| Alzheimer`s disease(AD) is a devastating, complex and irreversible progressive neurodegenerative disease. The typical neuropathological hallmarks of AD include extracellular senile plaques composed largely of amyloid-beta peptide(Aβ), intraneuronal neurofibrillary tangles caused by intracellular aggregates of hyperphosphorylated tau protein and cerebral atrophy due to neuronal and synapse loss. AD is the most common dementia in elderly people, and epidemiology predicted that there would be 24.3 million people with dementia in 2020 in the world. The clinical characteristic of AD is a progression from episodic memory and learning problems to a slow global deficit in cognitive function, and average living time of AD patients is 9 years after diagnosis with custodial care. Current drugs for AD targeting cholinergic and glutamatergic neurotransmission, including cholinesterase inhibitors and NMDA receptor antagonist, were thought to treat mild-to-moderate and moderate-to-severe AD respectively. But evidence from clinical treatment demonstrates to be unsatisfactory, especially for the long term efficacy. In addition, the other drugs including atypical antipsychotic, antidepressant, anticonvulsant, immunotherapy, β- or γ-secretase inhibitors, α-secretase activators, tau aggregation or hyperphosphorylation inhibitors, metal modulators and vitamin, are used to improve cognition function of AD patients, but evidence of their effectiveness is limited. Therefore, up till now, there is still no effective therapy to prevent, halt, or reverse AD.Liuwei Dihuang(LW) is a classical traditional Chinese medicine prescription with a long history of clinical application. Modern research indicates that LW, as a multi-target therapeutic prescriptions regulated the neuroendocrine-immune system, has antiamnestic and cognitive-enhancing activities. A double-blinded, placebo-controlled trial showed that LW derivatives enhanced cognitive ability in normal people, and the speed of information processing. In our laboratory, it was found that LW significantly improved cognitive impairments of senescence-accelerated mouse prone 8 strain(SAMP8), an AD animal model. Moreover, chronic administration of LW protects rodent against the D-galinduced spatial learning and memory impairment, and reverses scopolamine-, pchloroamphetamine- or cycloheximide-induced passive and active avoidance performance deficits. And also, LW has beneficial effects on streptozotocin- or ibotenic acid-induced spatial learning and memory impairment. These results indicated that LW possesses cognition-enhancing effect. However, the active fractions and ingredients, and the mechanism underlying the cognition-enhancing effect remains to be further studied and elucidated. The collaborative studies on chemistry and pharmacology of LW have been conducted in our institute since 1990 s. In isolation of the active fractions and ingredients from LW decoction, the idea and method of actibity-guided fractionation were employed. With this idea and method, three kinds of active fractions and components have been isolated from LW decoction, including polysaccharide fraction(LWB-B), glycosides fraction(LWD-b) and oligosaccharide fraction(CA-30), and a new formula(LW-AFC) consisting of the 3 fractions has been under developing. We hypothesis that LW-AFC may be a promising therapeutic medicine for AD, targeting neuroendocrine immunomodulation(NIM) network.In the present study, we investigated the effect of LW-AFC on cognitive behavioral consequences, histopathological changes, and biomarkers of AD mouse models. Pr Ph AβPPswe/PS1ΔE9(APP/PS1) mice and senescence-accelerated mouse prone 8 strain(SAMP8), classic AD animal models, were employed. And examining the anti-aging effects of LW-AFC on 12- and 24-month-old senescence-resistant mouse prone 1 strains(SAMR1). After long-term oral administration of LW-AFC, the conditions of neuroendocrine-immune system in the mice were investigated by measuring HPA and HPG axis, subsets and proliferation of splenocytes, cytokines in plasma.Part I. Effect of LW-AFC on central learning and memory1. The treatment of LW-AFC slowed aging process and improved cognitive impairments of SAMP8 miceThe male SAMP8 mice at six months old were separated into 7 groups at random, each group had 10~11 mice. LW-AFC was dissolved in distilled water at 80, 160 and 320 mg/m L, memantine to 1 mg/m L, and donepezil to 0.1 mg/m L. The mice in drug treated groups were given intragastric administration of memantine, donepezil or LW-AFC(0.1 m L/10 g body weight) once a day during the test for 150 days. SAMP8 mice as model group and the age-matched SAMR1 mice(10 males) as control group were given an equal volume of deionized water. After treated with LW-AFC, the grading score for evaluation of the degree of senescence in SAMP8 mice was decreased, and significantly reduced by medium dosage LW-AFC administration. The locomotor activity test was performed to investigate the spontaneous motor activity of mice. Results showed that the moving distance of SAMP8 mice were significantly increased by memantine and LW-AFC. The passive avoidance response in step down test, short and long term object recognition memory in novel object recognition test of SAMP8 mice were significantly ameliorated by medium dosage LW-AFC administration, active avoidance response in shuttle box test and spatial learning and memory in Morris water maze were significantly improved by memantine and LW-AFC(0.8g/kg and 1.6g/kg), These indicated that the treatment of LW-AFC had delaying effects on aging process and improvement effects on cognitive impairments of SAMP8 mice.2. LW-AFC treatment improved cognitive impairments, alleviated Αβ deposition and neuronal loss, decreased soluble Αβ levels of APP/PS1 miceNine month-old male APP/PS1 mice were separated into 4 groups at random, each group had 10~11 mice. LW-AFC was dissolved in distilled water at 160 mg/m L, memantine to 1 mg/m L, and donepezil to 0.1 mg/m L. The mice in drug treated groups were given intragastric administration of memantine, donepezil or LW-AFC(0.1 m L/10 g body weight) once a day. APP/PS1 mice as model group and the age-matched WT mice(15 males) as control group were given an equal volume of deionized water for 150 days. This study showed the treatment of memantine, donepezil, and LW-AFC had ameliorative effects on active avoidance response in APP/PS1 mice. Spatial learning and memory of APP/PS1 mice was significantly improved by memantine and LW-AFC, passive avoidance response and object recognition memory were significantly ameliorated by LW-AFC administration. LW-AFC treated mice had significantly less area of Αβ deposits in both whole brain and hippocampus, decreased the Nissl bodies and increased the density of healthy neurons in the hippocampus and CA3 region of APP/PS1 mice. The Αβ1-40 and Αβ1-42 levels in the hippocampus and plasma were measured by Alpha LISA. Statistical analysis showed that LW-AFC significantly reduced Αβ1-42 levels in the hippocampus and plasma of APP/PS1 as compared to the WT mice. These indicated that LW-AFC could improve learning and memory deficits, alleviate Αβ deposits, protecte against neuronal loss, and influence the Αβ1-42 levels in APP/PS1 mice.3. The treatment of LW-AFC slowed the aging process of old SAMR1 miceIn order to investigate the effects of LW-AFC on aging, we detected the influence of LW-AFC on the appearance of aging in 12- and 24-month-old SAMR1 mice. Results showed the treatment of melatonin and LW-AFC had improved effects on the grading score of senescence of 12- and 24-month-old SAMR1 mice, LW-AFC had improved effects on the life span of 12-month-old SAMR1 mice and average weight of both mice. The LW-AFC administration significantly improved spatial memory of 12- and 24-month-old SAMR1 mice. These indicated that the treatment of LW-AFC had delaying effects on aging process of normal aging mice.Part II. Mechanism of LW-AFC on central learning and memory1. Effect of LW-AFC on neuroendocrine immune system in SAMP8 miceIn order to investigate the mode of HPA and HPG axis in SAMP8 mice influenced by LW-AFC treatment, the concentration of Gn RH and CRH in hypothalamus, ACTH, FSH and LH in pituitary were measured by radioimmunoassary. The concentration of T and CORT in the plasma was measured by immunochemiluminescence assay and ELISA respectively. Results showed that in HPA axis, the concentration of CRH, ACTH and CORT were significant increase in SAMP8 mice compared with SAMR1 mice, while the level of CRH and ACTH were significantly decreased by 1.6 and 3.2 g/kg LW-AFC, 0.8 and 1.6 g/kg LW-AFC administration respectively in SAMP8 mice. In HPG axis, the concentration of Gn RH, FSH and LH were increased and T was deceased in SAMP8 mice compared with SAMR1 mice, while the level of Gn RH, FSH and LH was significantly decreased by LW-AFC(0.8, 1.6 g/kg) treatment, and T was increased by 0.8, 1.6 and 3.2 g/kg LW-AFC in SAMP8 mice. Additionally, treatment with memantine decreased the level of LH in pituitary of SAMP8 mice.To observe which immune cell is targeted by LW-AFC, Con A- and LPS-induced splenocytes proliferation was investigated by 3H-thymidine incorporation. Results showed that compared to the SAMR1 mice, splenocyte proliferation, Con A- and LPSinduced splenocyte proliferation in the SAMP8 mice was significantly decreased, while donepezil, and LW-AFC(0.8, 1.6 and 3.2 g/kg) treatment increased the spontaneous splenocyte proliferation, memantine, donepezil, and LW-AFC(0.8, 1.6 and 3.2 g/kg) treatment increased the Con A-induced splenocyte proliferation. But for LPS-induced splenocyte proliferation, memantine and donepezil had decrease effect, LW-AFC(0.8, 3.2 g/kg) had increase effect.In order to investigate the effect of LW-AFC on the lymphocyte subsets of SAMP8 mice, flow cytometry was employed. Results showed that CD3+ CD4+ T cells, CD3+ CD28+ T cells, and CD19+ B cells in SAMP8 mice were significantly less than that in SAMR1 mice and CD3+ CD8+ T cells, CD3+ CD25+Foxp3+T cells, and CD19+ CD80+ B cells were significantly more than that in SAMR1 mice. In SAMP8 mice, CD3+ CD4+ T cells was increased by memantine and LW-AFC(0.8 g/kg and 1.6 g/kg), CD3+ CD28+ T cells by donepezil and LW-AFC(0.8, 1.6 and 3.2 g/kg), CD19+ B cells by memantine, donepezil and LW-AFC(0.8, 1.6 and 3.2 g/kg), CD19+ CD80+ B cells by memantine, donepezil, and LW-AFC(0.8 and 1.6g/kg) treatment respectively, while CD3+CD8+ T cells were decreased by memantine and donepezil treatment. But CD3+CD25+Foxp3+ T cells in SAMP8 mice was increased by donepezil treatment and decreased by 1.6 g/kg LW-AFC treatment. Furthermore, the ratio of CD3+CD4+ T cells and CD3+ CD8+ T cells was significantly decreased in SAMP8 mice,while that was increased by memantine, donepezil, and LW-AFC(0.8 and 1.6g/kg) administration respectively.To observe the effect of LW-AFC on cytokine secretion, the multiplex bead analysis was employed. The treatment of memantine significantly decreased IL-23, TNF-β and eotaxin production, and donepezil only decreased IL-23 production in blood plasma of SAMP8 mice. The treatment of LW-AFC decreased IL-1β, IL-23, GM-CSF, IFN-γ, eotaxin, TNF-α and TNF-β, and increased MCP-1 production in 0.8, 1.6 and 3.2 g/kg dosage, and decreased IL-2, IL-6 and RANTES in 0.8 and 1.6 g/kg dosage, meanwhile, increased IL-5 production in 1.6 g/kg dosage.These results indicated that the NIM network of SAMP8 mice was abnormal. LWAFC administration had restorative effect on imbalance of HPA and HPG axis, enhancive effect on proliferation of splenocytes, corrective effect on the disorder of lymphocyte subsets, regulatory effect on abnormal production of cytokine, and then restored the aberrant NIM network in SAMP8 mice.2. LW-AFC had restorative effect on imbalanced NIM network in APP/PS1 miceResults showed that in HPA axis, the concentration of CRH, ACTH and CORT were significantly increased in APP/PS1 mice compared with WT mice. The level of CRH was significantly decreased by LW-AFC, while ACTH was reduced by LW-AFC and memantine. In HPG axis, the concentration of Gn RH, FSH and LH were increase and T was no significant difference in APP/PS1 mice compared with WT mice. The concentration of Gn RH was significantly decreased by LW-AFC, while the levels of FSH and LH were reduced by LW-AFC and memantine in APP/PS1 mice. The expression of CD3+ CD28+ T cells were increased by memantine, donepezil and LW-AFC treatment, while expression of CD3+CD25+Foxp3+ T cells was decreased memantine and LW-AFC treatment. The treatment of memantine significantly decreased IL-1β, IL-6, IL-23, GMCSF and TNF-β production, and donepezil only decreased IL-1β production in blood plasma of APP/PS1 mice. The levels of IL-1β, IL-2, IL-6, IL-23, GM-CSF, TNF-α, TNF-β, and eotaxin were decreased, IL-4 and G-CSF were elevated by LW-AFC administration. These results indicated that long-term oral administration of LW-AFC restored unbalanced neuroendocrine system, corrected disordered lymphocyte subsets, and rebalanced abnormal cytokine secretion in APP/PS1 mice.3. Reversal of immunosenescence in old SAMR1 mice by LW-AFCThe treatment of LW-AFC significantly increased spontaneous splenocyte proliferation of 12-month-old SAMR1 mice, and Con A- and LPS-induced splenocyte proliferation in 12- and 24-month-old SAMR1 mice.The CD3+ T cells, ratio of CD4+ and CD8+ T cells, CD3+ CD25+Foxp3+T cells of 24-month-old SAMR1 mice were improved by LW-AFC administration, CD3+ CD4+, CD3+ CD8+ T cells and CD19+B cells of both old SAMR1 mice were also reversed by LW-AFC. After treatment with LW-AFC and melatonin, B cells of both old SAMR1 mice were increased compared with control groups.After being administered with LW-AFC, 11 of 13 pro-inflammatory cytokines(eotaxin, GM-CSF, IFN-γ, IL-1β, IL-2, IL-6, IL-17, RANTES, TNF-α, IL-23, TNF-β) and all anti-inflammatory cytokines(G-CSF, IL-4, IL-5, IL-10) examined of 12-monthold SAMR1 mice were ameliorated, 12 of 13 pro-inflammatory cytokines(eotaxin, GMCSF, IFN-γ, IL-1β, IL-2, IL-6, IL-17, MCP-1, RANTES, TNF-α, IL-23, TNF-β) and no anti-inflammatory cytokines of 24-month-old SAMR1 mice were modulated. Melatonin was able to regulate 9 of 13 pro-inflammatory cytokines(eotaxin, GM-CSF, IFN-γ, IL-1β, IL-2, IL-6, IL-17, RANTES, TNF-α) and all anti-inflammatory cytokines(G-CSF, IL-4, IL-5, IL-10) examined of 12-month-old SAMR1 mice, and 8 of 13 pro-inflammatory cytokines(GM-CSF, IFN-γ, IL-2, IL-6, IL-17, RANTES, TNF-α, TNF-β) and 1 of 4 antiinflammatory cytokines(IL-4) of 24-month-old SAMR1 miceThese data demonstrated that LW-AFC possessed anti-aging activity and this effects might be through reversing immunodeficiency and decreasing chronic inflammation.4. The targets of LW-AFC underlying cognitive impairment in AD mouse modelsIn order to find endocrine hormone and cytokine contributing to cognitive impairments in SAMP8 mice and APP/PS1 mice, Pearson correlation analysis was performed. Results showed the cognition capability were significantly correlated with most endocrine hormones and cytokines in SAMP8 and APP/PS1 mice. For clarifying the underpinnings of cognition deficits by zooming out to the molecular level to search for the key causal molecules in many contributions of NIM network. Stepwise multiple linear regression analysis was performed among all the endocrine hormone and cytokine. Results showed that LH, T and Gn RH in HPG axis was important in SAMP8, while T and LH in HPG, ACTH in HPA axis in APP/PS1 mice. For cytokines contributing to cognitive impairment, pro-inflammatory factor IL-23, cytokines secreted by Th1 cell(GM-CSF, IFN-γ, IL-2, TNF-α) and chemotactic factors(eotaxin, RANTES) contributed to cognitive impairment in SAMP8, while cytokines secreted by Th2 cell(GM-CSF, IL-6) and chemotactic factors(eotaxin) in APP/PS1 mice. These results indicated that the unbalanced state of NIM network had been associated with cognitive decline, and LWAFC improved cognitive deterioration by effecting different targets in NIM network of SAMP8 and APP/PS1 mice.Conclusions:1. LW-AFC had ameliorative effects on passive and active avoidance, object recognition memory, spatial learning and memory impairments in SAMP8 and APP/PS1 mice,meanwhile, slowed the aging process of old SAMR1 mice. These indicated that long-term oral administration of LW-AFC had improvement effects on cognitive impairments and delaying effects on aging process.2. Long-term oral administration of LW-AFC could restore unbalanced HPA and HPG axis, correcte disordered lymphocyte subsets, rebalance abnormal cytokine secretion of SAMP8 and APP/PS1 mice. Moreover, LW-AFC had improved and modulatory effects on immune function of old SAMR1 mice. These indicated that long-term oral administration of LW-AFC had restorative effect on endocrine and immune dysfunction of AD mouse models.3. The unbalanced state of NIM network had been closely associated with cognitive decline. LW-AFC improved cognitive deterioration of SAMP8 mice mainly by effecting on Gn RH, LH, T, GM-CSF, IFN-γ, IL-2, TNF-α, IL-23, eotaxin and RANTES. Regarding APP/PS1 mice, mainly effecting on ACTH, LH, GM-CSF, IL-6 and eotaxin. These data indicated that LW-AFC improved the central learning and memory by restoring the aberrant NIM network.4. The ability to rebalance, restore and maintain the NIM network of LW-AFC is different from current drugs for AD. And the beneficial effects on cognitive deterioration and aging process of LW-AFC make it being a potential preventive or therapeutic agent for AD. |
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