Protective Effects Of Resveratrol On Lipopolysaccharide-induced Apoptosis In PC12Cells

Neurodegenerative diseases are a center nerve tissue retrograde degeneration diseases which effect life quality seriously and mutilation,the prevalence grows increasingly with human population aging.The mechanism of the diseases is not completely represented,infering be concerned with cell exitotoxicity、cell apoptosis and oxidative stress,so drugs to prevent and treat the diaeases are not good effect at present.The diseases include alzheimer disease、Parkinson disease、Huntington’s disease、amyotrophic lateral sclerosis and so on.The pathology characteristic is that definite functional nucleus dry up and neuron lose.The neuron lose usually accompany with astrocyte proliferation neuroglia excessive and specified nerve pathology mark,for example lewy body of PD. In clinical,there have not effective measure to control course of diseases,the patient will loss viability even death.The rate and amount of aging population are increasing along with social progress,the diseases have been becoming important problem influencing population health level and life quality.During the past decade study had discovered that Sirtuins are close correlated with the diseases, activation and inhibition may participate regulation of NDD process. Lipopolysaccharide is generally known as inflammation origin of creating inflammation,it is bacterial endotoxin,is component on gram-negative bacteria externa,and which regulates innate immunity by coordinating inflammation modulators’production to stimulate inflammatory factor and oxygen radicals creations promoting degenerative diserses.PC12is rats pheochromocytoma as neuron model to study Nervous Systerm Diseases in internationnal. Apoptosis is a programly style of cell dead,is a major signal path which is concerned with pathpphysiology of degenerative diserses. NF-KB/COX-2、Bax、BCL-2、Caspase-3、Hsp70participate PC12cells apoptosis process of induced by LPS,but the mechanism is not completely represented.PC12cells strain originates from pheochromocytoma,cultures generally,because of the nature is similar with pheochromocytoma, it has neurosecretory cell and neuron nature,had been used as nerve cells’s dead style and neurotoxic study.Enzymes、membrane receptor and synthetic transmitter of PC12cells,we select PC12cells as neuronal cell model.Though PC12cells cannot replace neuro cells,it supply new idea for basis study and clinic cure.Histone deacetylases (HDACs) are divided into three chasses(Ⅰ-Ⅱ andⅢ) according to homology of yeasts transcribe inhibitor. The catalytic core of Class Ⅰ and Class Ⅱ are obviously parallel,and is homologous with yeasts deacetylase Rpd3p and Hdalp.But HDACsⅢ has no sequence similarity with Class Ⅰ and Class Ⅱ,it is homologous with yeasts transcribe inhibitor——Silent information regulator2called Sirtuins.Sirtuins have been found in bacteria to eukaryotes. The family is divided into five classes (Ⅰ-Ⅳ and U) on the basis of a phylogenetic analysis. The human genome encodes seven sir-tuins, with representatives from classes Ⅰ-Ⅳ.Sirtuins are NAD+-depending Histone deacetylases Ⅲ,they work by deacetylases activityThe mammalian Sir2gene family (Sirtuins) are consists of seven members: SIRT1-7. Seven Sirtuins protein settle in different functions in different subcellular locations. SIRT1is an important nuclear protein, which can deacetylate H4K16and a large number of non-histone targets, such as P53, Ku70and PPARy, PGC-1α, NF-κB, HIF-2α, XPA and a number of FOXO subtypes. SIRT1is included in a variety of important cellular processes, including proliferation, DNA repair mechanisms and apoptosis, and is thought to play an important role in cancer and glucose homeostasis. In addition, increasing SIRT1may regulate energy constraints on the processes of life studies in lower organisms. The SIRT2settles in the cytoplasm as a microtubule deacetylase, but it also exists in the nucleus and interaction with histone. Although SIRT2as a potential tumor suppressor, recent studies show that simultaneous inhibition of SIRT1and SIRT2in P53-mediated apoptosis of cancer cells is essential. SIRT3, SIRT4, SIRT5, are a mitochondrial protein, and which are related with the regulation of cell metabolism process. For example, SIRT3deacetylates and thus activates the mitochon-drial complex I, acetyl-CoA synthase2(AceCS2) as well as glutamate dehydrogenase (GDH) which triggers production of ATP via the citric acid cycle. SIRT4is no deacetylase activity of the substances identified so far against the effect of SIRT3right glutamic acid ADP ribosylation. SIRT4-/-rat significantly increased insulin levels in the stimulation of glucose, whereas in rat of overexpression SIRT4insulin secretion is significantly reduced. Recent find-ings on SIRT4-dependent fatty-acid oxidation in liver and muscle cells also support the idea that inhibition of SIRT4may be beneficial for the treatment of diabetes. By deacetylating carbamoyl-phosphate synthetase1(CPS1) SIRT5controls the initial step in the urea cycle and thus elimination of toxic ammo-nia. SIRT6is a nuclear protein, it deacetylates the adjustment of the end of the centromere chromatin H3K9. Knockout the SIRT6leads to a lack of chromatin and thus lead to the symptoms of premature aging and increased triglyceride synthesis and the formation of fatty liver and metabolic disorders. The SIRT6also regulates DNA repair mechanisms to contribute to the stability of the genome. SIRT7is thought to be involved in tran-scription by associating with RNA polymerase I (Pol Ⅰ) in the nucleus and was shown to be important for cell viability. SIRT7may ensure tissue homeostasis to prevent cardiac aging. Sirtuins and neurodegenerative disease is gaining attention.The Sirtuins activator include resveratrol、fistein、butein and so on, mainly through the activation of histone deacetylase activity to extend the life of the cell. The Sirtuins inhibitors include nicotinamide、sirtinol、splitomicin. The presence form of resveratrol:cis-resveratrol, trans-resveratrol and cis-resveratrol glucoside and trans-resveratrol glucoside. The latter two forms of glycosidases in the gut can be decomposed to release resveratrol, to play its pharmacological effects. Among them, the physiologically active of trans-isomer is stronger than the cis-isomer, the monomer activity greater than the glucoside. Trans-resveratrol under UV irradiation can be transformed into the cis-isomer. Resveratrol is widely present in the seed plants, at least12families and31genera,72species were found. The grapes, peanuts and Chinese medicine Polygonum cuspidatum are resveratrol-rich plants, in particular, the highest content in the fresh grape skin, and trans-dominant is predominance. Epidemiological survey found that the Frenchman of coronary heart disease and other cardiovascular disease morbidity and mortality were much lower than the Anglo-American countries, namely,(Franch. Poradox). The reason may be drinking the right amount of red wine effect. Further studies showed that play a major role in the ingredients of resveratrol in red wine. RES connected through the N-terminal and SIRT1to increase its activity to play its role. RES as the "longevity" drugs received extensive attention because of the RES and cell survival and survival. In this paper, the role of RES in neuronal apoptosis and provide evidence for the above hypothesis. In order to observe the SIRT1whether to participate in the role of PC12cells during apoptosis induced by LPS and the role of RES in the process, this article will be the following two-part experiment.Part I:Role of SIRT1protein in apoptosis of lipopolysaccharide-induced PC12cellsObjective:Explore the role of SIRT1in the LPS-induced apoptosis in PC12cells. Methods:The first set of experiments (to determine the concentration of the LPS injury)①Normal group:PC12cells were cultured normally, joined normal saline;②Experimental group:PC12cells were induced by LPS (concentrations of200μg/ml,250μg/ml,500μg/ml,1000μg/ml,1250μg/ml) for24h.The survival of PC12cells was analyzed by MTT.The second set of experiments (to observed the contaction of SIRT1and cell apoptosis)①Normal group:PC12cells cultured normal, normal saline;②Experimental group:PC12cells was indued by LPS (the concentration of1000μg/ml) for1/2h,2h,18h,24h,48hCell apoptosis was observed by Hoechst staining, flow cytometry at different time points; Expression of SIRT1was measured by Western blotting technologyResults:1.MTT results showed that different concentrations of LPS-stimulated PCI2cells can lead to decreased cell survival,200μg/ml and250μg/ml concentration on survival was not statistically significant (P>0.05). The cell viability decreased gradually as concentration increased, the survival rate (0.6267±0.02)%when concentrations of LPS was100μg/ml,it was the lowest survival rate (0.4533± 0.05)%when concentrations of LPS was1250ug/ml. Based on the results of this experiment the writer selected1000μg/ml concentration follow-up test.2.Hoechst staining results suggest that PC12cells undergo apoptosis since1/2h, showing pyknosis, nuclear fragmentation, apoptotic bodies increased from18h, peaked at24h,48hours and then declined;3.Flow cytometry measurement results show that the PC12cell apoptosis rate in each experimental group and control group were statistically significant (P<0.05), the apoptosis rate gradually increased with time, peaked24h,48h and then decline, consistent with Hoechst staining results of apoptosis in PC12cells4. Measurement of SIRT1expression was by Western blotting at each time point, the control group, the expression of (1.84±0.04), since the the1/2h start SIRT1protein expression has reduced (1.17±0.09),24h was the lowest (0.62±0.03), the expression of pick-up (0.77±0.02)since48h,difference of groups compared with the control group was statistically significant (P<0.05);Part Ⅱ:Role of resveratrol in LPS-induced apoptosis in PC12cellsObjective:The role of resveratrol in LPS-induced apoptosis in PC12cells.Methods:The experiment was divided into six groups, Group1of normal saline; the concentration of LPS was lmg/ml of group2;Concentration of LPS was lmg/ml, RES concentration was5μM in group3;LPS concentration was lmg/ml, RES concentration was10μM In group4;Concentration of LPS was lmg/ml, RES concentration was25μM in group5;DMSO concentration was the same of3,4,5group dissolved Res required for DMSO.1.Flow cytometry to observe the6cell apoptosis;2.Quantitative expression of SIRT1by Western botting technology Results:1.Flow cytometry results for2and3group was no significant difference,1and6were not statistically significant, comparison among the other groups were statistically significant.2.Western blotting was quantified by measurement of SIRT1expression at each time point, the difference of2and3was not statistically significant, the difference of1and6was not statistically significant, comparison among the other groups were statistically significant.Conclusion:RES (concentration of5μM) was no protective effect in LPS-induced apoptosis in PC12cells, the concentration (10μM,25μM) has a protective effect,the protective effect enhanced with the concentration of increased.