| Alzheimer’s disease is a progressive neurologic disease that results in the irreversible loss of neurons, particularly in the cortex and hippocampus. The disease is neuropathologically characterized by the presence of both neurofibrillary tangles and neuritic plaques. The amyloid β-peptide (Aβ) is the main component of the cholinergic neural functional disturbance in AD and is generated from P-amyloid precursor protein (APP) through sequential cleavages first by β-secretase and then by y-secretase complex. Alternatively, APP can be cleaved by a-secretase within the Aβ domain to release soluble APPa and preclude Aβ generation. In addition, the family of transcription factors NF-κB/Rel can specifically recognize APP κB sites, which are present in the5-regulatory region of the APP gene. The APP κB sites interact specifically with a complex which contains P50the subunits of the family, and that they act as positive modulators of APP gene transcription. The key to prevent and treat AD is inhibiting the production of APP and reducing the deposition of Aβ. Therefore, a-, P-, y-secretase and transcription factors NF-κB/Rel have long been regarded as therapeutic targets for AD in the development of inhibitor drugs for reduction of Aβ.Panax notoginseng saponins (PNS) is the active compound that is extracted from the root of Panax notoginseng (Burk.) F. H. Chen, family Araliaceae. Our previous study showed that PNS could effect a significant improvement in learning and memory performance in AD model rats, inhibit the deposition of AP1-40and Aβ1-42and reduce the content of APP in the brains of SAMP8, which itself was achieved by down-regulating the expression of the APP gene. However, the mechanism of the inhibition Aβ and APP by PNS is still unknown.OBJECT In this study, we aimed to investigate whether transcription factors NF-κB/P50, α-, β-and γ-secretase-dependent pathways are involved in Aβ generation in SAMP8treated with PNS, thereby providing a new opportunity for research with regard to the pharmaceutical prevention and cure of Alzheimer’s disease.METHODS1. SAMP8were randomly divided into four groups:PNS high-dosage group (200mg·kg-1), PNS low-dosage group (100mg·kg-1), huperzine A group (0.03mg·kg-1) and control group,15mice in each group. Moreover,10mice were used to test whether the model was successful by determining the capacity of spatial learning and memory before drug administration. All by intragastric administration, for8consecutive weeks.2. Morris water maze was used to determine the capacity of spatial learning and memory of SAMP8. Hematoxylin-eosin (HE) staining was used to evaluate the damage of hippocampus neuron of SAMP8.3. Transcriptional levels of genes involved with a-, P-and y-secretases and NF-κB/P50following induction of PNS were measured using real-time PCR assay.4. A fluorimetric assay and ELISA assay were used to investigate the effects of PNS on α-, β-and γ-secretases respectivly after treatments of PNS. 5. Effects of PNS on protein expressions related to α-,β-and γ-secretases were determined using western blot and immunohistochemistry.6. The activation of microglia cells in the brain of SAMP8was investigated using HE staining.7. ELISA assay was used to explore the serum level of cytokines IL-1β RESULTSPart I Effects of PNS on the capacity of spatial learning and memory of SAMP81. In place navigation trail, the results showed with increasing days, in mice treated with PNS and huperzine A, mean escape latencies were decreased and the residence time was prolonged. The percentages of total swimming distance in the platform quadrant in treatment groups were significantly increased than that in control group (P<0.01or P<0.05). The results of spatial probe test showed that mice treated with PNS could significantly prolong the residence time and distance of the original platform quadrant and swim more times in platform quadrant. These results showed that PNS could improve the mice’s capacity of learning and memory. Moreover, indicators in the model group were better than the control group, suggesting the mice’s learning and memory abilities declined with age and SAMP8model was successful.2. The histopathologic findings indicated that the pyknosis neurons in hippocampus decreased significantly compared to control group. The cone cells demonstrated in chaos and low density and the volumes of cells were decreased and nucleolus was shrinked in control group. While in treatment group, the neurons were increased and arranged orderly. These results showed that PNS plays a protective role in the damage of neurons in hippocampus.Part II Effects of PNS on the transcription of APP in the brain of SAMP81. The results of HE staining showed that microglia cells in hippocampus in control mice were activated and strongly stained. Moreover, the volumes were expanded. Nevertheless, following PNS treatment, there were nonactivated microglia cells in hippocampus. The cells were weakly stained and smooth with elongated axonal.2. The result of ELISA demonstrated that the content of IL-1β in blood serum was decreased after treatment swith200mg·kg-1PNS (P<0.01)3. The result of real time PCR showed that the transcriptional level of NF-κB p50mRNA was down-regulated after treatment with200mg·kg-1PNS (P<0.05)4. The results of western blot and immunohistochemistry showed that following treatment with PNS the immunopositive neurons and expression level of NF-κB p50were reduced compared to control group (P<0.01)Part Ⅲ Effects of PNS on the secretase related to A β metabolic pathways in the brain of SAMP81. Results of fluorimetric assay showed that PNS treatments significantly enhanced RFU values of a-secretase, as compared to control group (P<0.01). However, the values of RFU of β-secretase were significantly reduced in treatment groups, as compared to control group (P<0.01), while the values of y-secretase activity showed no significant difference between treated groups and control group (P>0.05)2. Steady-state mRNA levels of genes involved with α-(ADAM9, ADAM10and ADAM17), β-(BACE1) and γ-(PS1, PS2and Aph-1) secretases were measured using real-time PCR assay.①For α-secretase, transcriptional level of ADAM9was significantly up-regulated after rescue by high dosage PNS(200mg-kg-1(P<0.01). However, ADAM10and ADAM17levels were down-regulated following treatment with low dosage PNS (100mg·kg-1). On the contrary, we did not observed altered transcriptional levels of ADAM10and ADAM17after treatments with high dosage PNS as compared to control group (P>0.05)②For β-secretase, the transcriptional level of BACE1was remarkably down-regulated following the induction of PNS (P<0.01)③For γ-secretase, following PNS treatments, Aph-1was the only regulated gene showing a significant down-regulation compared with control group (P<0.05)3. Western blot assay and immunohistochemistry assay were used to detect the levels of α-(ADAM9, ADAM10and ADAM17), β-(BACE1) and γ-(PS1, PS2and Aph-1) secretase proteins in the brains of SAMP8.①ADAM9expression was dramatically enhanced after PNS treatment, as compared to control group (P<0.01). However, PNS treatment induced a significant reduction of ADAM10level (P<0.01). On the other hand, treatment with200mg·kg-1PNS decreased ADAM17level (P<0.05)②BACE1expression was significantly decreased after treatments with PNS, as compared to control group (P<0.01)③Treatment of PNS induced a significant increase of PS2level and had a dramatically reduction of Aph-1level (P<0.01or P<0.05)CONCLUSION1. PNS can improve the capacity of learning and memory of SAMP8, and PNS has a better curative effect of the capacity of learning and memory with a prolonged training period.2. PNS can decrease the damage of neurons in hippocampus of SAMP8.3. PNS can reduce the number of activated microglia cells, which results in the decreased level of IL-1β, and the decreased level of IL-1β recedes the stimulation of APP κB binding site which results in decreased the transcriptional and protein level of NF-κB/p50.4. PNS increases the transcriptional and protein level of ADAM9, which may lead to elevated activity of a-Secretase.5. PNS decreases the transcriptional and protein level of BACE1, which leads to reduced activity of P-Secretase.6. PNS treatment induces a significant reduction of Aph-1protein expression level via down-regulating Aph-1gene expression and the level of PS2expression is increased. Moreover, the levels of PS1protein and gene expression are not regulated by PNS. However, PNS does not have effects on y-secretase activity partially because y-secretase activity is dependent on the interaction with all four essential y-secretase components, even though PNS regulates Aph-1and PS2protein levels.Our studies demonstrate that the reduction of Aβ generation of SAMP8induced by PNS treatment is via down-regulating the expression of transcription factors NF-κB/p50which results in the decreased generation of APP, elevating the activity of α-secretase and reducting the activity of β-secretase. |
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