The Effect Of Icariin On Lymphocyte-mediated Immune Inflammation Response In Alzheimer’s Disease

BackgroundAlzheimer’s disease(AD)is the most common neurodegenerative disorder and the main cause of dementia,which is characterized by progressive memory loss and cognitive deterioration.It is estimated that there is a new case every 3 s in the world,and this community is likely to rise to about 152 million people by 2050.The neuropathological hallmarks of AD include the extracellular aggregation of amyloid-β(Aβ)peptide in plaques and the formation of intraneuronal neurofibrillary tangles(NFTs)by hyperphosphorylated tau protein.Although the mechanisms causing the neuropathological lesions are largely unknown,increasing documents initially underlined that neuroinflammation may be a major driver in the pathogenesis of AD.Growing evidence indicates that deposition of Aβ induces an inflammatory response by activating microglia in the brain.T cells have been identified in neuritic plaques and microglia in the post-mortem AD brain.T cells from peripheral immune system may play a critical role in driving the neuroinflammatory changes and the associated neuronal degeneration.Approximately,over 80%of the T lymphocytes in the cerebrospinal fluid are CD4+cells.Accumulating evidence suggests increased intracerebral infiltration and enhanced peripheral CD4+T cell responses to Aβ in patients suffering from AD.Regulatory Tcells(Trees)have been proven to play a beneficial role in the pathophysiology of AD,by modulating microglial response to Aβ.Regulation of CD4+T cells differentiation may be a feasible way to affect the pathophysiological changes of AD.Our previous study found that human cord blood-derived stem cells could alter Tregs and alleviate neuroinflammation in AD transgenetic mice.Despite current advances in the underlying mechanisms of AD,there remains no effective treatment to cure the intractable disease in clinical practice.Icariin(ICA),a natural flavonoid glucoside from Epimedium,has been proved to have various pharmacological activities,such as anti-inflammatory,and neuroprotective effects.It presents an activity to prevent neuronal degeneration in AD animal models,by inhibiting neuronal apoptosis and Aβ deposition.Moreover,ICA negatively regulates the activation of T lymphocytes and pro-inflammatory cytokine profiles in CD4+T cells.Meanwhile,ICA plays a regulatory effect on T-helper(Th)17 and Tregs function.These findings indicate that ICA has a potent immune regulating function to prevent chronic neuroinflammatory and might be a promising therapeutic agent for AD.Transgenic animal models of AD can simulate the behavioral and pathological changes in AD patients,thus are considered as an optimal alternative for testing novel therapeutics.The APP/PS1 mouse model,first developed from a cross between the mutant amyloid precursor protein(APP)transgenic line Tg2576 and mutant presenilin 1(PS1)transgenic mice,presents substantial,age-related neuron loss in the pyramidal layer of the hippocampus,which has been widely used to explore underlying AD pathophysiology.Thus,in the present study,we attempted to investigate the specific effect of ICA on Aβ deposition and CD4+T lymphocyte-related immuno-inflammatory responses in peripheral immune system and brain parenchyma in double transgenic APP/PS1 mice.Method1.ReagentICA(18760,purity)98%via HPLC)was purchased from Beijing Solarbio Science&Technology Co.,Ltd.(Beijing,China).2.MiceC57BL/6 mice(n=16,male,4 months old)and mutant C57BL/6J-TgN(APP/PS1)double transgenic mice(n=16,male,4 months old)were purchased from Beijing HFK Bio-Technology,Institute of Laboratory Animal Science(Beijing,China).All of the mice were housed in a thermostatic 12-hr light/dark cycle condition with free access to water and food.Animal protocols and procedures have been approved and in accordance with the guidelines of the Ethical Committee for Animal Experiments of Shandong University.3.AdministrationAll mice were randomly divided into four groups(each n=8),which were wild type(WT),WT+ICA,APP/PS1 mice(Tg),and Tg+ICA.ICA was dissolved in distilled water.After 2 weeks of adaptive feeding,animals were orally administered by gavage with ICA(60 mg/kg/d)or distilled water for 8 months.Excluding death and severe weak laboratory animals,6 experimental animals in each group were randomly selected for the subsequent experiment.4.Morris water maze testThe Morris water maze test was performed to assess spatial learning and memory deficits.The pool(120 cm in diameter and 50 cm in height)was filled with water(opaqued with powdered milk)and maintained at 23±1℃.The test was performed 1 week after the last gavage.During the next 5 days,the navigation test was conducted 4 trials per day at 4 different start points,to find the hidden platform located under the water 1.0 cm.Each trial lasted until the animal found the platform or 60 s.Animals failing to find the platform within 60 s were guided to the hidden platform,and stayed for 15 s.Escape latency and the number of platform crossing times were recorded during the platforam trials.The escape latency was recorded as 60 s.On the sixth day,the spatial probe test was performed,and the timein the target quadrant was measured.An automatic photographic recording and analysis system(Smart3.0,Panlab)was used to track and analyze behavioral parameters.5.Hippocampus Ap measurement by ELISAAfter the behavioral test,mice were anesthetized using chloral hydrate(4 mL/kg).After harvesting peripheral blood,the brains were removed on ice immediately,and hippocampus was separated.Then,the hippocampus tissue was transferred to liquid nitrogen for storage.The levels of soluble and insoluble Ap40 and Ap42 in hippocampus were quantified according to previous procedure and were determined by ELISA kit(BioLegend)in accordance with the manufacturer’s instructions.Each experimental sample was run in duplicate.The data were recorded at 620 nm using a microplate reader(Biorad,USA).6.T lymphocyte subtypes detected by flow cytometryPeripheral blood was harvested in tubes with heparin sodium through angular veins.After centrifluging at 1000 rpm for 30 mins at 4oC,the supermatant plasma was collected for cytokine array.For flow cytometry,the resuspended cells with PBS were isolated by mouse lymphocyte separation medium(Dakewe,China)according to the instruction.Then,cells were incubated with antibodies against CD4+T,CD8+T,Thl,Th2,Thl7,and Tregs.The antibodies(BioLegend Inc)used in the experiment included anti-mouse FITC-conjugated CD4(identifying CD4+T cells),anti-mouse APC/CY7-conjugated CD8(identifying CD8+T cells),anti-mouse PE/CY7-conjugated IFN-γ,anti-mouse PE-conjugated IL-4,antimouse APC-conjugated IL-17,anti-mouse AF647-conjugated CD25,anti-mouse PE-conjugated Foxp3.Thl cells were defined as CD4+T cells expressing IFN-γ,Th2 cells were defined as CD4+T cells expressing IL-4,Thl7 cells were defined as CD4+T cells expressing IL-17,and Treg cells were defined as CD4+T cells expressing the molecules CD25 and FOXP3.Cells were resuspended with PBS and were analyzed by a flow cytometer(Beckman CytoFLEX)using CytExpert software.7.Cytokine array analysisIn the present study,multiple cytokine levels in the brain and serum were measured using LEGENDPlex assay(BioLegend,Beijing Ltd.)according to the instructions of the manufacturer.8.Statistical analysisThe data were presented as meann±SEM.Graphs were obtained with GraphPad Prism 7,and comparisons between groups were evaluated by two-way ANOVA followed by Fishery LDS multiple comParsions.P-value less than 0.05 was considered to be statistically significant.Result1.ICA treatment alleviates cognitive deficits in APP/PSI miceThe Morris water maze test was used to examine the effect of ICA on the amelioration of spatial learning and cognitive deficits in APP/PSI mice.In the place navigation test,APP/PS1 mice presented an increased escape latency to find the hidden platform compared with WT mice,but ICA treatment could significantly decrease the escape latency(Figure 1A,P<0.05).APP/PS1 mice with ICA treatment required less time to locate the original platform position than those with saline lavage in the fourth and fifth training days(Figure IB,P<0.05).Time spent in the target zone by APP/PS1 mice was significantly shorter than that by WT mice,while ICA treatment could significantly increase the time spent in the target section relative to APP/PS1 mice(Figure 1C,P<0.05).APP/PS1 mice showed obviously decreased number of platform crossing times relative to WT mice(Figure ID,P<0.05).APP/PSI mice under ICA treatment moderately increased the crossing times compared with untreated mice,but no significance was observed(Figure ID,P>0.05).These findings demonstrated that ICA treatment may alleviate cognitive and memory deficits in APP/PS1 mice.2.ICA treatment reduces hippocampus Aβ deposition in APP/PSI miceGiven that Ap deposition is a major component in AD brain and hippocampus is the key region for memory and cognitive function,we performed ELISA assay to detect both soluble and insoluble Ap40 and Ap42 levels in the hippocampus.The levels of insoluble Aβ40 and Aβ42 in the hippocampus of APP/PS1 mice were higher than that in the WT group(Figure 2A and B,P<0.05),and ICA treatment remarkably lowered the level of insoluble Ap40 and Ap42 in Tg mice(Figure 2 A and B,P<0.05).Soluble Ap40 and Ap42 in the Tg group were significantly increased relative to that in the WT group,while treatment with ICA for 8 months remarkably decreased soluble Aβ40 deposition only(Figure 2C and D,P<0.05).ICA treatment moderately decreased the level of soluble Aβ42 in Tg mice,but no significance was observed(Figure 2D,P>0.05),that might because Aβ42 aggregates more easily to form an insoluble state.Our results indicated that the deposition of Aβ in APP/PS1 mice could be ameliorated under ICA treatment.3.ICA treatment modulates the differentiation of CD4+T cells in peripheral blood ofAPP/PSI miceTo observe the potential of ICA in the regulation of T cell subtypes,we performed a flow cytometry to test the proportion of CD4+ and CD8+T cells.The results showed that the rate of CD4+T cells in the Tg group was significantly increased compared with that in the WT group(Figure 3A and F,P<0.05),but ICA treatment did not change the proportion of total CD4+cells in both Tg and WT mice(Figure 3 A and F,P>0.05).There was no statistical significance in the proportion of CD8+T cells among four groups(Figure 3 A and G,P>0.05).Th1,Th2,and Th17 cells were determined by measuring the intracellular cytokines IFN-γ,IL-4 and IL-17,respectively,in CD4+T cells.The data indicated that Thl cell proportion was markedly increased in the Tg group compared with that in the WT group,and ICA treatment could decrease the polarization of Thl in Tg mice(Figure 3B and H,P<0.05).There was no significant difference for Th2 cell proportion among four groups(Figure 3C and I,P>0.05).Tg mice presented significantly increased Thl7 cell proportion compared with WT mice,and ICA treatment could decrease the proportion of Th17 cells in Tg mice(Figure 3D and J,P<0.05),but not in WT mice(Figure 3D and J,P>0.05).Tregs(CD4+CD25+Foxp3+)in APP/PS1 mice were tested according to our previous study.As shown in Figure 3E and K,we observed that there was no statistical difference of the percentage of Tregs between the Tg and the WT groups(P>0.05).Whereas,ICA treatment could increase the percentage of Tregs both in the Tg and the WT groups(Figure 3E and K,P<0.05).4.ICA treatment alleviates neuroinflammation and regulates the release of inflammatory cytokines in the plasma and brainCytokine induction is a critical pathologic event in inflammatory and anti-inflammatory responses in AD.To further study the effect of ICA on immune-inflammatory response,the secretion of several pro-inflammatory cytokines(IL-1α,IL-1β,IL-6,IL-10,IL-12p70,IL-17A,IL-23,IL-27,MCP-1,IFN-β,IFN-γ,TNF-a,and GM-CSF)was detected in both plasma and brain tissues using the multiple detection kit.The plasma results showed the levels of IL-1β,IFN-γ,IL-6,MCP-1,IL-10,and IL-17A in Tg mice were significantly higher than that in WT mice(Figure 5,P<0.05).ICA treatment could remarkably decrease the levels of IL-1β,TNF-α,IFN-γ,MCP-1,IL-17A,and GM-CSF in Tg mice(Figure 5,.P<0.05).There was no statistical difference in the level of TNF-α,GM-CSF,IL-1α,IFN-β,IL-23,IL-12-p70,and IL-27 between the Tg and the WT groups(Figure 5,P>0.05).ICA treatment had no significant effect on cytokines IL-6,IL-10,IL-1α,IFN-β,IL-23,IL-12-p70,and IL-27(Figure 5,P>0.05).In brain tissues,the data showed that the level of IL-1β,TNF-α,IL-6,IL-17A,and GM-CSF were higher in Tg mice than that in WT mice(Figure 6,P<0.05).ICA treatment could significantly lower the levels of IL-1β,IL-17A,IL-12p70,and MCP-1(Figure 6,P<0.05).Interestingly,ICA treatment could elevate the IL-10 level in the brain tissues of Tg mice(Figure 6,P<0.05).There was no statistical significance in the levels of IL-23,IL-12p70,MCP-1,IL-10,IL-1α,IFN-γ,IFN-β,and IL-27 between WT and Tg mice(Figure 6,P>0.05).ICA treatment had no regulation role for TNF-α,IL-1α,IFN-γ,IFN-β,GM-CSF,and IL-27 in Tg mice((Figure 6,P>0.05)ConclusionCD4+T cell-mediated immuno-inflammatory response has been proven to participate in the pathological process of AD.ICA appears to be a promising drug to treat chronic inflammatory responses.Thus,there is practical value to explore the therapeutic potential of ICA in AD.The following conclusions can be drawn from the our research results1.The decrease of cognitive function,deposition of A β in the brain and pathological changes of chronic inflammation in APP/PSI mice were closely related to the changes in CD4+T lymphocyte subsets.2.Long-term ICA treatment could improve the cognitive function of APP/PS1 mice and reduce the deposition of A p in the brain of APP/PS1 mice.3.Long-term ICA therapy could regulate the proportion of CD4+T lymphocyte subtypes such as Thl,Th17 and Tregs in APP/PS1 mice.4.Long-term ICA therapy could regulate the level of inflammatory cytokines in the peripheral and brain tissues of APP/PS1 mice.ICA has the potential to treat AD.