fatty acids were detected in the livers of both groups. Among the fatty acids investigated, pentadecanoic acid (C15∶0), γ-linolenic acid (C18∶3n6), and elaidic acid (C18∶1n9t) cannot be detected by the methods reported in the literature. By contrast, the method developed in this study could separate the isomers of oleic acid (elaidic acid, C18∶1n9t; oleic acid, C18∶1n9c) and linolenic acid (linolelaidic acid, C18∶2n6t; linoleic acid, C18∶2n6c). In conclusion, the developed method is simple and can detect a large number of fatty acids using small sample amounts and few reagents. More importantly, it could successfully separate fatty acid isomers. These findings indicate that the developed method is suitable for the detection of fatty acid composition and contents in the liver in clinical and experimental research.

Two new organic acids from Portulaca oleracea L. and their anti-inflammatory and anticholinesterase activities.

Two new organic acids, identified as (7E,9E,12E)-pentadecyl-7,9,12-trienoic acid, named Oleraceacid A (1), and 6,7-dihydroxy-4-(4-hydroxy-3,5-dimethoxyphenyl)-2-naphthoic acid, named Oleraceacid B (2), were isolated from Portulaca oleracea L.. The structures were verified by spectroscopic methods, including UHPLC-ESI-QTOF/MS, 1 D and 2 D NMR. Both Oleraceacid A (1) and Oleraceacid B (2) at 20 µM inhibited the inflammatory factor, IL-1ß in the RAW 264.7 cells induced by LPS, moreover, Oleraceacid A (1) can inhibit cholinesterase activity.

N6-methyladenosine regulates ATM expression and downstream signaling.

N6-methyladenosine (m6A) is the most abundant modification in eukaryotic mRNAs, which plays an important role in regulating multiple biological processes. ATM is a major protein kinase that regulates the DNA damage response. Here, we identified that ATM is a m6A-modificated gene. METTL3 (a m6A "writer") and FTO (a m6A "eraser") oppositely regulated ATM expression and its downstream signaling. Mechanically, m6A "readers" YTHDFs and eIF3A suppressed ATM expression in the post-transcriptional levels. We also revealed the oncogenic potential of METTL3 and YTHDF1 related to ATM modulation. This is the first report that ATM, a master in the DNA damage response, is modified by m6A epigenetic modification, and METTL3 disrupts the ATM stability via m6A modification, thereby affecting the DNA-damage response.

Bradykinin, as a Reprogramming Factor, Induces Transdifferentiation of Brain Astrocytes into Neuron-like Cells.

Kinins are endogenous, biologically active peptides released into the plasma and tissues via the kallikrein-kinin system in several pathophysiological events. Among kinins, bradykinin (BK) is widely distributed in the periphery and brain. Several studies on the neuro-modulatory actions of BK by the B2BK receptor (B2BKR) indicate that this neuropeptide also functions during neural fate determination. Previously, BK has been shown to induce differentiation of nerve-related stem cells into neuron cells, but the response in mature brain astrocytes is unknown. Herein, we used rat brain astrocyte (RBA) to investigate the effect of BK on cell transdifferentiation into a neuron-like cell morphology. Moreover, the signaling mechanisms were explored by zymographic, RT-PCR, Western blot, and immunofluorescence staining analyses. We first observed that BK induced RBA transdifferentiation into neuron-like cells. Subsequently, we demonstrated that BK-induced RBA transdifferentiation is mediated through B2BKR, PKC-δ, ERK1/2, and MMP-9. Finally, we found that BK downregulated the astrocytic marker glial fibrillary acidic protein (GFAP) and upregulated the neuronal marker neuron-specific enolase (NSE) via the B2BKR/PKC-δ/ERK pathway in the event. Therefore, BK may be a reprogramming factor promoting brain astrocytic transdifferentiation into a neuron-like cell, including downregulation of GFAP and upregulation of NSE and MMP-9 via the B2BKR/PKC-δ/ERK cascade. Here, we also confirmed the transdifferentiative event by observing the upregulated neuronal nuclear protein (NeuN). However, the electrophysiological properties of the cells after BK treatment should be investigated in the future to confirm their phenotype.

The COX-2-derived PGE2 autocrine contributes to bradykinin-induced matrix metalloproteinase-9 expression and astrocytic migration via STAT3 signaling.

BACKGROUND: The matrix metalloproteinase-9 (MMP-9) is up-regulated by several proinflammatory mediators in the central nervous system (CNS) diseases. Increasing reports show that MMP-9 expression is an inflammatory biomarker of several CNS disorders, including the CNS inflammation and neurodegeneration. Bradykinin (BK) is a common proinflammatory mediator and elevated in several brain injury and inflammatory disorders. The raised BK may be detrimental effects on the CNS that may aggravate brain inflammation through MMP-9 up-regulation or cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) production in brain astrocytes. However, the relationship between BK-induced MMP-9 expression and COX-2-derived PGE2 release in brain astrocytes remains unclear. METHODS: Herein we used rat brain astrocytes (RBA) to investigate the role of the COX-2/PGE2 system in BK-induced MMP-9 expression. We used zymographic, RT-PCR, EIA, and Western blotting analyses to confirm that BK induces MMP-9 expression via a COX-2/PGE2-dependent pathway. RESULTS: Our results show activation of native COX-2 by BK led to PGE2 production and release. Subsequently, PGE2 induced MMP-9 expression via PGE2 receptor (EP)-mediated c-Src, Jak2, ERK1/2, and then activated signal transducer and activator of transcription 3 (STAT3) signaling pathway. Finally, up-regulation of MMP-9 by BK via the pathway may promote astrocytic migration. CONCLUSION: These results demonstrated that a novel autocrine pathway for BK-induced MMP-9 protein expression is mediated through activation of STAT3 by native COX-2/PGE2-mediated c-Src/Jak2/ERK cascades in brain astrocytes. Video Abstract.

Zinc oxide nanoparticles modulate the gene expression of ZnT1 and ZIP8 to manipulate zinc homeostasis and stress-induced cytotoxicity in human neuroblastoma SH-SY5Y cells.

Zinc ions (Zn2+) are important messenger molecules involved in various physiological functions. To maintain the homeostasis of cytosolic Zn2+ concentration ([Zn2+]c), Zrt/Irt-related proteins (ZIPs) and Zn2+ transporters (ZnTs) are the two families of proteins responsible for decreasing and increasing the [Zn2+]c, respectively, by fluxing Zn2+ across the membranes of the cell and intracellular compartments in opposite directions. Most studies focus on the cytotoxicity incurred by a high concentration of [Zn2+]c and less investigate the [Zn2+]c at physiological levels. Zinc oxide-nanoparticle (ZnO-NP) is blood brain barrier-permeable and elevates the [Zn2+]c to different levels according to the concentrations of ZnO-NP applied. In this study, we mildly elevated the [Zn2+]c by ZnO-NP at concentrations below 1 µg/ml, which had little cytotoxicity, in cultured human neuroblastoma SH-SY5Y cells and characterized the importance of Zn2+ transporters in 6-hydroxy dopamine (6-OHDA)-induced cell death. The results show that ZnO-NP at low concentrations elevated the [Zn2+]c transiently in 6 hr, then declined gradually to a basal level in 24 hr. Knocking down the expression levels of ZnT1 (located mostly at the plasma membrane) and ZIP8 (present in endosomes and lysosomes) increased and decreased the ZnO-NP-induced elevation of [Zn2+]c, respectively. ZnO-NP treatment reduced the basal levels of reactive oxygen species and Bax/Bcl-2 mRNA ratios; in addition, ZnO-NP decreased the 6-OHDA-induced ROS production, p53 expression, and cell death. These results show that ZnO-NP-induced mild elevation in [Zn2+]c activates beneficial effects in reducing the 6-OHDA-induced cytotoxic effects. Therefore, brain-delivery of ZnO-NP can be regarded as a potential therapy for neurodegenerative diseases.

Effects of Hericium erinaceus Mycelium Extracts on the Functional Activity of Purinoceptors and Neuropathic Pain in Mice with L5 Spinal Nerve Ligation.

Neuropathic pain is a serious clinical problem that is difficult to treat. Purinoceptors (P2Rs) transduce pain perception from the peripheral to the central nervous system and play an important role in the transmission of neuropathic pain signals. We previously found that the crude extracts of Hericium erinaceus mycelium (HE-CE) inhibited P2R-mediated signaling in cells and reduced heat-induced pain in mice. The present study explored the effects of HE-CE on neuropathic pain. We used adenosine triphosphate (ATP) as a P2R agonist to generate Ca2+ signaling and neuronal damage in a cell line. We also established a neuropathic mouse model of L5 spinal nerve ligation (L5-SNL) to examine neuropathic pain and neuroinflammation. Neuropathic pain was recorded using the von Frey test. Neuroinflammation was evaluated based on immunohistofluorescence observation of glial fibrillary acidic protein (GFAP) levels in astrocytes, ionized calcium-binding adaptor molecule1 (iba1) levels in microglia, and IL-6 levels in plasma. The results show that HE-CE and erinacine-S, but not erinacine-A, totally counteracted Ca2+ signaling and cytotoxic effects upon P2R stimulation by ATP in human osteosarcoma HOS cells and human neuroblastoma SH-SY5Y cells, respectively. SNL induced a decrease in the withdrawal pressure of the ipsilateral hind paw, indicating neuropathic pain. It also raised the GFAP level in astrocytes, the iba1 level in microglia, and the IL-6 level in plasma, indicating neuroinflammation. HE-CE significantly counteracted the SNL-induced decrease in withdrawal pressure, illustrating that it could relieve neuropathic pain. It also reduced SNL-induced increases in astrocyte GFAP levels, microglial iba1 levels, and plasma IL-6 levels, suggesting that HE-CE reduces neuroinflammation. Erinacine-S relieved neuropathic pain better than HE-CE. The present study demonstrated that HE inhibits P2R and, thus, that it can relieve neuropathic pain and neuroinflammation.

Rottlerin, a natural polyphenol compound, inhibits upregulation of matrix metalloproteinase-9 and brain astrocytic migration by reducing PKC-δ-dependent ROS signal.

BACKGROUND: Upregulation of matrix metalloproteinase-9 (MMP-9) has been indicated as one of the inflammatory biomarkers. In the central nervous system (CNS), the MMP-9 is induced by several proinflammatory mediators and participates in the CNS disorders, including inflammation and neurodegeneration. In addition, protein kinase Cs (PKCs) has been shown to be involved in regulation of various inflammatory factors like MMP-9 by several stimuli in many cell types. Several phytochemicals are believed to reduce the risk of several inflammatory disorders including the CNS diseases. The rottlerin, a principal phenolic compound of the Kamala plant Mallotus philippinensis, has been shown to possess an array of medicinal properties, including anti-PKC-δ, antitumor, anti-oxidative, and anti-inflammatory activities. METHODS: Herein, we used rat brain astrocytes (RBA) to demonstrate the signaling mechanisms of phorbol 12-myristate 13-acetate (PMA)-induced MMP-9 expression by zymographic, RT-PCR, subcellular isolation, Western blot, ROS detection, and promoter reporter analyses. Then, we evaluate the effects of rottlerin on PMA-induced MMP-9 expression in RBA and its influencing mechanism. RESULTS: We first demonstrated that PMA stimulated activation of various types of PKC, including PKC-δ in RBA. Subsequently, PMA induced MMP-9 expression via PKCδ-mediated reactive oxygen species (ROS) generation, extracellular signal-regulated kinase 1/2 (ERK1/2) activation, and then induced c-Fos/AP-1 signaling pathway. Finally, upregulation of MMP-9 by PMA via the pathway may promote astrocytic migration, and the event could be attenuated by rottlerin. CONCLUSIONS: These data indicated that rottlerin may have anti-inflammatory activity by reducing these related pathways of PKC-δ-dependent ROS-mediated MMP-9 expression in brain astrocytes.

Proinflammatory S100A8 Induces PD-L1 Expression in Macrophages, Mediating Tumor Immune Escape.

S100A8 is a damage-associated molecular pattern protein released by monocytes, playing a decisive role in the development of inflammation. Nonresolving inflammation is viewed as a driving force in tumorigenesis, and its role in tumor immune escape also attracted attentions. PD-1/PD-L1 axis is a critical determinant of physiological immune homeostasis, and anti-PD-1 or PD-L1 therapy has becoming the most exciting field of oncology. Multiple regulation mechanisms have been contributed to PD-L1 expression modulation including inflammatory mediators. In this study we reported that S100A8 significantly induced PD-L1 expression in monocytes/macrophages but not in tumor cells. S100A8 induced PD-L1 transcription through the TLR4 receptor and multiple crucial pathways of inflammation process. S100A8 modulated the histone modification of the PD-L1 promoter in monocytes/macrophages. S100A8-pretreated macrophages had immunosuppressive function and attenuated the antitumor ability of CTLs both in vitro and in vivo. A highly positive correlation existed between S100A8 expression and PD-L1 expression in human cancer specimens. To our knowledge, our study uncovers a novel molecular mechanism for regulating PD-L1 transcription by an inflammatory mediator S100A8, and reveals the importance of comprehensive understanding the role of inflammation in tumorigenesis as well as in tumor immune escape.

A significant role of transcription factors E2F in inflammation and tumorigenesis of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a malignant tumor from head and neck with characteristics in remarkable geographic and racial distribution worldwide, which has the important features of vigorous proliferation and inflammatory cells infiltration. By analyzing the expression profile data of NPC, we found that the E2F-related gene sets were highly enriched in NPC tissues. E2F transcription factor family is an important cycle regulator, which can promote the malignant proliferation and tumorigenesis. Here, we showed that E2Fs accelerated malignant phenotypes of NPC cells. RNA sequencing revealed that E2Fs can significantly up-regulate the inflammatory pathways in NPC cells. E2F1, as a transcription factor, can active the transcription activity of IL-6 promoter, and modulate macrophage function through a microenvironment manner. Thus, this study characterized a significant role of E2Fs in inflammation and tumorigenesis of NPC, which provided a promising anti-tumor target in NPC, since E2Fs are highly expressed and activated in NPC.

Lactoferrin deficiency induces a pro-metastatic tumor microenvironment through recruiting myeloid-derived suppressor cells in mice.

Lactoferrin, an innate immunity molecule, is involved in anti-inflammatory, anti-microbial, and anti-tumor activities. We previously reported that lactoferrin is downregulated in specimens of nasopharyngeal carcinoma and negatively associated with tumor progression and metastasis of patients with nasopharyngeal carcinoma. However, the relationship between lactoferrin and the pro-metastatic microenvironment has not been reported yet. Here, by using the lactoferrin knockout mouse, we found that lactoferrin deficiency facilitated melanoma cells metastasizing to lungs, through recruiting myeloid-derived suppressor cells (MDSCs) in the lungs. Mechanistic studies showed that in the lung microenvironment of the lactoferrin knockout mice, the TLR9 signaling was the most repressed signaling. Lactoferrin can induce MDSCs differentiation and apoptosis, as well as upregulate TLR9 expression. TLR9 agonist or lactoferrin treatment can rescue this phenotype in the tumor metastasis mouse model. Our results suggest a protective role of lactoferrin in cancer metastasis, along with a deficiency in certain components of the innate immune system, may lead to a pro-metastatic tumor microenvironment.

Ultraviolet B irradiation induced Nrf2 degradation occurs via activation of TRPV1 channels in human dermal fibroblasts.

Ultraviolet (UV) irradiation causes cellular oxidative stress. Under redox imbalance, Keap1-dependent Nrf2 degradation is minimal. In this study, we examined the role of Ca2+ in Nrf2 homeostasis after UVB irradiation using human dermal fibroblasts. UVB irradiation stimulates 12-lipoxygenase and the product 12-hydroxyeicosatetraenoic acid then activates TRPV1 increasing the cell's cytosolic Ca2+ concentration. UVB irradiation induced reactive oxygen species generation and apoptosis are inhibited in the absence of Ca2+ or in the presence of either a 12-lipoxygenase inhibitor or a TRPV1 inhibitor during and after UVB irradiation. Thus, the Ca2+ increase via TRPV1 is a critical factor in UVB irradiation induced oxidative stress. UVB irradiation induces a Ca2+ dependent Nrf2 degradation and thus activation of TRPV1 with 12-hydroxyeicosatetraenoic acid also decreasing Nrf2 levels. UVB irradiation induced Nrf2 degradation is inhibited by co-treatment of cells with W-7, cyclosporin A, SB-216763 or MG-132, which are inhibitors of calmodulin, calcineurin, GSK3ß and the proteasome, respectively. Furthermore, UVB irradiation in parallel induces GSK3ß dephosphorylation in a Ca2+ dependent manner. Co-immunoprecipitation showed that UVB irradiation induces an increase in Nrf2 phosphorylation, an increase in the binding of ß-TrCP and Nrf2, and an increase in Nrf2 ubiquitination; these effects are all Ca2+ dependent. These findings suggest that UVB irradiation induced GSK3ß activation in a Ca2+ dependent manner, which then stimulates the phosphorylation and ubiquitination of Nrf2 via ß-TrCP. Indeed, silencing of ß-TrCP was found to inhibit UVB irradiation-induced oxidative stress, Nrf2 degradation and apoptosis, while it had no effect on the Ca2+ increase. Taken together, our results suggest that a Ca2+ influx via TRPV1 is responsible for UVB irradiation-induced Nrf2 degradation and that modulation of the Ca2+-calmodulin-calcineurin-GSK3ß-Nrf2-ß-TrCP-Cullin-1 pathway may explain Ca2+ dependent Nrf2 degradation.

Baicalein inhibits matrix metalloproteinase 1 expression via activation of TRPV1-Ca-ERK pathway in ultraviolet B-irradiated human dermal fibroblasts.

Increased matrix metalloproteinase 1 (MMP-1) expression is a feature of photo-aged skin. We investigated the effects of baicalein and sulphoraphane on ultraviolet B (UVB) irradiation-induced MMP-1 expression and apoptosis using human dermal fibroblasts. UVB irradiation not only increased MMP-1 expression, but also caused apoptosis. Both baicalein and sulphoraphane protected cells from UVB irradiation-induced apoptosis, but only baicalein inhibited MMP-1 expression. UVB irradiation activated 12-lipoxygenase, and its product, 12-hydroxyeicosatetraenoic acid, activated TRPV1 channels. The resulting UVB irradiation-induced Ca2+ increase was blocked by the 12-lipoxygenase inhibitor baicalein and the TRPV1 blocker capsazepine, but not by the Nrf2 inducer sulphoraphane. UVB irradiation also increased ROS generation and decreased Nrf2 protein levels. UVB irradiation-induced MMP-1 expression was blocked by the Ca2+ chelator BAPTA, by capsazepine and by TRPV1 silencing. However, induction was unaffected by the antioxidant N-acetylcysteine. ERK phosphorylation and JNK phosphorylation were induced by UVB irradiation, but only ERK phosphorylation was Ca2+ sensitive. Increased MMP-1 expression was blocked by PD98059, but not by SP600125. Thus, increased MMP-1 expression is mediated by increased cytosolic Ca2+ and ERK phosphorylation. UVB irradiation-induced ROS generation is also Ca2+ sensitive, and UVB irradiation-induced apoptosis is caused by increased ROS. Thus, baicalein, by blocking the UVB irradiation-induced cytosolic Ca2+ increase, protects cells from UVB irradiation-induced MMP-1 expression and apoptosis. In contrast, sulphoraphane, by decreasing cellular ROS, protects cells from only UVB-induced apoptosis. Thus, targeting 12-lipoxygenase may provide a therapeutic approach to improving the health of photo-aged human skin.

Epstein-Barr virus miR-BART3-3p promotes tumorigenesis by regulating the senescence pathway in gastric cancer.

Epstein-Barr virus-associated gastric cancer (EBVaGC) accounts for about 10% of all gastric cancer cases and has unique pathological and molecular characteristics. EBV encodes a large number of microRNAs, which actively participate in the development of EBV-related tumors. Here, we report that EBV-miR-BART3-3p (BART3-3p) promotes gastric cancer cell growth in vitro and in vivo Moreover, BART3-3p inhibits the senescence of gastric cancer cells induced by an oncogene (RASG12V) or chemotherapy (irinotecan). LMP1 and EBNA3C encoded by EBV have also been reported to have antisenescence effects; however, in EBVaGC specimens, LMP1 expression is very low, and EBNA3C is not expressed. BART3-3p inhibits senescence of gastric cancer cells in a nude mouse model and inhibits the infiltration of natural killer cells and macrophages in tumor by altering the senescence-associated secretory phenotype (SASP). Mechanistically, BART3-3p directly targeted the tumor suppressor gene TP53 and caused down-regulation of p53's downstream target, p21. Analysis from clinical EBVaGC samples also showed a negative correlation between BART3-3p and TP53 expression. It is well known that mutant oncogene RASG12V or chemotherapeutic drugs can induce senescence, and here we show that both RASG12V and a chemotherapy drug also can induce BART3-3p expression in EBV-positive gastric cancer cells, forming a feedback loop that keeps the EBVaGC senescence at a low level. Our results suggest that, although TP53 is seldom mutated in EBVaGC, its expression is finely regulated such that EBV-encoded BART3-3p may play an important role by inhibiting the senescence of gastric cancer cells.

Neuroprotective Effects of Dehydroepiandrosterone Sulfate Through Inhibiting Expression of Matrix Metalloproteinase-9 from Bradykinin-Challenged Astroglia.

Dehydroepiandrosterone sulfate (DHEAS), one of the most important neuroactive steroids, is produced in the adrenals and the brain. DHEAS is believed to play a critical role in modulating different forms of cellular control, including processes associated with human neural systems. Its production rate and level in serum, adrenals, and brain gradually decrease with advancing age. The decline of DHEAS level was associated with age-related neuronal dysfunction and degeneration, most probably because the steroids protect the central nervous system (CNS) neurons against neurotoxic challenges. Moreover, increasing studies show that matrix metalloproteinases (MMPs), MMP-9 especially, are upregulated by proinflammatory mediators in the CNS disorders. The increased MMP-9 as an inflammatory biomarker of several CNS disorders that may participate in the CNS inflammation and neurodegeneration. Herein, we investigate the effects of DHEAS on brain inflammation by the model we have defined of bradykinin (BK)-induced MMP-9 expression in rat brain astrocyte (RBA) and its mechanism. The results showed that DHEAS significantly reduce MMP-9 induced by BK. Pretreatment with DHEAS can inhibit BK-stimulated phosphorylation of c-Src and PYK2. Moreover, DHEAS attenuated BK-stimulated NADPH oxidase (Nox)-derived reactive oxygen species (ROS) production, suggesting that DHEAS has an antioxidative effect. We further demonstrated that DHEAS blocked activation of ERK1/2, Akt, and c-Fos/AP-1 by BK. Finally, DHEAS decreased MMP-9-related events including RBA migration and neuronal apoptosis. The results will provide new insights into the anti-inflammatory action of DHEAS, supporting that DHEAS may have a neuroprotective effect in the improvement of the CNS disorders by reducing neuroinflammation.

Epstein-Barr Virus MicroRNA miR-BART5-3p Inhibits p53 Expression.

Epstein-Barr virus (EBV) is the first human virus found to encode many microRNAs. It is etiologically linked to nasopharyngeal carcinoma and EBV-associated gastric carcinoma. During the latent infection period, there are only a few EBV proteins expressed, whereas EBV microRNAs, such as the BamHI-A region rightward transcript (BART) microRNAs, are highly expressed. However, how these BART miRNAs precisely regulate the tumor growth in nasopharyngeal carcinoma and gastric carcinoma remains obscure. Here, we report that upregulation of EBV-miR-BART5-3p promotes the growth of nasopharyngeal carcinoma and gastric carcinoma cells. BART5-3p directly targets the tumor suppressor gene TP53 on its 3'-untranslated region (3'-UTR) and consequently downregulates CDKN1A, BAX, and FAS expression, leading to acceleration of the cell cycle progress and inhibition of cell apoptosis. BART5-3p contributes to the resistance to chemotherapeutic drugs and ionizing irradiation-induced p53 increase. Moreover, BART5-3p also facilitates degradation of p53 proteins. BART5-3p is the first EBV-microRNA to be identified as inhibiting p53 expression and function, which suggests a novel mechanism underlying the strategies employed by EBV to maintain latent infection and promote the development of EBV-associated carcinomas.IMPORTANCE EBV encodes 44 mature microRNAs, which have been proven to promote EBV-associated diseases by targeting host genes and self-viral genes. In EBV-associated carcinomas, the expression of viral protein is limited but the expression of BART microRNAs is extremely high, suggesting that they could be major factors in the contribution of EBV-associated tumorigenesis. p53 is a critical tumor suppressor. Unlike in most human solid tumors, TP53 mutations are rare in nasopharyngeal carcinoma and EBV-associated gastric carcinoma tissues, suggesting a possibility that some EBV-encoded products suppress the functions of p53. This study provides the first evidence that a BART microRNA can suppress p53 expression by directly targeting its 3'-UTR. This study implies that EBV can use its BART microRNAs to modulate the expression of p53, thus maintaining its latency and contributing to tumorigenesis.