Protective Effect Of Lentinan On SH-SY5Y Cells Damaged By Hydrogen Peroxide

Alzheimer's disease (AD) is the most frequent neurodegenerative disease and the most common cause of dementia in humans. It is clinically associated with cognitive impairment, loss of language and motor skills, and changes in behavior. The disease is pathologically characterized by the presence of extracelluar senile plaques and intracelluar neurofibrillary tangles as well as the selective loss of neurons and synaptic connections. Several hypotheses have been proposed to explain Alzheimer's disease pathogenesis. But there is accumulating evidence that suggests a key role of oxidative stress in the pathophysiology of Alzheimer's disease.Lentinan (LNT), which is extracted from the lentinus, has been reported to have various pharmacological effects including the enhancement of immune function, anti-cancer, anti-virus, hepato-protective, antioxidative and anti-inflammation. Lentinan has been used in the adjuvant treatment of cancer and hepatitis, but its role in Alzheimer's disease remains to be further studied.In the present study, we examined the protective effect of lentinan on SH-SY5Y cells injured by hydrogen peroxide (H2O2) which is an exogenous free radical generating system. The results contribute to better understanding of the application of lentinan in Alzheimer's disease.1. Effect of lentinan on the cell morphology of SH-SY5Y cells injured by H2O2The cell morphology was observed by the inverted microscope. The SH-SY5Y cells had spindle-shaped, triangulate or polygonal cell body as well as short axons and dendritic-like apophysis. But the cellular body treated by H2O2 was corrugativus and even round. Compared with the model group, the morphology of cells protected by lentinan tended to be normal.2. Effect of lentinan on the viability of SH-SY5Y cells injured by H2O2The cell viability was measured by MTT assay. H2O2 decreased the viability of SH-SY5Y cells significantly, while lentinan relieved the cell damage induced by H2O2. The result suggested that lentinan protected SH-SY5Y from oxidative damage. 3. Effect of lentinan on the leakage of LDH in SH-SY5Y cells injured by H2O2The level of lactate dehydrogenase (LDH) in the cultured cell supernatant and lysate was measured by kit. Exposure to H2O2 for 12 h markedly increased cell membrane permeability and LDH leakage from cells into culture medium, while treatment of SH-SY5Y cells with various concentrations of lentinan inhibited H2O2-induced LDH leakage.3. Effect of lentinan on MDA content and SOD activity in SH-SY5Y cells injured by H2O2The malondialdehyde (MDA) content and superoxidase dismutase (SOD) activity were measured by kit. The MDA content of SH-SY5Y cells significantly increased after incubated with H2O2, which could be attenuated by the treatment of lentinan. But the SOD activity of SH-SY5Y cells notably decreased after treated with H2O2, while lentinan attenuated the H2O2-induced decrease of SOD activity. The results suggested that the protective effect of lentinan was exerted by alleviation of the oxdation damage.4. Effect of lentinan on the nuclear morphology of SH-SY5Y cells injured by H2O2The nuclear morphology was obseverd by the fluorescence microscope after using the chromatin dye Hoechst 33258. Cells injured by H2O2 obviously exhibited nuclear condensation, while the nuclear morphology of cells with the protection of lentinan tended to be normal.5. Effect of lentinan on the apoptosis rate of SH-SY5Y cells injured by H2O2The percentage of apoptosis was monitored by flow cytometry analysis after Annexin V-FITC/PI staining. The rate of apoptosis increased after treatment with H2O2, while lentinan reduced the apoptosis rate. The result indicated the protective effect of lentinan from cell apoptosis.6. Effect of lentinan on the expression of caspase-3 in SH-SY5Y cells injured by H2O2The expression of Caspase-3 was investigated by Western Blot method. The result showed that the level of Caspase-3 is significantly elevated in the H2O2 model group, while lentinan significantly decreased the level of Caspase-3. This indicated that H2O2 up-regulated the expression of Caspase-3, while lentinan inhibited it, which suggested that the anti-apoptosis effect of lentinan was exerted by inhibition of the expression of Caspase-3.