Alzheimer disease (AD) is a progressive neurodegenerative disease, accounting for 50–75% of all dementia patients worldwide (http://www.alz.co.uk/). However, their diagnoses are often missed or delayed due to the lower prevalence of dementia in younger people and large variability in etiologies [17, 18].. And, non-effective drugs against AD in market approved accumulate the serious situation, which puts the prevention and treatment into an impending place . Then, early this year, FDA proposed that the drug development and diagnosis of AD should be focused on the early stage before the onset of overt dementia .
TCM has a long history in oriental countries. Preventing Disease, an essential consideration of TCM treatment strategy, emphasizes the conception of prevention and early treatment of diseases. Based on this thinking, for the past ten years, we have been tried to find and explain the neuroprotective role of TCM in AD. According to previous studies, in vitro, we found that GP and PNS combination alleviated the damage of ischemia/reperfusion neurons by promoting the secretion of active substances from brain microvascular endothelial cells (BMECs) ; under oxygen/glucose-deprivation condition, the combination protected both primary rat hippocampal neurons and BMECs from cell death ; again, under formaldehyde stress, TLJN inhibited the cell apoptosis by modulating the expression of Bcl-2, P53, caspase 3 and caspase 9, and increasing the activity of intracellular superoxide dismutase and glutathione peroxidase (9, 10). These data suggest that it can prevent neurons from cell death, but also can protect BMECs. In vivo, to further verify its efficacy on learning and memory, we used a rat model and three other different AD Tg mice. In rat, after Aβ25−35 injection into the bilateral hippocampus CA1 areas, we found that GP and PNS prolonged a cavity delitescence, decreased arm entries in Y-maze test, and reduced amyloid plaques in rat brain by up-regulating insulin-degrading enzyme and neprilysin levels ; in APP/V717I Tg mice, GP and PNS improved their spatial memory deficits in both Water-Maze test and inhibitory avoidance test (Step-down test) at ten-month age ; in APP/PS1 Tg mice, we found that it improved the spatial learning and memory in Water-Maze test and reduced amyloid plaques in brain at 10-month age (data have not published yet); in APP23 Tg mice, we observed an inhibitory effect of the combination on amyloidogenic APP processing by down-regulating the cleavage enzymes BACE1 at twelve-month age . These evidence showed that GP and PNS can improve learning and memory in different AD Tg mice, and reduce the deposition of amyloid plaques.
Based on these studies, we continued to investigate its neuroprotective role in an early stage. We used APP/PS1 Tg mice. These mice express human APP (695 Swedish mutation) and a mutant human presenilin1 (PS1-dE9). Both mutations are associated with early-onset AD. They develop beta-amyloid deposits in brain by six to seven months of age , but their memory deficits usually occurred at the age of nine-month and older . APP/PS1 Tg mouse is one of the most widely used AD-like animal, due to the extensive Aβ deposition and synaptic deficit with aging, accompanied with memory deficits. At 4-months old of APP/PS1 Tg mice, there is an approximately 30% reduction in spine density as well as about 20% decrease in both dendritic area and dendritic diameter in the transgenic mice as compared with wild-type littermates . In addition, Aβ accumulation in APP mutant neurons reduces PSD-95 and AMPA receptor (GluR1) in synapses . Then, by using young APP/PS1 Tg mice, we focused on the prevention started from one-month age, lasting for four-month age, which is before formation of amyloid deposition.
At the early stage, we firstly detected the production of Aβ. Both Aβ1−42 and Aβ1−40 are significantly increased in APP/PS1 Tg mice at four-month age. The ratio of Aβ1−42/Aβ1−40 is significantly increased as well compared with WT group. Secondly, to detect its morphology in hippocampus CA1 areas, we found the pyramidal cell layer is severe condensed with uneven thickness in Tg group compared with WT mice. in addition, according to clinical studies, the loss of synapses has been considered as one of the most reliable index of cognition deficits in both postmortem and biopsied AD brain ; and amyloid deposition or Aβ peptide (either insoluble Aβ or soluble Aβ species) is associated with synaptic abnormalities, such as dendritic spine loss , breakage of neuronal branches, spine dysfunction and collapse . We further investigated the change of synaptic plasticity. We found that the number of dendritic spines is significantly reduced in Tg mice at four-month age; again, the protein level of PSD95, a synaptic-related protein, is significantly reduced; in hippocampus DG areas, the number of AMPA receptor positive cells is significantly reduced as well. These data suggest that APP/PS1 Tg mice exhibited a neurodegeneration processing at an early age (four-month age) before the formation of amyloid plaques and cognition impairment occurred. Then, under the early treatment, GP + PNS significantly decreased the ratio of Aβ1−42/Aβ1−40 ratio in both hippocampus and cortex. Interestingly, in hippocampus, the production of Aβ1−42 was reduced under TLJN treatment; while in cortex, it was the production of Aβ1−40 significantly increased. Secondly, we focused on the changes of synaptic plasticity. We found that the number of dendritic spines in hippocampus CA1 areas was significantly increased in GP + PNS group compared with Tg group. In addition, due to detect synaptic plasticity, we examined the expression level of synaptic-related protein, such as PSD-95. Our data showed that the combination has an impact on increasing the protein level of PSD-95. These data suggest that GP + PNS can maintain the delicate balance of Aβ production in APP/PS1 Tg mice to reduce the ratio of Aβ1−42/Aβ1−40 in both hippocampus and cortex; and it can enhance synaptic plasticity at four-month age of APP/PS1 Tg mice (Fig. 5).
In this study, we also used Aricept as a positive control group, since it is the first drug approved by FDA to treat AD in clinic. Aricept (also called donepezil), as a cholinesterase inhibitor, leads to a temporary slowdown in the loss of cognitive function by decreasing cholinesterase activity, resulting in higher acetylcholine (Ach) levels and improved brain function with mild-to-moderate AD patients [27, 28]. However, we did not observe evident effects of Aricept on APP/PS1 Tg mice at four-month age. Firstly, Aricept did not significantly reduce the ratio of Aβ1−42/Aβ1−40 in both hippocampus and cortex; secondly, in terms of synaptic plasticity, Aricept did not significantly increase the number of dendritic spines in hippocampus CA1 areas compared with Tg mice; and, the protein level of PDS-95 was further decreased compared with Tg mice. There might be there reasons: 1) Aricept is a cholinesterase inhibitor. In this study, we did not focus on the change of Ache level. But in terms of Aβ production, at an early age of APP/PS1, before the formation of amyloid plaques, Aricept did not show an evident effect to maintain the balance of Aβ production; 2) again, in terms of synaptic plasticity, before the cognition impairment occurred, Aricept did not show a positive role to enhance the health of synapses; 3) according to clinic studies, Aricept cannot stop the progressive loss of neurons or halt the progressive deterioration of cognitive faculties in AD patients . This may provide us a hint that this limitation might be a hinder to its efficacy in an early stage. In addition, the side effects of Aricept should be matters, since it significantly increases ratings for vigour and anxiety symptoms .
To summary, preventing Disease is an essence strategy in TCM for treatments, especially in chronic disease and complicated diseases. Base on previous studies, in this experiment, we highlights the neuroprotective role of GP and PNS combination in an early stage. Before the formation of amyloid plaques, we found that GP and PNS can maintain the delicate balance of Aβ production to reduce the ratio of Aβ1−42/Aβ1−40 in both hippocampus and cortex at four-month age of APP/PS1 Tg mice; before the cognition impairment occurred, the combination can enhance synaptic plasticity at an early age. These results suggest that the administration of GP and PNS in an early stage of APP/PS1 Tg mice can protect neuron damage from Aβ accumulation and promote the health of synapses.