The Effects Of Low Molecular Weight Chondroitin Sulphate In Preventing And Curing Amyloid Beta (Aβ)-induced Neurotoxicity And The Mechanisms

Alzheimer’s disease (AD) is a progressive, age-related, neurodegenerative disorder characterized by observable memory impairment or cognitive decline. The distinctive pathophysiology of this disease is the accumulation of amyloid beta (AP) in senile plaques (SP), the presence of intracellular neurofibrillary tangles (NFTs), and a reduction in brain volume due to neuronal loss. It is in agreement with that Aβ is the main component of SP, a characteristic neuropathologic hallmark in AD. A large number of evidence has demonstrated that Aβ induced neurotoxicity in vitro and in vivo, thus, Aβ plays roles in the drug discovery of AD.Low molecular weight chondroitin sulphate (LMWCS), derived from chondroitin sulphate (CS) after oxidative degradation, was concentrated by ultrafiltration using membranes with molecular weights cut-off of 2000 and 5000 Da. The fractions were named as LMWCS-A, B, C (the fractions of M<2000 Da、 2000-5000 Da、>5000 Da) and D (the total fraction after oxidative degradation). The average molecular weights of them were 1173 Da,3928 Da,6146 Da and 5846 Da.As a candidate of anti-AD agents, the aim of the present paper was to investigate the protective effects of LMWCS on Aβ-induced damage in neural cells and AD model mice, and to explore the underlying molecular mechanisms of its neuroprotective action.1. Study on the protective effects and mechanisms of LMWCS on the nerve cell injuries induced by AP25-35In the experiments, the effects of different LMWCS fractions on the neurodamage induced by Aβ25-35 in pheochromocytoma PC12 cells and neuroblastoma SH-SY5Y cells were evaluated with the MTT assay. The viabilities of PC 12 and SH-SY5Y cells were significantly decreased compared with the control groups after exposure to A025-35-LMWCS inhibited the damage induced by Aβ25-35 in both PC12 and SH-SY5Y cells. When the cells were pretreated with LMWCS of different concentrations (50,100 and 200 μg/ml) for 24 h, and then exposed to Aβ25-35 for 24 h, the damage decreased, and the cell viabilities were both significantly dose-dependently increased. In particular, LMWCS-B caused the highest cell viability compared with other fractions. These datas suggested that LMWCS was able to block Aβ25-35-induced cell damage in a dose-dependent manner, and LMWCS-B was the most effective fraction. Therefore, LMWCS-B was used to post-experiment.In addition, the effect of LMWCS on the neurotoxicity induced by Aβ25-35 was evaluated by three treatment modes with LMWCS, pre-treatment with LMWCS, treatment with LMWCS followed by addition of AP25-35, incubation with both LMWCS and Aβ325-35-It was found that LMWCS increased the cell viability, and cell viability of the pre-treatment with LMWCS was higher than that of other treatments in the same concentration. These results showed that LMWCS might inhibit the neurotoxicity of Aβ25-35 via different pathways.The above observation revealed that Aβ25-35 triggered the neurotoxicity in PC 12 and SH-SY5Y cells. To examine whether the Aβ25-35-induced cell death of PC12 and SH-SY5Y cells in our studies occurred by an apoptotic-like mechanism, flow cytometry analysis was used to measure the relative numbers of cells. The results showed that Aβ25-35 significantly increased apoptotic death of PC 12 and SH-SY5Y cells, and pre-treatment of cells with LMWCS for 24 h prior to Aβ25-35 exposure dose-dependently decreased the apoptotic rate. The anti-apoptosis mechanisms of LMWCS on the apoptosis induced by Aβ25-35 were further investigated. The results showed that pre-treatment with LMWCS for 24 h reduced the increased intracellular calcium concentration level and the generation and accumulation of free radicals induced by Aβ25-35 remarkably. LMWCS also improved the mitochondrial membrane potential stimulated by Aβ25-35 because LMWCS reduced the percentage of low mitochondrial membrane potential cells remarkably. Furthermore, LMWCS reversed the reduction of Bcl-2, an anti-apoptotic protein, and increased the expression of Bax and Caspase-9,-3, the promoting-apoptotic proteins. Thus, studies have demonstrated that the neuroprotection of LMWCS against Aβ might be through inhibiting the apoptotic pathway.2. Study on the protection effects and mechanisms of LMWCS on the learning and memory loss induced by Aβ1-40During in vivo experiments, to evaluate the anti-Aβ effect of LMWCS, open field test (OFT) and Morris water maze (MWM) were used to measure spatial learning and memory in mice induced by injecting Aβ1-40 into the cerebral ventricles. Moreover, the possible molecular mechanisms of LMWCS were investigated on cellular and molecular levels in the Aβ1-40-treated mice. The results showed that the spatial learning and memory of mice was damaged by Aβ1-40, however, the administration of LMWCS (50,150 and 450 mg/kg) increased the numbers of rearing, grooming behaviour and locomotor activity in the OFT, and shortened the escape latency, swimming distance, first time to original platform, latency in the probe trial, distance and times in the target quadrant in the MWM. Thus, these results demonstrated that LMWCS improved the ability of spatial learning and memory in Aβ1-40 injected mice.The mice were killed by decapitation immediately after the MWM. Brains were quickly removed on ice, and the cerebral hippocampus and the cortexes were homogenized in tubes using a homogenizer. A spectrophotometry method was used to assay the level of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), Na+/K+ATPase and acetylcholinesterase (AChE), choline acetyltransferase (ChAT), according to the description of the assay kits. The results showed that LMWCS increased the level of ChAT, SOD,GSH-Px and Na+/K+ATPase, and decreased the level of MDA and AChE in the mouse brain. These results suggested that LMWCS could improve the oxidative stress and cholinergic system level induced by Aβ1-40 in mice and maintain the stability of the energy metabolism in mice.Furthermore, obvious hippocampal histopathological damage was observed in the Aβ1-40 injected mice. The pyramidal layered structure was disintegrated, and neuronal loss was found in the CA1 region. However, LMWCS decreased the density of pyramidal cells of CA1 regions, reduced the hippocampal histopathological abnormalities induced by the Aβ peptide. Moreover, immunohistochemical staining suggested that LMWCS reduced the protein expression of Caspase-9,-3 and decreased the number of positive cells in the hippocampus of mouse brain. Moreover, Western blotting results showed that LMWCS had anti-apoptotic effect, LMWCS decreased the apoptotic-promoting protein expression of Bax, Caspase-9,-3, increased the anti-apoptotic protein expression of Bcl-2 in the hippocampus of mice, and ultimately reduced apoptosis induced by Aβ1-40.Thus, the neuroprotective mechanisms of LMWCS may be related to reducing neurotoxic effects and inhibit Aβ1-40 induced apoptosis.In conclusion, this is the first report to evaluate the protective effects of LMWCS against apoptosis induced by Aβ both in vitro and in vivo. LMWCS protected the PC 12 and SH-SY5Y cells from AP25-3s-induced neurotoxicity and reversed cell apoptosis by decreasing the intracellular calcium concentration level, inhibiting production of free radicals and mitochondrial dysfunction, and blocking the expression of apoptosis-promoting proteins. During in vivo experiments, it was observed that LMWCS improved the cognitive impairment in behavioural tasks, increased ChAT, SOD, GSH-Px and Na+/K+ ATPase activity, reduced AChE activity and MDA level, increased the number of pyriform cells in the hippocampus of AD model mice, and reduced the concentration of apoptotic proteins in the hippocampus of mouse brains. These findings suggest that LMWCS may be a promising new therapeutic agent for the treatment of AD.