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OBJECTIVE To study the neuroprotective effects of Shenzao jianna o oral liquid （SZJN）on Alzheimer ’s disease （AD）model mice and its mechanism. METHODS The mice were randomly divided into sham operation group ，model group ， Donepezil hydrochloride tablet group （0.65 mg/kg），SZJN low-dose ，medium-dose and high-dose groups （0.3，1.5 and 7.5 g/kg， calculated by crude drug quantity ），with 12 mice in each group ，half male and half female. Each group was given relevant medicine（intragastric administration of water at constant volume in sham operation group and model group ），twice a day ，for consecutive 28 d. On the 15th day of administration ，intracerebroventricular injection of β-amyloid 1-42（Aβ1-42）combined with intraperitoneal injection of scopolamine hydrobromide were used to induce AD model. Morris water maze was used to detect the learning and memory ability of mice. HE staining and Nissl staining were used to evaluate the pathological changes of brain tissue in mice. The levels of MDA and SOD in brain tissue of mice were detected. The phosphorylation level of cyclic adenosine monophosphate response element binding protein （CREB） and expression of brain-derived neurotrophic factor （BDNF） in hippocampal tissues were detected by Western blot. RESULTS Compared with sham operation group ，the escape latency of the model group was significantly prolonged ，and the number of crossing the platform and the percentage of residence time in the target quadrant were significantly reduced （P＜0.01）. The level of SOD in brain tissue ，the phosphorylation level of CREB and the expression level of BDNF in hippocampus decreased significantly （P＜0.01），while the level of MDA increased significantly （P＜ 0.01）. In hippocampal CA 1 area and cortical tissue ，nerve cells showed significantly decreased number ，the disordered arrangement and large gap ；the shape of nucleus was irregular and deeply stained ，and Nissl body was blurred ，loosely arranged and the number decreased. Compared with model group ，the escape latency of mice in each dose group of SZJN was significantly shortened ，and the times of crossing the platform and the percentage of residence time in the target quadrant were significantly jing- increased（P＜0.01）. Above indexes of brain tissue in mice were reversed sig nificantly in SZJN high-dose group （P＜0.01），and pathological damage of brain tiss ue was improved. CONCLUSIONS SZJN can significantly improve the learning and memory ability of AD model mice ，and alleviate the pathological injury and oxidative stress of brain tissue ，which may be related to the activation of CREB/BDNF signaling pathway.
RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: According to the theory of traditional Chinese medicine (TCM), Alzheimer's disease (AD) is identified as "forgetfulness" or "dementia", and is mainly caused by "kidney essence deficiency" which ultimately induces "encephala reduction". Therefore, herbal formulas possessing the efficacy of nourishing kidney essence or replenishing brain marrow are commonly served as effective strategies for AD treatment. Shenzao jiannao oral liquid (SZJN), a traditional Chinese preparation approved by the China Food and Drug Administration (CFDA), is used for the treatment of insomnia and mind fatigue at present for its efficacy of nourishing kidneys. In present study, we found that SZJN could improve cognitive function of AD-like mice. AIMS OF STUDY: This study aims to investigate the effects of SJZN on ameliorating cognitive deficits of AD-like mouse model, and to illuminate the underlying mechanisms from the perspective of neuroprotection and neurogenesis. MATERIALS AND METHODS: Kunming mice (28 ± 2 g) were randomly allocated into seven groups: control, sham, model, donepezil and SZJN groups (low, middle and high). The AD mouse model was established by Aß42 combined with scopolamine. SZJN were intragastrically administrated at doses of 0.3, 1.5 and 7.5 g/kg for 28 days. Morris water maze (MWM) test was applied to determine the cognitive function. Hematoxylin eosin (HE) and Nissl staining were carried out to evaluate pathological damages in the cortex and hippocampal tissues. To explore the protective effects of SZJN on multiple pathogenic factors of AD, protein levels of Aß42, glial fibrillary acidic protein (GFAP), Bax, Bcl-2, Caspase-3, synaptophysin (SYP), brain-derived neurotrophic factor (BDNF), and neurogenesis related proteins were assessed using Immunofluorescence (IF) and western blot analysis. In vitro, the AD cell model was established by transduction of APP695swe genes into Neural stem cells (NSCs) isolated from the hippocampal tissues of neonatal C57BL/6 mice. Cell viability assay and neurosphere formation assay were carried out to verify the efficacy of SZJN on proliferation of NSCs. RESULTS: Our results demonstrated that SZJN (1.5 g/kg and 7.5 g/kg) treatment significantly ameliorated cognitive deficits of AD-like mice. SZJN (7.5 g/kg) treatment significantly retarded the pathological damages including neuronal degeneration, neuronal apoptosis, Aß peptides aggregation and reaction of astrocytes in AD-like mice. In addition, SZJN (7.5 g/kg) increased the expression of BDNF and SYP, and restored the abnormal level of MDA and SOD in the brain of AD-like mice. Furthermore, SZJN treatment for 28 days remarkably increased the proliferation of NSCs evidenced by more Nestin+ and BrdU+ cells in the hippocampal DG regions, and increased the amount of mature neurons marked by NeuN both in the cortex and hippocampal DG regions. In vitro, SZJN treatement (16, 32, 64 mg/ml) promoted the proliferation of NSCs evidenced by the increased amount and enlarged size of the neurospheres (p < 0.05). CONCLUSIONS: Our findings indicated that SZJN could ameliorate cognitive deficits by protecting neurons from death and triggering endogenous neurogenesis. Therefore, SZJN may be considered as a promising agent to restore neuronal loss and deter the deterioration in AD patients.
