The major finding of the present study was that pretreatment with the CDT prevented the spatial memory impairment and neuronal damage induced by i.c.v. injection of Aβ25-35. The mechanism of these neuroprotective effects may relate to elevated ChAT, RACK1 levels, and restored the balance between cytokines (IL-6 and TNF-α) and neurotrophins (BDNF) in the brain.
In Alzheimer’s disease, amyloid β (Aβ) protein is the main composite of senile plaques. Intracerebroventricular infusion of the active fragment of Aβ protein, Aβ25-35, has been shown to induce spatial learning and memory deficits in AD animal models [20, 21]. Our results are consistent with previous reports that i.c.v. injection of Aβ25-35 induced significant spatial memory impairment measured by Morris water maze test . In this study, we found that the escape latency was longer, and both of the time spent in the target quadrant and the numbers of platform crossings were shorter in the model group than those in the control group. Treatment with LCDT not only decreased the mean escape latency but also increased the time spent in the target quadrant and the numbers of platform crossings. Thus, it was reasonable to believe that LCDT could ameliorate the spatial memory impairment in Aβ25-35 treated mice.
The central cholinergic system plays an important role in learning and memory processes . Loss of cholinergic neurons due to the neurotoxicity of Aβ, especially in hippocampus, is the major neuropathological feature that is associated with memory loss in AD [24, 25]. Currently, the main clinical strategy is to increase ACh levels, modulate glial activation, cerebral blood flow, the amyloid cascade, and tau phosphorylation in brains with AD disease by acetylcholinesterase inhibitors . However, choline acetyltransferase (ChAT) is one of the specific cholinergic marker proteins for monitoring the functional state of cholinergic neurons in the central nervous systems is ChAT, the biosynthetic enzyme for Ach . Activation of ChAT could ultimately lead to synthesis sufficient Ach, may serve as a strategy for the treatment of memory impairment. In consideration of the main composition of CDT, Savia miltiorrhiza, is the novel acetylcholinesterase inhibitors . The current work displayed that LCDT not only has a notable neuroprotective effect in Aβ25-35 treated mice, especially in CA3 of hippocampus and cortex via inhibiting the neuron damages but also significantly increased the ChAT protein level in hippocampus and cortex of Aβ25-35 induced mice. Thus, these results indicate that LCDT could be an effective approach for attenuating the neurotoxicity induced by Aβ25-35 via modulation of ChAT protein.
Neuroinflammatory responses of the central nervous system (CNS) are well-known features of Alzheimer’s disease (AD). Aβ not directly cause neuronal cell death but activate microglial cells to produce inflammatory factors. In turn, proinflammatory cytokines such as TNF-α and IL-6 had also been shown as an amplifier in the amyloid cascade process [28, 29]. Overproduction of IL-6 and TNF-α were related to memory impairment [30, 31], which were considered a histopathological hallmark of various neurological diseases in the brain . Since hippocampus is more susceptible to these cytokine-induced inflammations , we analyzed the mRNA and protein levels of IL-6 and TNF-α in hippocampus and found that CDT (0.81 g/kg) administration prominently inhibited the production of pro-inflammatory mediators such as IL-6 and TNF-α. Thus, we suggest LCDT as a potential neuroprotective agent to prevent and treat neuroinflammation.
Recently, increasing evidence has showed that neuroinflammation may trigger neuroprotection or neurodegeneration through the neurotrophic system, such as BDNF in acute and chronic neurodegenerative. In AD, over-stimulated microglia but suppressed astrocyte functions resulted in the decrease of BDNF . So imbalance of cytokines and neurotrophins has been put forward as the new mechanism of AD . On the other hand, receptors for activated C kinase1 (RACK1), a family of proteins involved in anchoring activated PKCs to relevant subcellular compartments, is also deficient in the brain of alzheimer’s disease patients . Presently study indicated that nuclear RACK1 localizes at the promoter IV region of the BDNF gene to regulate the expression of the BDNF gene . To certain the effect of CDT on RACK1/BDNF signal pathway in AD mice, we further studied the mRNA and protein expression of RACK1 and BDNF in hippocampus of Aβ25-35-induced mice. Evidently, contrary to the anti-inflammatory effects, RACK1 and BDNF were shown to be lowered by Aβ25-35 treatment compared to the levels observed in sham mice. CDT treatment (0.81 g/kg) greatly rescued the Aβ25-35-induced RACK1/BDNF signal pathway in hippocampus. Thus, LCDT-mediated the modulation of RACK1/BDNF in hippocampus might contribute to neuroprotective effects of LCDT. Simutaneously, in present study, we found 0.81 g/kg CDT treatment significantly rescued the imbalance of cytokines (IL-6 and TNF-α) and neurotrophins (RACK1 and BDNF). We think this is due to over-expression cytokines (IL-6 and TNF-α) over activated microglia and resulted in the decrease of RACK1/BDNF signal pathway. Therefore, CDT restoration of RACK1/BDNF levels by inhibiting the cytokines levels.