| BackgroundAlzheimer’s disease (AD), also known as senile dementia, is a degenerative disease of the central nervous system, which was susceptible to the disease in the elderly. According to the World Alzheimer’s Disease Association research, a population of people with AD will reach115million to2050in the world. AD, one of the three major killers, is a threat to the health of the elderly. Its characteristic is insidious onset and course of chronic progressive. The main symptoms are progressive memory impairment, cognitive dysfunction, personality changes, and the language barrier and other neuropsychiatric symptoms. Pathological changes characteristic of AD are mainly formed by deposition of amyloid β extracellular senile plaques and tau protein hyperphosphorylation formed nerve cells neurofibrillary tangles, as well as the main cholinergic neurons of the central nervous cell degenerate and loss associated with glial cell hyperplasia, and so on. Previous studies showed that oxidative stress directly involved in the pathogenesis of Alzheimer’s and other neurodegenerative diseases, and which is a vital cause in nerve cell damage. However, the etiologies of the above process are still not fully uncovered.Homocysteic Acid (HCA), identified as an N-methyl-D-aspartic acid (NMDA) receptor agonist, is an oxidized metabolite of Homocysteine which could induce neuronal death through activating the NMDA receptor subtype and an excitatory amino acid analogue of glutamate. HCA could not been prodeced and automatically generated in the body, which must be transformed from the food or Hey. Studies showed that the level of HCA and Hey in the body could be reduced though supplementary folic acid or vitamin B nutrients, but only partially reduced. Previous researches showed that plasma HCA levels increase the risk of AD, but the underlying mechanisms of HCA-induced toxicity is not yet fully understood.Berberine, also named as berberine hydrochloride, is a common alkaloids of isoquinoline in nature, and widespread in Berberis, Rutaceae and other plants. Not only has berberine hydrochloride been widely used in the treatment of gastroenteritis, dysentery and so on, but also it has a significantly efficacy in treatment of the tuberculosis, scarlet fever, tonsillitis and acute respiratory infections.. Previous studies showed that berberine may improve cognitive function in dementia risk populations, which may be mainly through the protective effect of oxidative stress in the central nervous cells to achieve. However, the effect of berberine on HCA neurotoxicity has not been reported. HCA has an obiviously neurotoxicity in nerve cells, but the mechanism of toxicity and whether berberine could inhibit the toxicity of HCA are unknown.ObjectiveThis research is divided into two parts. The first part of experiment was to investigate the effect of HCA to hippocampal neuronal cell viability at the cellular level. The mechanisms of toxicity of HCA was discussed by analyzing HCA-induced apoptosis and necrosis, the extent of oxidative stress, multifaceted impact on Bcl-2/Nrf2/caspase3signaling pathway and so on. The second part of experiment was to screen the protective Chinese monomers against HCA cytotoxicity, and discussed the mechanisms of protection of effective herbal monomer.MethodsPart1The immortalized mouse hippocampal neurons HT22cells were selected. To investigate the viability and morphology in HT22cells with or without HCA (0.1-2mM), the MTT assay was carried out at indicated time-points (6-24h). Detection of HCA-induced cell death pathways combine with MTT assay. Annexin V-FITC/PI assay kit for flow cytometric analysis, Hochest33258/PI fluorescent staining and western blotting of caspase3/Cleave-caspase3, Bcl-2/Bax pathway were used to detect apoptosis and necrosis. The degree of oxidative stress damage cells was determined by H2DCF-DA and DHE fluorescent dye, and western blotting of Nrf2/HO-1. The level of intracellular GSH was measured by a commercial glutathione assay. Mitochondrial membrane potential (ΔΨ) was monitore using Rhodamine123by flow cytometric analysis. Effect of different pathway inhibitors (SP600123, SB203580, PD98059) on HCA-induced cytotoxicity was detected by MTT assay and western blotting assay.Part2Common used monomers for anti-oxidative damage from Chinese herbs, including Berberine, Ligustrazine, Cinnamic acid, Cinnamaldehyde, Coffee acid and Coffee acid phenethyl ester, were selected by MTT assay. According to the literature concentration, HT22cells were pretreated with these monomers for30min and then incubated with1mM of HCA. A traditional Chinese medicine monomer with the significant protective effect was screened by MTT assay. Dose-effect relationship of effective medicine monomer will be analyzed further. The effect of Chinese medicine monomers inhibit the production of the intracellular oxygen free radicals by HCA-induced, which was measured by fluorescent dye. The level of intracellular GSH was measured by a commercial glutathione assay. Mitochondrial membrane potential (ΔΨ)was monitored using Rhodamine123by flow cytometric analysis. The effect of monomer combined with different pathway inhibitors restrained HCA-induced cytotoxicity was measured by MTT assay, and then western blot assay showed that proteins Bcl-2, JNK, Nrf2, Akt appear to a role in berberine-mediated neuroprotection.ResultsPart1This part of the study found that HCA decreased HT22cells viability in a dose-and time-dependent manner, but did not activate Caspase-3. Additionally, HCA increased ROS production, depleted GSH, inactivated the Nrf2/HO-1pathway, decreased mitochondrial membrane potential and increased the ratio of Bax/Bcl-2, two apoptosis-related proteins. Next, HCA significantly increased the levels of p-JNK and p-c-Jun and its toxicity dramatically attenuated by SP600125, a specific JNK pathway inhibitor.Part2This part of the study found that berberine, a chinese monomer, extremely attenuated the cytotoxicity of HCA in a dose-dependent manner. Berberine had no cytotoxicity at the concentration of100μM in HT22cells but it only inhibited cells growth. Berberine significantly attenuated the cytotoxicity of HCA by decreasing the production of ROS, but failed to increase intracellular the GSH level. The ratio of Bax/Bcl-2, the expression of Nrf2and HO-1, and the level of p-c-Jun and p-JNK were not altered after pretreatment with berberine in the presence of HCA in HT22cells, but the expression of p-Akt was activated.Conclusion1. Our studies confirmed that HCA induced HT22cells death in concentration-and time-dependent manners by inducing oxidative stress in hippocampal neurons lead to cells death and the pathological changes of plurality of oxidative stress-related proteins. The changes of proteins are likely closely related to the degree of prevalence of AD.2. The results demonstrated that HCA might induce neuronal apoptosis or necrosis via increasing the level of intracellular ROS and leading to oxidative stress damage, depleting of intracellular GSH, accumulating the oxygen free radical in cells, decreasing the ratio of Bcl-2/Bax; Simultaneously, it also made the classic antioxidant pathway-Nrf2/HO-1inactivation, decreased expression of Nrf2/HO-1. In addition, HCA activated the JNK pathway and increased expression of p-JNK/c-Jun.3. Berberine significantly inhibited the neurotoxicity of HCA, the mechanism of protection of berberine might be inhibiting the level of intracellular ROS, reducing oxidative stress damage to cells, activating the pathway of Akt. However, the exact mechanism is unclear. |
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