Effect Of Nicotinic Agonist And Antagonist On Nerve Cell Injury Induced By β-Amyloid Peptides

The incidence and prevalence of Alzheimer's disease(AD) are increasing rapidly along with ageing in the world.β-amyloid peptides play a critical role in AD, and result in the lost of neurons, especially the pre-synapse neurons, meanwhile, nicotinic receptors are decreased obviously too. Some researchs found that there is interaction betweenβ-amyloid peptide and a7 nicotinic acetylcholine receptor(a7nAChR) with high affinity, and it could serve as a pathogenic factor in the formation of amyloid deposit in neurons. At present, cholinesterase inhibitor is the most important medicine in treating AD by increasing concentration of acetylcholine in brain through suppressing it's catabolism. However, its effect disappeared with extending use, and is not better than placebo at last. As a nicotinic agonist, nicotine show neuroprotection in short-term use, but this effects is uncertain in the long-term use. Generally, nicotinic antagonist is considered to have no neuroprotection in a short-term use, and the research about it's long-term effects is seldom. Both AD and chronic heart failure (HF) possesed the similar characters of receptor over-activation and apoptosis and down-regulation of receptors. Adrenergic beta-agonists can improve the symptom of heart failure in short-term, but it can deteriorated the heart function and increase mortality latter. On the contrary, adrenergic beta-antagonists become the most important treatment for HF. Now then there were some problems with nicotinic agonist treating with AD chronically, so what kind of effect can be produced by nicotinic antagonist with long-term use. Therefore, this study will investigate ifα7 nicotinic antagonist has neuroprotection in long-term use in PC12 cells and rat neurons.Part I Effect of Nicotinic Agonist and Antagonist on PC12 Cells Injury Induced byβ-Amyloid PeptidesObjectiveTo investigate the effect of niconitic agonist and antagonist on PC12 cells injury induced by Aβ25-35 and to explore the neuroprotective effects of a7 nicotinic antagonist in the long-term.Methods1. The PC12 cell injury model was established by treating cells with Aβ25-35, and nicotine and methyllycaconitine were used to pretreat before Aβ25-35 injury.2. The experimental groups were divided as follows:Control; Aβ25-35; Nicotine+Aβ25-35; Methyllycaconitine+Aβ25-35. 3. The viability of PC12 cells was detected by MTT chromatometry at 36,48,60,72 and 84 h after treatment.4. The apoptosis of PC12 cells were detected at 36 and 84h with Hoechst staining and assay of flow cytometry.Results1. The viability of cells in Methyllycaconitine+Aβ25-35 group increased relatively, but the viability of cells in nicotine+Aβ25-35 group decreased along with the extending use.2. Methyllycaconitine demonstrated a suppressive effect of apoptosis, but the anti-apoptotic effect of nicotine gradually disappeared along with the extending use.ConclusionsMethyllycaconitine had no protective effect on PC12 cells used in a short period, but it had a tendency of protection by bloking the binding between Aβ25-35 andα7nACR in a long time. In spite of protective effect of nicotine on PC12 cells by anti-injury of Aβ25-35 through other ways early, but it's protection gradually disappeared along with the extending use.PartⅡEffect of Nicotinic Agonist and Antagonist on nAChR Function in PC12 Cells Injury Induced byβ-Amyloid PeptidesObjectiveTo investigate the effect of nicotinc agonist and antagonist on nAChR function in PC12 cells injury induced by Aβ25-35 in extending use on the basis of previous study.Methods1. Experimental groups were divided as before. 2. Detections were carried out at 36 and 84h after different treatments. Confocal microscopewere used to assay the basal and activated concentration of Ca2+in PC 12 cells after eluting themedcines and calcium staining with Fluo 3/AM.Results1. Aβcaused Ca2+ overload at 36h, however the concentration of Ca2+ decreased potentially due to the worsen condtion of cells at 84h in PC12 cells; simultaneously nAChR function was decreased obviously.2. Methyllycaconitine reduced Ca2+ overload at 36h and 84h, simultaneously increased nAChR function, and this effect is superior to nicotine.3. Nicotine didn't reduce Ca2+ overload at 36h and 84h, and promoted the development of Ca2+ overload lately. Nicotine can enhanced the nAChR function, but showed lower effect than methyllycaconitine lately.ConclusionsMethyllycaconitine had the effect of relieveing Ca2+ overload and enhanced nAChR function through effect of protective on PC12 cells or upregulaion of nAChRs. Nicotine didn't reduce Ca2+ overload, even promoted the development of Ca2+ overload lately. Although nicotine can enhance nAChR function, probably through other ways of protection on PC12 cells or upregulaion of nAChRs, but its effect was lower than methyllycaconitine lately.PartⅢMechanism of Nicotinic Agonist and Antagonist on PC12 Cells Injury Induced byβ-Amyloid PeptidesObjectiveTo explore the mechanism underlied the neuroprotective ofα7 nicotinic antagonist by studying the regulation ofα7 nAChR and the signal pathway of apoptosis in PC12 cells.Methods1. Experimental groups were divided as before.2. Analysis of mitochondrial membrane potential was used to detect apoptosis of PC12 cells. qRT-PCR was used to detect the mRNA levels of bcl-2/bax and caspase 3, and western Blot was used to detect Bcl-2/Bax andα7 nAChR at 36h and 84h after different treatments.Results1. The effect on PC12 cell injury induced by Aβ25-35 and the anti-apoptotic effect of nicotine and methyllycaconitine were relevant with mitochondrial apoptosis pathway.2. The proteinic expression ofα7 nAChR was positively correlation with Bax at the presence of Aβ25-35 which suggests that PC12 cell injury induced by Aβ25-35 was possibly mediated throughα7 nAChR.3. Methyllycaconitine inhibited the expression of caspase 3 at mRNA level at 36h and 84h, and increased the mRNA expression of bcl-2/bax at 84h. Nicotine had no effect with the the expression of bcl-2/bax and increased the mRNA expression of caspase 3 at 84h.4. Methyllycaconitine increased the expression of Bax-2/Bax at 36h and 84h. Both of methyllycaconitine and nicotine downregulated the expression ofα7nAChR, and methyllycaconitine showed a more obvious effect.ConclusionsPC12 cells injury induced by Aβ25-35 was possibly mediated throughα7nAChR, and methyllycaconitine probably inhibited apoptosis of PC12 cells by improving bcl-2/bax and lowering caspase 3 through down-regulation ofα7 nAChRs and blocking the injury of Aβ25-35.PartⅣVerification of Effect of Nicotinic Agonist and Antagonist against Injury induced byβ-Amyloid Peptides in Rat NeuronsObjectiveTo explore the neuroprotective effect ofα7nicotinic antagonist in rat neurons and further verify the results in PC12 cell researchs.Methods1. The injury models were carried by Aβ25-35 in rat neurons, and methyllycaconitine and nicotine were used to pretreatment.2. Experimental groups were divided as before.3. The viability of rat neurons was detected by MTT chromatometry at 3,7,14,21 day after treatment.4. The apoptosis and necrosis of rat neurons were detected at 21 day.Results1. Nicotine increased the viability of rat neurons at 3 and 7 day and has no effect on neuron viability at 21 day, but methyllycaconitine showed the effect of improving viability of rat neurons at 21 day.2. Nicotine inhibited the apoptosis and necrosis of rat neurons at day 3 and 7 day and has no effect on the apoptosis and necrosis of rat neurons at 21 day, but methyllycaconitine showed the inhibition of apoptosis and necrosis at day 21 day.ConclusionsMethyllycaconitine had no protection on rat neurons in a short period, but it had a protection with in the long-term by suppressing apoptosis and necrosis obviously. Then the reversal is for nicotine.