Novel Acetylene Phenolic Compound Selaginellin: Anti-aging Of Endothelial Cells And Neurobiology Research

Atherosclerosis (AS) is recognized as one of the major causes to threaten the life of people. It is considered that pathogenesis of AS is involved in many factors, such as blood fat, smoking, hypertension, diabetes, infection and induced pathological changes of vascular biology. Homocysteine (Hcy) can promote atherogenesis, and plasma level of Hcy are positively correlated with morbidity and mortality from coronary heart diseases, suggesting that Hcy is an independent risk factor for AS.Endothelial dysfunction is recognized as all early event in the pathogenesis of AS, which is related to senescence and damages of endothelial cells. Also, the decreased expression of anti-senescent molecules contributes to progression of cell senescence. The down-regulation of Sirtl, an enzyme to remove acetyl groups from specific proteins, has been shown to mediate oxidative stress-induced endothelial senescence. It has been reported that pro-atherogenetic properties of Hcy are related to inducing cell senescence.Selaginella Tamariscina (Beauv) spring can lower blood pressure, decrease the level of serum glucose, enhance immune function and inhibit oxidant production and so on. Selaginellin, a compound with novel chemical structure, was extracted from Selaginella Tamariscina. Our preliminary data showed that selaginellin had strong anti-oxidative activity. Based on these data mentioned above, in the present study, we investigated the effects of selaginellin on Hcy-induced senescence of endothelial cells and its underlying mechanisms.METHODSHuman umbilical vein endothelial cells (HUVECs) were cultured and used for all these studies. Cell viability was analyzed by using MTS assay. Cell senescence was evaluated by usingβ-galactosidase staining, telomerase activity detected by real time PCR and cell cycle distribution determined by staining DNA with propidium iodide. The level of intracellular reactive oxygen species (ROS) was determined by using fluorescent ROS detection kit and the level of Sirtl mRNA was detected by real time PCR.RESULTS(1) Treatment with Hcy (0.5 M) for 48 h markedly decreased the viability of endothelial cells. Pretreatment with selaginellin for 1 h prior to exposure to homocysteine concentration-dependently reversed the decrease in cell viability induced by Hcy.(2) Treatment with Hcy (0.5 M) for 48 h markedly induced the senescence of endothelial cells shown by the decrease in telomerase activity, the increase inβ-galactosidase activity and the increase in the percentage of cells in G0/G1 phage, which could be attenuated by pretreatment with selaginellin in a concentration-dependent manner.(3) Treatment with Hcy (0.5 M) significantly increased the level of intracellular ROS in endothelial cells. However, selaginellin could significantly inhibit Hcy-induced elevation of intracellualr ROS level.(4) The mRNA expression of Sirt1, an important anti-senescent protein, was markedly decreased in Hcy-incubated endothelial cells. However, selaginellin could significantly upregulate the mRNA expression of Sirtl in the absence or presence of Hcy.CONCLUSIONSelaginellin attenuates Hcy-induced senescence of endothelial cells, and such effects may be related to inhibiting oxidative stress and up-regulating Sirtlexpression. L-glutamate, an excitatory amino acid, has been identified as the principal transmitter mediating excitatory synaptic responses and neuronal development via glutamate receptor activation in the central nervous system. However, excessive excitatory transmission can be transformed into an implement of neuronal destruction resulting in CNS disorder, such as cerebral ischemia, hypoxia, alcoholism, autoimmune encephalomyelitis, and Alzheimer's, Huntington's and Parkinson's diseases. Glutamate can induce cell death by two different pathways: excitotoxicity and oxytosis. The excitotoxicity of glutamate is mediated through the activation of several types of excitatory amino acid receptors, and subsequently massive influx of extracellular Ca2+. The oxytosis of glutamate is mediated by competitive inhibition of cystine uptake. Selaginellin is a component extracted from S.pulvinata (Hooket Grev.) Maximo. Selaginellin contains hydroxy group, we therefore presumed that it would have the ability of binding free radicals. In the present study, we therefore studied the protective effect of selaginellin on glutamate-induced cytotoxicity and apoptosis in differentiated rat pheochromocytoma (PC 12) cells. Because Klotho, an anti-aging protein with anti-oxidative property, has been shown to have anti-apoptotic function in a variety of cell types, we also investigated whether the anti-apoptotic effect of selaginellin on PC 12 cells is related to regulation of Klotho gene expression.METHODSAfter cultured with the serum-free medium for 24 h, the differentiated PC 12 cells were pretreated with various concentrations (10-7,3×10-7 or 10-6 M) of selaginellin for 1 h prior to exposure to L-glutamate. Cell viability was analyzed by using MTS assay. Cell injury was evaluated using observation of morphologic changes, cell viability and LDH release. Cell apoptosis was determined by Hoechst staining and caspase-3 activity. The level of intracellular reactive oxygen species (ROS) was determined using fluorescent ROS detection kit and the level of Klotho mRNA was detected by real time PCR. (1) Treatment of PC 12 cells with 100 ng/mL nerve growth factor for 1 week successfully induced differentiation as shown by round cell bodies with fine dendritic networks.(2) MTS assay showed that the cell viability was inhibited by glutamate in a concentration-dependent manner. Glutamate at 10 mM led to about 45% inhibition of cell viability. We therefore chose 10 mM of glutamate for the subsequent experiments.(3) Under a phase-contrast microscopy, normal differentiated PC 12 cells showed round cell bodies with fine dendritic networks, and the cell edges were intact and clear. In contrast, incubation of cells with 10 mM of L-glutamate for 24 h induced shrinkage of the cell bodies, disappearance in cell reticular formation and disruption of the dendritic networks. Pretreatment with selaginellin dramatically alleviated morphological manifestations of cell damage in a concentration-dependent manner. Glutamate significantly increased the release of LDH and decreased the cell viability. These effects of glutamate were reversed by selaginellin in a concentration-dependent manner.(4) Hoechst 33342 staining assay showed that treatment with glutamate (10 mM) for 24 h significantly increased the ratio of cells with a profile of cell shrinkage, chromatin condensation and fragmented fluorescent nuclei. The caspase-3 activity, a marker of apoptosis, was also found to be increased in glutamate-treated cells. The number of Hoechst 33342-positive cells and the activity of caspase-3 induced by glutamate were significantly reduced by selaginellin in a concentration-dependent manner.(5) Glutamate increased intracellular ROS generation as indicated by the increase in 2',7'-dichlorofluorescein fluorescence. Selaginellin significantly reduced the increase in ROS generation induced by glutamate in a concentration-dependent manner.(6) Incubation of PC 12 cells with glutamate (10 mM) for 24 h led to a significant decrease in Klotho mRNA expression. In contrast, the decreased mRNA expression of Klotho was reversed by pretreatment with selaginellin in a concentration-dependent manner.CONCLUSIONSelaginellin protected against L-glutamate induced cell injury and apoptosis in differentiated PC 12 cells through regulation of ROS/Klotho gene pathway. BACKGROUNDAlzheimer's disease (AD) is a progressive neurodegenerative disorder. The major neuropathological hallmarks of AD are the formation of senile plaques following neurofibrillary tangles and neurons loss. Although the mechanisms of AD pathology are still undefined, lipoprotein dysbolism has been recently reported to be involved in the progress of AD.Low-density lipoprotein (LDL) exists within the brain and is highly vulnerable to oxidative modifications. Once formed, oxidized LDL (ox-LDL) is capable of eliciting neurons apoptosis involving in reactive oxygen species (ROS). It has been demonstrated that neurons apoptosis takes part in the progress of AD. In vitro experiments have shown that ox-LDL can induce many kinds of cell apoptosis via combining with its receptors.Lectin-like oxidized low density lipoprotein receptor-1 (LOX-1), a novel ox-LDL receptor, was firstly found to be expressed in bovine aortic endothelial cells. Ox-LDL activates LOX-1 followed by the production of intercellular reactive oxygen species (ROS), wherein ROS play an important role in a variety of biological processes. We presumed that LOX-1 might be involved in ox-LDL-induced neurons apoptosis. NADPH oxidases is the major source of ROS in diffirent cell types. Studies have shown that NADPH oxidases are involved in ROS-induced neuronal damage in the AD pathogenesis.Selaginellin, a component extracted from S.pulvinata (Hooket Grev.) Maximo. has the ability of binding free radicals. In the present study, we therefore studied the protective effect of selaginellin on ox-LDL-induced apoptosis in differentiated rat pheochromocytoma (PC 12) cells. We also investigated whether the anti-apoptotic effect of selaginellin on PC 12 cells is related to regulation of LOX-1/NADPH oxidase/ROS pathway.METHODSAfter cultured with the serum-free medium for 24 h, the differentiated PC 12 cells were pretreated with various concentrations (10-7,3×10-7 or 10-6 M) of selaginellin for 1 h prior to exposure to ox-LDL. Cell viability was analyzed by using MTS assay. Cell apoptosis was determined by Hoechst staining, flow cytometry with annexin V-FITC/PI double staining and caspase-3 activity. The level of intracellular reactive oxygen species (ROS) was determined using fluorescent ROS detection kit. The level of LOX-1 mRNA was detected by RT-PCR. The activity of NADPH oxidase was measured using cell NADPH oxidase detection kit and the expression of NOX-1, gp91phox and p47phox mRNA was detected by real time PCR. RESULTS(1) MTS assay showed that the cell viability was inhibited by ox-LDL in a concentration-dependent manner. Ox-LDL at 100μg/mL led to about 40% inhibition of cell viability. We therefore chose 100μg/mL of ox-LDL for the subsequent experiments.(2) Ox-LDL significantly decreased the cell viability. These effects of ox-LDL were reversed by selaginellin in a concentration-dependent manner.(3) Hoechst 33342 and annexin V-FITC/PI staining assay showed that treatment with ox-LDL (100μg/mL) for 24 h significantly increased the ratio of cells with a profile of cell shrinkage, chromatin condensation and fragmented fluorescent nuclei. The caspase-3 activity was also found to be increased in ox-LDL-treated cells. The number of Hoechst 33342-positive cells and annexin V-FITC/PI staining cells as well as the activity of caspase-3 induced by ox-LDL were significantly reduced by selaginellin in a concentration-dependent manner.(4) Ox-LDL increased intracellular ROS generation. Selaginellin significantly reduced the increase in ROS generation induced by ox-LDL in a concentration-dependent manner.(5) Incubation of PC 12 cells with ox-LDL (100μg/mL) for 24 h led to a significant increase in LOX-1 mRNA expression. In contrast, the decreased mRNA expression of LOX-1 was reversed by pretreatment with selaginellin in a concentration-dependent manner.(6) Ox-LDL activated NADPH oxidase, which was reversed by selaginellin. Also, as shown by the results of real time PCR, incubation of PC 12 with ox-LDL for 24 h led to a significant increase in NOX-1, gp91phox and p47phox mRNA expression. In contrast, the increased mRNA expression of NOX-1, gp91phox and p47phox was reversed by pretreatment with selaginellin. Co-incubation with anti-LOX-1 could completely block ox-LDL-induced the activation of NADPH oxidase.CONCLUSIONSelaginellin protected against ox-LDL apoptosis in differentiated PC 12 cells through regulation of LOX-1/NADPH oxidase/ROS pathway. BACKGROUNDAlzheimer's disease (AD) is a progressive neurodegenerative disorder. It is considered that pathogenesis of AD is involved in many factors, such as genetic factor, cell apoptosis, oxidation and aluminium poisoning. Inflammation has been recently reported to be involved in the progress of AD. Astrocytes express an array of inflammatory mediators that are involved in the initiation of inflammatory reactions within CNS parenchyma. It is documented that toll-like receptor 4 (TLR4) plays a key role in the program of AD pathology. TLR4 can activate nuclear factor-κB (NF-κB) signal pathway, which mediate the pro-inflammatory effects of many kinds of inflamatory cytokines. Selaginellin, a compound with novel chemical structure, was extracted from Selaginella Tamariscina. Our preliminary data showed that selaginellin had strong anti-oxidative activity, which inhibited L-glutamate-and ox-LDL-induced apoptosis of differentiated PC 12 cell. Therefore, we presumed that selaginellin had neuronal protective effects. The aim of the present study was to investigate the effect of selaginellin on inflammation of astrocytes and whether anti-inflammatory effect of selaginellin was related to TLR4-NF-κB pathway. METHODSCell viability was analyzed by using MTS assay. The expression of TLR4, TNF-α, MCP-1, IL-6 and NF-κB p65 mRNA was detected by real time PCR. The levels of TNF-α, MCP-1 and IL-6 in culture medium were assayed by enzyme linked immunosorbent assay (ELISA). NF-κB DNA-binding activity was determined by electrophoretic mobility shift assay (EMSA).RESULTS(1) Treatment with LPS (1μg/mL) for 24 h markedly decreased the activity of astrocytes. Pretreatment with selaginellin for 1 h prior to exposure to LPS concentration-dependently reversed the decrease in cell viability induced by LPS.(2) In cultured astrocytes, incubation with LPS significantly up-regulated the mRNA expression of inflammatory factors tumor necrosis factor-α(TNF-α), MCP-1 and IL-6, which could be attenuated by pretreatment with selaginellin in a concentration-dependent manner.(3) The levels of TNF-α, MCP-1 and IL-6 in cultured medium were increased after incubated with LPS for 24 h. In contrast, selaginellin decreased the levels of TNF-α, MCP-1 and IL-6 in a concentration-dependent manner. (4) The mRNA expression of TLR4 was markedly increased in LPS-incubated astrocytes. In contrast, selaginellin could significantly downregulate the mRNA expression of TLR4 in the presence of LPS.(5) Incubation of astrocytes with LPS for 24 h led to a significant increase in NF-κB p65 mRNA expression. In contrast, the increased mRNA expression of NF-κB p65 was reversed by pretreatment with selaginellin in a concentration-dependent manner. Also, as shown by the results of EMSA, LPS activated NF-κB, which was reversed by selaginellin. Co-incubation with TLR4 antagonist could completely block LPS-induced the activation of NF-κB.CONCLUSIONSelaginellin can decrease inflammatory cytokines TNF-a, MCP-1 and IL-6 synthesis and release in astrocytes induced by LPS via inhibiting TLR4-NF-κB signaling pathway.