| Risk factors for Parkinson’s disease (PD, a common age-related debilitating neurodegenerative disorder), include genetic predisposition (susceptibility gene-parkin),age, exposure to agents such as pesticides, metals, infectious agents,oxidative stress, mitochondrial dysfunction, excitatory transmitter, apoptosis and dysfunction of ubiquitin proteasome system,and so on, but the concrete mechanisms are still unclear. In recent years, the potential pathogenic mechanisms involving neuroinflamation have been studied, especially several lines of evidence, from epidemiological studies, findings of post-mortem PD brains or PD animal models, have confirmed the hypothesis that sustained activation of glia (macroglia and microglia) is believed to contribute to the progressive DA neurodegeneration via neuroinflammation. Microglia as specialized macrophages in central nervous system against injury and infections, respond to slight disturbances in the microenvironment and readily become activated. Activated microglia, release various proinflammatory and neurotoxic factors such as tumor necrosis factor-alpha(TNF-a), interleukin-1(IL-1), reactive nitrogen and the fatty acid cents such as oxygen-free radicals, the vast majority of which are deemed to contribute to the injury of neurons, and sometimes trophic factors.Sirtuins, NAD+-dependent class III histone deacetylases, regulate cell functions in genetic modification, age, supersession oxidative stress and so on. Mammalian sirtuin family features seven members S1RT1-SIRT7, with the nuclear SIRT1being the closest to Sir2, and the best understood. The first sirtuin was reported as Sir2from Saccharomyces cerevisiae. SIRT1is widely found in adult human and rodents, and the deacetylation activity of it is involved in the effect of the elevated [NAD+]/[NADH] ratio. Its activators include polyphenols such as resveratrol, butein, fistein, and so on, and inhibitors of it are nicotinamide, sirtinol, splitomicin and so on. Many of its nonhistone substrates have been identified, including p53, NF-κB, forkhead transcription factor (FOXO), peroxisome proliferator activated receptor-r, peroxisome proliferator-activated receptor-g coactivator-la (PGC-la), Ku70, and liver X receptor (LXR), all of which have a close relationship to the genes regulating cell life and survival. Due to its deacetylation on above substrates, SIRT1regulates various biological functions including cell proliferation, differentiation, DNA repair, glucose metabolism, lipid metabolism, mitochondrial biogenesis, apoptosis, and inflammation, and plays an important role in delaying the progression of neurodegenerative diseases.Lipopolysaccharide (LPS), bacterial endotoxin, is generally known as inflammation origin of creating inflammation. It activates glia via multiple mechanisms involving NF-κB, AP-1, and IRF3. A variety of stimulus and accumulation of proinflammatory and neurotoxic factors of microglia are believed to have relation to the progressive DA neurodegeneration. BV-2cells were derived from raf/myc-immortalised murine neonatal microglia and its morphology, and main features are similar to those of primary microglia. Because of their ease of culture, BV-2cells are the most frequently used substitute for primary microglia, in pharmacological, phagocytosis studies, or immunological discoveries. PC12cell line, the neural crest-derived, rat pheochromocytoma cell, can synthesize and store dopamine. Not only synthetic transmitter of PC12cells, but also its enzymes and membrane receptors are similar to those of midbrain dopamine neurons, so it has been widely used as an established dopaminergic neuronal model of neuron death or neurotoxic study. Though the use of PC12cells could not replace that of dopamine neurons, it supplies a new method for basic study.In the present work, the PD model of PC12injured by microglial-derived factors induced by LPS, was used to investigate the possible preventive mechanisms of SIRT1on injury of dopaminergic neurons in an inflammation/immunity model.Part I Role of SIRT1in regulation of Lipopolysaccharide-induced secretion of IL-6and TNF-a in activated BV-2cellsObjectiveTo observe the effect of lipopolysaccharide (LPS) on the cell morphology of BV-2cells and the expressions of interleukin-6(IL-6) and tumor necrosis factor-a (TNF-a) so as to detect the role of silence information regulator1(SIRT1) in regulation of LPS-induced proinflammatory cytokines production in activated BV-2cells.Methods1. BV-2cells were divided into the control group with normal culture medium, and the treatment groups that were cultured with different concentrations of LPS(0.5、1、10、50、100μg/mL),SIRT1activator resveratrol (10、25、50、100μmol/L) or SIRT1inhibitor sirtinol (1、2.5、5、10、25、50μmol/L), respectively. The MTT assay was employed to identify the cell viability after the inducement in24h.2.Based on the above MTT results, the cells were then grouped into the control group, LPS group, resveratrol+LPS group, and sirtinol+LPS groups, with the suitable concentrations of LPS (1μg/mL), resveratrol (10、25μmol/L) and sirtinol (25μmol/L) determined. The levels of IL-6and TNF-a were measured with ELISA at6,12h,24h after the inducement.3. The expression of SIRT1was detected by western blotting at24h in groups as followed, the control group, LPS group, resveratrol+LPS group, and sirtinol+LPS groups,with the suitable concentrations of LPS (1μg/mL), resveratrol (25μmol/L) or sirtinol (25μmol/L)Results1. No changes in cell viability measured by MTT test were observed for untreated-group, LPS-treated group, and BV-2cells’ culture medium-treated group. Compared to the control group, the BV-2cells of LPS treated group looked like the forms of activated microglias, with increased cell number, hypertrophier cell body, and shorten cell processes. The cell survival rate increased with increasing concentrations of LPS (P<0.05), from0.5μg/ml to100mg/ml.2. An obvious reduction of cell viability only started from50μM resveratrol concentration (P<0.05). So in the next experiments, we decided to choose the non-toxic resveratrol concentration of10,25μM to activate SIRT1.Sirtinol had no significant toxicity to BV-2cells from1μM to25μM,and it was toxic from50μM clearly (P<0.05). We therefore planted to use sirtinol concentration of25μM to inhibit SIRT1at the subsequent step.3. TNF-a and IL-6secretion was strongly increased by LPS, especially at the time piont of6h,12h or24h. Preincubation (2h) with resveratrol as positive control significantly reduced LPS-stimulated TNF-a and the result was dose-dependent. Preincubation with resveratrol at the concentration of10or25μM, significantly reduced LPS-stimulated IL-6secretion at6h,12h,24h. On the contrary, as the similar effects in the result of TNF-a, IL-6secretion had a significant increase in sirtinol-treated group, compared to LPS-treated group at6h,12h,24h (P<0.05)4. The results in Westerrn blot showed that, treament of LPS, reduced the level of SIRT1in BV-2cells, compared to the controls. The SIRT1expression had an increase in cells treated by resveratrol and a decrease in cells treated by sirtinol, compared to that in cells incubated with LPS only (P<0.05)ConclusionLPS can change the morphology of BV-2cells, and induce the levels of proinflammatory cytokines production; Impairment of SIRT1may contribute to such progress obviously.Part II Protection of SIRT1on toxicity of activated BV-2cells induced by LPS to PC12cellsObjectTo observe the effects of SIRT1on toxicity of activated BV-2to PC12cells and the possible mechanisms involved.Methods1. BV-2cells were divided into the control group and a treatment group coincubated with1μg/ml LPS. Afterwards, culture medium was harvested at15hour, respectively. PC12cells were divided into groups as follows:control (no treatment), LPS (LPS alone), BV-2cells (culture medium of BV-2only, harvested at the15-hour time piont above), and activated BV-2cells (culture medium of BV-2cocultivated with LPS, harvested at the15-hour time piont in the previous step). After24h of treatment, cell viability was measured by MTT test.2. PC12cells were divided into the control group with normal culture medium, and the treatment groups that were cultured with different concentrations of SIRT1 activator resveratrol (5、10、25、50、100μM) or SIRT1inhibitor nicotinamide (5%10,25,50mM), respectively. MTT assay was employed to identify the cell viability after the inducement in24h. PC12cells were divided into groups as followed:the control group, activated BV-2cells’culture medium-treated group, resveratrol-treated group and nicotinamide-treated group (pretreated with resveratrol or nicotinamide for2h, and then subjected to culture medium of activated BV-2cells, in the presence of resveratrol or sirtinol forl8h). Hoechst33258staining was used to study the apoptosis of PC12cells at6h. The cell viability, cell apoptosis rate, expressions of SIRT1, acetyl-p53, total p53, caspase-3, cleaved caspase-3, were measured by OD value in MTT assay, Flow cytometry, and western blot at18h respectively.ResultsIn PC12cells,treament with culture medium of activated BV-2cells, decreased the expression of SIRT1and caspase-3, and increased acetyl-p53and cleaved caspase-3expression, an effect that reduced the cell viability and increased the apoptosis, compared with the controls (P<0.05), whereas treatment with resvertral inhibited this effect. Conversely, addition of nicotinamide contributed to a decrease of SIRT1and caspase-3and an increase of acetyl-p53and cleaved caspase-3(P<0.05), an effect that increased the rate of cell-killing of PC12cells injured by activated BV-2-derived factors. On other hand, there was no change observed in total p53content among all groups of our study.ConclusionSIRT1can inhibit toxicity of activated BV-2cells to PC12cells, the mechanism of which is partly via p53-caspase-3-dependent mechanism of apoptosis. |
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