| Senile dementia is Alzheiner’s disease (AD), it is a neurodegenerative disease of the central nervous system with primary clinical feature of progressive cognitive and memorial dysfunctions. The hallmark pathologic feature of AD are neuronal loss senile plaques (SP) which is out of nerve cells and neurofibrillary tangles (NFT) which is in nerve cells. The prevalence of AD significantly increased with advanced age and its specific pathogenesis is still not clear. At present, there has no effective treatment method, so more deeply studies about the pathogenesis and the mechanism of anti-senile dementia drugs are required for new effective drugs to cure AD.Objective:1. In vitro, studies the influence of TEN on apoptosis, Tau phosphorylation and the ubiquitin-proteasome pathway (UPP) in PC12cell model induced by Aβ1-40.2. In vivo, studies the influence of TEN on learning-memory, apoptosis and the ubiquitin-proteasome pathway (UPP) in AD rats induced by AP1-40.Method:1. In vitro, PC12cell model was made by Aβ1-40(25μmol·L-1) and then co-cultured intervention with TEN(18.5,37.0,74.0mg/kg) for24hours. The expression of apoptosis related proteins (Bax,Bcl-2,Cyt-C,Caspase-9,Caspase-3), Tau protein phosphorylation-related protein (PKA, PP-2A, Tau(p-Ser396), total Tau) and UPP-related proteins (ubiquitinated protein) were detected by western blot; The levels of26S PSM were measured with enzyme-linked immunosorbent assay (ELISA).2. In vivo, the AD rat model was established by injection of Aβ1-40into the right hippocampus. Then the AD rats were given TEN (18.5,37.0,74.0mg·kg-1) by gavage for30days. Learning and memory ability was measured using Morris water maze; The apoptosis rate of cerebral neurons was detected by flow cytometry; The gene expression of bax,bcl-2,cyt-c,caspase-9and caspase-3was evaluated by RT-PCR; The levels of26S PSM were measured with ELISA; The expression of β-amyloid protein were assessed by immunohistochemistry; The expression of ubiquitinated protein and Tau(p-Ser396) was detected by western blot.Results:1. In vitro, compared with the normal control group, the expression of Bax, Cyt-C,Caspase-9and Caspase-3were increased while the expression of Bcl-2was reduced obviously(P<0.01) in the model group; Compared with the model group, the expression of Bax, Cyt-C, Caspase-9and Caspase-3were reduced while the expression of Bcl-2was increased in each dosage group after24h of treatment with TEN, and those differences were statistically significant(P<0.05, P<0.01, P<0.01).2. In vitro, compared with the normal control group, the expression of PKA, Tau(p-Ser396) and total Tau were increased while the expression of PP-2A was reduced obviously(P<0.01) in the model group; Compared with the model group, the expression of PKA, Tau(p-Ser396) and total Tau were reduced while the expression of PP-2A was increased in each dosage group after24h of treatment with TEN, and those differences were statistically significant(P<0.05, P<0.01, P<0.01).3. In vitro, compared with the normal control group, the expression of ubiquitinated protein and Tau(p-Ser396) were increased while the expression of26S PSM was reduced obviously(P<0.01) in the model group; Compared with the model group, the expression of ubiquitinated protein and Tau(p-Ser396) were reduced while the expression of26S PSM was increased in each dosage group after24h of treatment with TEN, and those differences were statistically significant(P<0.05, P<0.01, P<0.01).4. In vivo, compared with the normal control group, the average escape latency were obviously lengthened, the swimming distance was significantly increased and the times of passing through the platform were significantly decreased in the model group(P<0.01); Compared with the model group, the average escape latency were obviously shortened, the swimming distance was significantly decreased and the times of passing through the platform were significantly increased in the middle and high dose groups after30d of treatment with TEN(P<0.05, P<0.01), while the low dose group were no significant difference (P>0.05). 5. In vivo, compared with the normal control group, the apoptosis rate of cerebral neurons was significantly increased and the gene expression of bax, cyt-c, caspase-9and caspase-3was increased while the gene expression of bcl-2was reduced obviously(P<0.01) in the model group; Compared with the model group, the apoptosis rate of cerebral neurons was significantly decreased and the gene expression of bax, cyt-c,caspase-9and caspase-3was reduced while the gene expression of bcl-2was increased obviously in three treatment groups after30d of treatment with TEN(P<0.05, P<0.01, P<0.01).6. In vivo, compared with the normal control group, the expression of ubiquitinated protein, P-amyloid protein and Tau(p-Ser396) were increased while the expression of26S PSM was reduced obviously(P<0.01) in the model group; Compared with the model group, the expression of ubiquitinated protein, P-amyloid protein and Tau(p-Ser396) were reduced while the expression of26S PSM was increased in three treatment groups after30d of treatment with TEN(P<0.05, P<0.01, P<0.01).Conclusion:1. In vitro, TEN can regulate the expression levels of Bcl-2and Bax to change the mitochondrial membrane permeability, preventing Cyt-C from entering into cytoplasm, reducing the expression of Caspase-9and Caspase-3, and then protecting PC12cells from the toxic and apoptosis induced by Aβ1-40.2. In vitro, TEN can regulate the expression of PP-2A and PKA, reducing total Tau content and recovering the protein phosphorylation to the relatively low levels, and then protect ing PC12cells from the toxic induced by Aβ1-40.3. In vitro, TEN can relieve the inhibition of26S PSM, promoting ubiquitinated protein and hyperphosphorylation of Tau protein degradation, and then protect the ubiquitin-proteasome pathway from the toxic induced by Aβ1-40.4. In vivo, TEN can reduce nerve cells toxicity to improve learning and memory capacity in AD rats induced by Aβ1-40.5. In vivo, TEN can regulate the gene expression of bax and bcl-2, changing the mitochondrial membrane permeability, preventing cyt-c from entering into cytoplasm,reducing the gene expression of caspase-9and caspase-3, and then preventing nerve cells apoptosis through the mitochondrial apoptosis pathway.6. In vivo, TEN can relieve the inhibition of26S PSM, promoting ubiquitinated protein, β-amyloid protein and hyperphosphorylation of Tau protein degradation,and then protect the ubiquitin-proteasome pathway from the toxic induced by Aβ1-40. |
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