[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.

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.

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.

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.

A new copper ionophore DPMQ protects cells against ultraviolet B irradiation by inhibiting the TRPV1 channel.

Copper is more likely than iron to generate reactive oxygen species (ROS) in a redox reaction due to its higher electrochemical reactivity. This study examined the effect of a newly synthesized Cu2+ binding compound, (E)-2-(4-(dimethylamino)phenylimino)methyl)quinolin-8-ol (DPMQ), on ultraviolet B (UVB) irradiation-induced cytotoxicity in human dermal fibroblasts. DPMQ induced Cu2+ influx as effectively as disulfiram, a Cu2+ ionophore anticancer drug. However, disulfiram induced ROS generation, mitochondrial dysfunction, and apoptosis in fibroblasts in a Cu2+ -dependent manner, whereas DPMQ was not only nontoxic, but protected cells against UVB irradiation-induced apoptosis in a Cu2+ -independent manner. UVB irradiation induced a Ca2+ -dependent increase in ROS generation, a decrease in Nrf2 levels, and activation of the mitochondrial apoptotic pathway, and these effects were prevented by DPMQ, which also increased Nrf2 nuclear translocation in a Cu2+ -independent manner. UVB irradiation activated 12-lipoxygenase and 12-hydroxyeicosatetraenoic acid (12-HETE), a product of 12-lipoxygenase, activated the TRPV1 channel. DMPQ did not act as a Ca2+ chelator, but inhibited the cytosolic Ca2+ increase induced by 12-HETE or capsaicin, but not that induced by bradykinin or ATP. Blockade of Ca2+ influx by pharmacological inhibition or silencing of the TRPV1 channel or chelation of cytosolic Ca2+ inhibited the UVB irradiation-induced Nrf2 reduction, ROS generation, mitochondrial dysfunction, and apoptosis. Taken together, our results suggest that Ca2+ influx via the TRPV1 channel is responsible for UVB irradiation-induced cytotoxicity and that DPMQ protects cells against UVB irradiation by inhibiting the TRPV1 channel and stabilizing Nrf2, and could thus be a potentially useful compound for the treatment of free radical-induced diseases.

Lion's Mane Medicinal Mushroom, Hericium erinaceus (Agaricomycetes), Modulates Purinoceptor-Coupled Calcium Signaling and Murine Nociceptive Behavior.

Hericium erinaceus is well known for the neurotrophic effect it confers by promoting nerve growth factor biosynthesis. We discovered a novel bioactivity of H. erinaceus in its ability to suppress adenosine triphosphate (ATP)-induced calcium signaling in neuronal PC12 cells. ATP, known primarily as a neurotransmitter, also acts on purinoceptors (P2 purinergic receptor [P2R]) to generate the cellular calcium signaling and secretion that mediate P2R physiological manifestations, including pain. Chronic pain reduces quality of life. However, constant analgesic administration can cause liver and kidney injury, as well as loss of the analgesic effect because of desensitization. In this study we investigated the analgesic potential of H. erinaceus through measurements of ATP-induced Ca2+ signaling in cell lines and observation of pain behaviors in mice. In P2R-coupled Ca2+ signaling measurements, extracts of H. erinaceus mycelia (HEEs) blocked ATP-induced Ca2+ signaling in both rat PC12 cells and human HOS cells. HEEs completely blocked ATP-induced Ca2+ signaling in human HOS cells, suggesting that this effect of HEEs is exerted through the P2R subtypes present in HOS cells, which include the P2X4, P2X7, P2Y2, and P2Y4 subtypes. In observations of animal behavior during pain, HEEs significantly reduced heat-induced pain, including postponing both the tail-flick response to heat stimulation and the paw-lifting response to a hot plate. This study demonstrates novel characteristics of H. erinaceus in reducing nociceptive behavior and blocking the functional activity of P2R. Further studies are required to verify this linkage and its molecular mechanisms.

Dopamine elevates intracellular zinc concentration in cultured rat embryonic cortical neurons through the cAMP-nitric oxide signaling cascade.

Zinc ion (Zn2+), the second most abundant transition metal after iron in the body, is essential for neuronal activity and also induces toxicity if the concentration is abnormally high. Our previous results show that exposure of cultured cortical neurons to dopamine elevates intracellular Zn2+ concentrations ([Zn2+]i) and induces autophagosome formation but the mechanism is not clear. In this study, we characterized the signaling pathway responsible for the dopamine-induced elevation of [Zn2+]i and the effect of [Zn2+]i in modulating the autophagy in cultured rat embryonic cortical neurons. N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a membrane-permeable Zn2+ chelator, could rescue the cell death and suppress the autophagosome puncta number induced by dopamine. Dopamine treatment increased the lipidation level of the endogenous microtubule-associated protein 1A/1B-light chain 3 (LC3 II), an autophagosome marker. TPEN added 1h before, but not after, dopamine treatment suppressed the dopamine-induced elevation of LC3 II level. Inhibitors of the dopamine D1-like receptor, protein kinase A (PKA), and NOS suppressed the dopamine-induced elevation of [Zn2+]i. PKA activators and NO generators directly increased [Zn2+]i in cultured neurons. Through cell fractionation, proteins with m.w. values between 5 and 10kD were found to release Zn2+ following NO stimulation. In addition, TPEN pretreatment and an inhibitor against PKA could suppress the LC3 II level increased by NO and dopamine, respectively. Therefore, our results demonstrate that dopamine-induced elevation of [Zn2+]i is mediated by the D1-like receptor-PKA-NO pathway and is important in modulating the cell death and autophagy.

Ultraviolet B irradiation increases keratin 1 and keratin 10 expressions in HaCaT keratinocytes via TRPV1 activation and ERK phosphorylation.

In this study, we characterized the effect of ultraviolet B (UVB) irradiation with or without epidermal growth factor (EGF) on the regulation of keratinocyte differentiation under physiological concentration of Ca2+ (1.8 mM). In addition, growth factor deprivation used to measure signal transduction and kinase phosphorylation in many studies is physiologically unreal. Therefore, 1% of serum was also included in all experiment. We found that UVB irradiation Ca2+ dependently induced morphological differentiation and increased keratin 1 and 10 (K1/K10) expressions. Both were inhibited by treatment of cells with EGF. In quiescent cells, phosphorylation of ERK was stimulated by acute EGF treatment, while it rapidly desensitized in chronic EGF treatment or 1% serum exposure. UVB irradiation-induced keratinocyte differentiation required Ca2+ influx through TRPV1. Ca2+ -dependent phosphorylation of ERK was responsible for the expression of K1/10. Cotreatment of cells with EGF during UVB irradiation inhibits the UVB irradiation-induced differentiation by desensitizing ERK phosphorylation.

Saikosaponin d induces cell death through caspase-3-dependent, caspase-3-independent and mitochondrial pathways in mammalian hepatic stellate cells.

BACKGROUND: Saikosaponin d (SSd) is one of the main active triterpene saponins in Bupleurum falcatum. It has a steroid-like structure, and is reported to have pharmacological activities, including liver protection in rat, cell cycle arrest and apoptosis induction in several cancer cell lines. However, the biological functions and molecular mechanisms of mammalian cells under SSd treatment are still unclear. METHODS: The cytotoxicity and apoptosis of hepatic stellate cells (HSCs) upon SSd treatment were discovered by MTT assay, colony formation assay and flow cytometry. The collage I/III, caspase activity and apoptotic related genes were examined by quantitative PCR, Western blotting, immunofluorescence and ELISA. The mitochondrial functions were monitored by flow cytometry, MitoTracker staining, ATP production and XF24 bioenergetic assay. RESULTS: This study found that SSd triggers cell death via an apoptosis path. An example of this path might be typical apoptotic morphology, increased sub-G1 phase cell population, inhibition of cell proliferation and activation of caspase-3 and caspase-9. However, the apoptotic effects induced by SSd are partially blocked by the caspase-3 inhibitor, Z-DEVD-FMK, suggesting that SSd may trigger both HSC-T6 and LX-2 cell apoptosis through caspase-3-dependent and independent pathways. We also found that SSd can trigger BAX and BAK translocation from the cytosol to the mitochondria, resulting in mitochondrial function inhibition, membrane potential disruption. Finally, SSd also increases the release of apoptotic factors. CONCLUSIONS: The overall analytical data indicate that SSd-elicited cell death may occur through caspase-3-dependent, caspase-3-independent and mitochondrial pathways in mammalian HSCs, and thus can delay the formation of liver fibrosis by reducing the level of HSCs.

Baicalein increases keratin 1 and 10 expression in HaCaT keratinocytes via TRPV4 receptor activation.

In this study, we characterized the effect of baicalein on the regulation of keratinocyte differentiation and proliferation, which are abnormal in atopic dermatitis or psoriasis. Treatment of HaCaT keratinocytes with 10 µm baicalein slightly inhibited cell growth, caused morphological differentiation and increased expression of keratins 1 and 10 (K1/K10) without affecting ROS generation, cytochrome c release or apoptosis. Baicalein treatment caused growth arrest in G0 /G1 phase and also induced Ca(2+) influx via TRPV4 receptor activation. Phosphorylation of ERK, Akt and p38 MAPK, but not JNK, was increased by baicalein, and inhibition of phosphorylation of ERK, but not that of Akt or p38 MAPK, blocked the baicalein-induced increase in K1/K10 expression, suggesting that ERK activation is involved in this increase. Removal of extracellular Ca(2+) or blockade of Ca(2+) influx by pharmacological inhibition or silencing of the TRPV4 receptor did not affect growth arrest, ROS generation or apoptosis, but inhibited baicalein-induced ERK phosphorylation and K1/K10 expression. Thus, baicalein treatment increases differentiation, and decreases proliferation, of keratinocytes. The mechanism of differentiation of keratinocytes is distinct from that of proliferation, the former being Ca(2+) dependent and the latter Ca(2+) independent.

Baicalein Decreases Hydrogen Peroxide-Induced Damage to NG108-15 Cells via Upregulation of Nrf2.

Baicalein is a flavonoid inhibitor of 12-lipoxygenase. Here, we investigated its effect on hydrogen peroxide-induced damage to NG108-15 cells. Hydrogen peroxide activated the mitochondrial apoptotic pathway, decreased Nrf2 expression, increased reactive oxygen species (ROS) levels, reduced viability, and increased cell death after 2-24 h treatment of NG108-15 cells. Co-treatment with hydrogen peroxide and baicalein completely suppressed the activation of mitochondrial apoptotic pathway by upregulating Nrf2 expression and reducing ROS stress and partially inhibited the effects on cell viability and cell death. Silencing of 12-lipoxygenase had a similar protective effect to baicalein on hydrogen peroxide-induced damage by blocking the hydrogen peroxide-induced decrease in Nrf2 expression and increase in ROS levels. Neither protective effect was altered by addition of 12-hydroxyeicosatetraenoic acid, the product of 12-lipoxygenase, suggesting that hydrogen peroxide induced damage via 12-lipoxygenase by another, as yet unknown, mechanism, rather than activating it. Co-treatment of cells with hydrogen peroxide and N-acetylcysteine or the Nrf2 inducer sulforaphane reduced hydrogen peroxide-induced damage in a similar fashion to baicalein, while the Nrf2 inhibitor retinoic acid blocked the protective effect of baicalein. Silencing Nrf2 also inhibited the protective effects of baicalein, sulforaphane, and N-acetylcysteine and resulted in high ROS levels, suggesting ROS elimination was mediated by Nrf2. Taken together our results suggest that baicalein protects cells from hydrogen peroxide-induced activation of the mitochondrial apoptotic pathway by upregulating Nrf2 and inhibiting 12-lipoxygenase to block the increase in ROS levels. Hydrogen peroxide also activates a second mitochondrial dysfunction independent death pathway which is resistant to baicalein.

Saikosaponin a and saikosaponin d inhibit proliferation and migratory activity of rat HSC-T6 cells.

The proliferation and migration of hepatic stellate cells (HSCs) profoundly impact the pathogenesis of liver inflammation and fibrogenesis. As a perennial herb native to China, Bupleurum falcatum is administered for its anti-inflammatory, antipyretic, and antihepatotoxic effects. Saikosaponin a (SSa) and Saikosaponin d (SSd) are the major active components of triterpene saponins in Bupleurum falcatum. This study analyzes how SSa and SSd affect rat HSC-T6 cell line proliferation and migration. Experimental results indicate that, in addition to suppressing HSC-T6 proliferation, wound healing activity and cell migration in a time- and dose-dependent manner, SSa and SSd significantly induce apoptosis. Additionally, SSa and SSd decreased the expressions of extracellular matrix-regulated kinase 1/2 (ERK1/2), platelet-derived growth factor receptor 1 (PDGFR1), and subsequently transforming growth factor-ß1 receptor (TGF-ß1R), α-smooth muscle actin, TGF-ß1 and connective tissue growth factor. They also decreased phosphorylation of p38 (p-p38) and ERK1/2 (p-ERK1/2) of HSC-T6. Furthermore, both SSa and SSd can block PDGF-BB and TGF-ß1-induced cell proliferation and migration of HSC-T6. These results suggest that SSa and SSd may inhibit proliferation and activation of HSC-T6, and the modulated mechanisms warrant further study.

Dual effect of capsaicin on cell death in human osteosarcoma G292 cells.

Thirty percent of osteosarcoma patients die within 5 years. New agents that induce apoptosis of osteosarcoma cells might be therapeutically useful. Here, we characterized the apoptotic mechanism induced by capsaicin in G292 osteosarcoma cells. Our results show that capsaicin induces an increase in the cytosolic Ca(2+) concentration which is independent of the extracellular Ca(2+) concentration and depletes intracellular Ca(2+) stores, suggesting the presence of endoplasmic reticulum transient receptor potential vanilloid receptor type 1. Capsaicin also activates the mitochondrial caspase 3-dependent death cascade. Rapamycin, an inhibitor of mammalian target of rapamycin, evokes autophagy, as do capsaicin or thapsigargin, a sarco(endo)plasmic reticulum Ca(2+) ATPase inhibitor that causes Ca(2+) store depletion. Capsaicin-induced cell death is completely inhibited by co-treatment with the pan-caspase inhibitor Z-VAD-fmk and increased by the autophagy inhibitor 3-methyladenine, suggesting the existence of an autophagy-dependent anti-apoptotic mechanism. Capsaicin also induces ERK phosphorylation, which acts as a downstream effector of autophagy. 3-Methyladenine or PD98059, an ERK kinase inhibitor, restores capsaicin-induced cell death in the presence of Z-VAD-fmk, suggesting that inhibition of autophagy activates a second cell death pathway that is caspase-independent. Taken together, our data show that capsaicin causes Ca(2+) depletion of intracellular Ca(2+) stores and simultaneously activates the mitochondrial caspase-dependent death cascade and autophagy-dependent ERK activation and that the latter counteracts a second death signaling pathway that is caspase-independent.

Acute phorbol ester treatment inhibits thapsigargin-induced cell death in porcine aortic smooth muscle cells.

We have previously shown that, in porcine aortic smooth muscle cells, endoplasmic reticulum (ER) stressor thapsigargin simultaneously activate the mitochondrial caspase-dependent death cascade and an extracellular signal-regulated kinase (ERK)-dependent pathway, which inhibits the caspase-independent death pathway. Our aim in the present study was to examine the effect of the phorbol ester phorbol 12-myristate 13-acetate (PMA) on these processes. We found that thapsigargin induced autophagy, which led to cell death. Treatment of cells with PMA for 5min, which activates protein kinase C (PKC), partially inhibited thapsigargin-induced cell death, whereas PMA treatment for 24h, which downregulates PKC, did not. This protection after short PMA treatment was not due to inhibition of the thapsigargin-induced cytosolic calcium concentration increase, mitochondrial permeability transition pore (PTP) opening, or caspase-3 activation, but coincided with increased ERK phosphorylation and decreased autophagosome formation and the decreased autophagosome formation was prevented by the ERK kinase inhibitor PD98059. Thus, under conditions of ER stress caused by thapsigargin-induced disturbance of calcium homeostasis, PKC activation induced ERK phosphorylation, which inhibited autophagic, but not apoptotic, cell death. After acute PMA treatment, protection against thapsigargin-induced cell death was enhanced by the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone or the PTP blocker cyclosporin A, but decreased by PD98059 or the PKC inhibitor Go6983. Taken together, these results suggest that PKC activation alleviates ER stress and that this is attributable to enhanced ERK phosphorylation, which inhibits autophagic, but not apoptotic, cell death.

Demonstration of an olfactory bulb-brain translocation pathway for ZnO nanoparticles in rodent cells in vitro and in vivo.

ZnO nanoparticles (ZnO-NPs) are widely used in the engineering and cosmetic industries, and inhaled airborne particles pose a known hazard to human health; their translocation into humans is a recognized public health concern. The pulmonary-blood pathway for ZnO-NP toxicity is well documented, but whether translocation of these particles can also occur via an olfactory bulb-brain route remains unclear. The potential toxicity of ZnO-NPs for the human central nervous system (CNS) is predicated on the possibility of their translocation. Our study investigated translocation of ZnO-NPs both in vitro using the neuronal cell line PC12 and in vivo in a Sprague-Dawley rat model. Our findings indicate that the zinc-binding dye, Newport-Green DCF, binds ZnO stoichiometrically and that ZnO-NP concentration can therefore be measured by the fluorescence intensity of the bound dye in confocal fluorescence microscopy. Confocal data obtained using Newport-Green DCF-2 K(+)-conjugated ZnO-NPs along with the membrane probe FM1-43 demonstrated endocytosis of ZnO-NPs by PC12 cells. In addition, Fluozin-3 measurement showed elevation of cytosolic Zn(2+) concentration in these cells. Following in vivo nasal exposure of rats to airborne ZnO-NPs, olfactory bulbs and brains that were examined by Newport-Green fluorescence and TEM particle measurement clearly showed the presence of ZnO-NPs in brain. We conclude that an olfactory bulb-brain translocation pathway for airborne ZnO-NPs exists in rats, and that endocytosis is required for interneuron translocation of these particles.