The Effect Of Evodiamine On The HMGB1/TLR4/NF-κB Signaling Pathway Of The Rats With Alzheimer’s Disease

Objectives: Alzheimer’s disease(AD) is a progressively neurodegenerative disease of the central nervous system, the major clinical manifestations are progressive loss of memory and other cognitive functions, and the changes of emotion, personality and behavior. The pathogen and mechanism of AD are not quite clear now and it has been the forth disease that causes death, what’s more, the morbidity and mortality of AD is gradually increased, which ADI(Alzheimer’s Disease International, ADI) estimates the incidence of AD in our country will increase in a proportion of 336%, and China’s dementia patient will be equal to the sum of developed country’s AD patient(more than 20 million). Therefore the research of prevention and treatment of AD has become hot topic around the world.Now there are several different theories tries to elucidate its etiology: Cholinergic hypothesis, Amyloid hypothesis and Tau hypothesis and so on. With the development of research, numerous scholars consider that the inflammation induced by Aβ1-42 deposit is the main mechanism of AD. It will be an important approach for treating AD to alleviate the inflammation lesion.High-mobility group box 1(HMGB1), which originally is characterized as a nuclear DNA-binding protein with a highly conserved structure in several species, participates in the formation of nucleosome and the regulation of gene transcription. But when cells became injured or underwent necrosis, HMGB-1, which normally residing in nuclei, translocated to the cytoplasm and/or extracellular space. Recently, HMGB1 has been described as a key cytokine to play an extracellular role when it is involved in cellular activation and proinflammatory responses. HMGB1 itself may signal through the receptor for advanced glycation end products(RAGE) and toll-like receptors like TLR2 and TLR4. The importance of this protein in physiologic and pathologic states is underlined by the activation of these receptors resulting in the activation of nuclear factor κB(NF- κB), which induces the up-regulation of leukocyte adhesion molecules and the production of proinflammatory cytokines in both hematopoietic and endothelial cells, thereby, promoting inflammation.Evodiamine(Evo), an alkaloidal component extracted from the fruit of Evodiae-fructus(Evodia rutaecarpa Benth, Rueaceae) has vast pharmacological activities. Including anti-inflammation, antitumor, retarding developing of atherosclerosis and antinociceptive effects. For treatment, multiple researches reveal Evo can improve AD patient’s cognitive function. It is an inhibitor of nuclear transcription factor k B(N F- k B) which has antiinflammation bioactivities thus inhibiting the transcription of COX-2 m RNA. As a result, it inhibits the occurrence, development and deterioration of the inflammatory response. However, whether Evodiamine has the neuroprotection by down-regulating the HMGB1, TLR4 and NF-κB is not clear. The purpose of this study was to evaluate the potential neuroprotection of Evodiamine and the possible mechanisms in rats thus to probe the mechanism of AD. In this study, we builded the AD rats model by bilateral hippocampus injection of Aβ1-42 and observe the rats behavior changes and the express of HMGB1, TLR4 and NF-κB in the rats hippocampus.Methods: Thirty-two healthy male SD rats were randomly assigned to four groups according to the random figure chart: the Sham group, the Sham +Evo group, the AD group and the AD+Evo group. Every group contained 8 rats. The rats were adapted for one week prior to the present experiment. The Evodiamine treatment group were treated Evodiamine by gavage once a day for 3 weeks, the other two groups were treated with equal saline. After 3 weeks, the AD group and the AD+Evo group were injected into the rats’ bilateral hippocampus with 10μg Aβ1-42, and the Sham group, the Sham +Evo group with equal saline. All the rats were trained on a Morris water maze test on day 7 after the injection of Aβ1-42, to identify learning and memory ability of the rats. The rats were sacrificed after the MWM test and the brain tissue was obtained for pathological observation and intracellular staining. Hematoxylin and eosin(HE) staining was adopted to observe the different macroscopic pathological features in the hippocampus. The activation of microglia was detected by immunostaining. Western Blot was used to detect the expression of TLR4, TRAF6 and NF-κB.Results:1 The result of Morris water maze test showed that each group’s latent period of escape was not same(F=38.432, P<0.05) and either does the frequency of crossing platform on 7 days after modeling(F=4.388, P<0.05). Compared with the Sham group, the latent period of escape in AD group was significantly longer(P<0.05). In the space probe test, the mean number of crossing platform of the AD+Evo group showed significant higher than did the AD group( P <0.05).2 HE staining: The pyramidal cells of the rats’ hippocampal CA1 area were in large quantity, regularly arranged, the cellular nuclei were clear and homogeneously stained in the Sham group. Compared with the Sham group, sparsely formed cells and deeply stained nuclei were obviously demonstrated in the AD group. By contrast, in the AD+Evo group, the neurons and the nuclei just looked the same as in the Sham group. The number, arrangement and morphology of the neurons were all recovered in a degree.3 The expression of HMGB1,TLR4 and NF-κB: Compared with the Sham group, the AD group significantly increased the protein level and the number of positive cells of HMGB1,TLR4 and NF-κB. The AD+Evo group was significantly decreased their expression compared with the AD group(P<0.05).Conclusions:1 Through injecting of Aβ1-42 into the rat’s bilateral hippocampus,we successfully established AD model.2 There were plentiful of activated microglias in the hippocampus of the AD group rats, with the increase of HMGB1,TLR4 and NF-κB, which indicated HMGB1,TLR4 and NF-κB anticipated the inflammation reaction in AD.3 Evodiamine can improve the learning and memory abilities of the AD rats and decrease the loss of neurons, which indicates the neuroprotective effect of Evodiamine.4 Evodiamine decreased the expression of HMGB1, TLR4 and NF-κB to a certain degree thus inhibiting HMGB1/TLR4/ NF-κB signal pathway.