| BackgroundAlzheimer's disease(AD)is the most common type of neurodegenerative disease,and has the characteristics of high incidence rate and great harm.At present,there is not any effective treatment or prevention.Microglia is a cell that plays an important role in the initiation of AD,and is considered to be the third pathological feature of amyloid protein deposition and neurofibrillary tangle.Under the stimulation of amyloid plaques,microglia specifically activate NLRP3,and inflammatory body mediated chronic neuroinflammation is associated with the pathogenesis of AD.In Traditional Chinese Medicine,the symptoms of AD can be classified into "daze syndrome","forgetfulness","Wenchi" and other diseases.The pathogenesis of AD is based on the deficiency of viscera and the pathogenesis of phlegm and blood stasis.Deficiency of kidney essence and brain marrow filling are the major causes of the disease.Previous studies of our group found that moxibustion can improve the oxidative stress state in the brain,down regulate the apoptosis pathway,delay aging,and moxibustion has been proved to be able to regulate the immune system,showing good anti-inflammatory effect,which suggests that moxibustion may also play a regulatory role in AD cerebral neuritis.Therefore,based on previous studies,we proposed a hypothesis:moxibustion can regulate the neuroinflammatory response of Alzheimer's disease brain tissue by affecting the NLRP3 inflammasome mediated microglia polarization,so as to play an anti-inflammatory and anti-AD role.ObjectiveTo observe the effects of moxibustion on learning and memory ability,amyloid deposition,microglia polarization and NLRP3 inflammasome in AD model mice,and to explore the mechanism of Moxibustion in preventing and treating AD from the perspective of neuroinflammation.Methods24 6-month-old APP/PS1 double transgenic mice were randomly divided into model group and moxibustion group,and 12 wild-type C57BL/6 mice were used as control group.Mice in the model group and the control group were grabbed and fixed in the mouse fixator every day and exposed to the indoor air;mice in moxibustion group were grabbed and moxibustion was applied at Guanyuan point(RN4)with moxa stick.All interventions were 20 minutes a day,6 times a week,for a total of 8 weeks.Morris water maze,Y maze,elevated plus maze and open field test were carried out at the 9th week.At the end of the behavioral experiment,after deep anesthesia,the cerebral cortex and hippocampus were removed,and the amyloid plaques in hippocampus were detected by Congo red staining.CD11b,iNOS and Arg-1 were detected by double immunofluorescence staining.Quantitative real-time PCR was used to detect the markers of M1 and M2 microglia,such as iNOS,IL-1?,Arg-1 and CD206.The relative expressions of TLR4,NLRP3,ASC,Caspase-1,IL-1? and c-fos were observed by immunohistochemistry and Western blot.In addition,24 6-month-old APP/PS1 mice were randomly divided into moxa smoke group and cigarette group to explore the mechanism of moxa combustion products(MCP).Mice were grabbed daily and intervened in moxa smoke or cigarette smoke of 5-15 mg/m3 concentration for 20 min,6 times a week,for a total of 8 weeks.Congo red staining,Western blot,immunohistochemistry,ELISA were used to compare the effects of moxa smoke and cigarette intervention on hippocampal NLRP3 inflammasome and serum inflammatory factors such as TNF-? in APP/PS1 model mice.Results1.The results of behavioral experiment of mice in each groupMorris water maze:compared with C57BL/6 mice of the same age,the learning and memory ability of 8-month-old APP/PS1 mice decreased significantly.In the space exploration test,the activity track was disordered,and there was no purposeful exploration in the target quadrant and platform area.The distance and time of swimming in the platform area were significantly lower than those of the other two groups(P<0.05).Compared with the model group,the performance of mice in moxibustion group,such as escape latency,times of crossing the platform area and swimming time in the platform area,were significantly better than that in the model group(P<0.05).Y maze:The frequency of continuous entry into three arms and the spontaneous alternation rate of mice from high to low were the control group>moxibustion group>model group and there were significant differences between the control group and moxibustion group and model group(P<0.05).The heat map showed that the exploration time in the three arms of the blank group was more balanced,the moxibustion group spent more time in the open arm,and the model group spent more time in the middle area of the intersection of the three arms and the new arm.Elevated plus maze:Compared with the control group,the number and time of exploring the open arm in the model group were significantly decreased(P<0.05),but the activity speed in the open arm was higher than that in the other two groups(P<0.05).Mice in the moxibustion group performed better than the model group in this experiment.The activity trajectory and heat map showed that mice in the control group and moxibustion group still had more activities to explore the open arm,while mice in the model group were more inclined to move in the closed arm and less to explore the open arm,and the speed was accelerated when the open arm was used.Open field test:Total distance model group mice(P<0.05),the most in the central area through the central frequency and time are lower than the other two groups(P<0.05),from the point of activity track and action heat maps,the control group and moxibustion group mice activities throughout the observation box,model group mice activity,the more the scope of activity in the walls and around the corner,less activity to explore the central area.2.Congo red staining and microglia polarizationCongo red staining results:There were more A? precipitate particles stained orange in the hippocampus of model group mice,and the cells were scattered and irregular,with more space visible,and the apoptosis and necrosis of cells were serious.In the moxibustion group,the pathology of mice was less than that of the model group,A? particles were scattered in the hippocampus,cells were arranged neatly,and the gaps caused by the absence of neurons were occasionally seen.In the control group,cells in the hippocampal tissue were clear,normal and orderly,and there were fewer orange A? precipitation particles.Results of microglia polarization:? The expression of CD11b,iNOS and Arg-1 immunofluorescence in each group was observed by laser confocal microscopy.Compared with control group,the expression of iNOS in model group was increased.Compared with the model group,the fluorescence expression of iNOS in moxibustion group decreased.Compared with the control group,the fluorescence expression of Arg-1 was increased in the model group.Compared with the other two groups,the fluorescence expression of Arg-1 in moxibustion group was significantly increased.?RT-PCR results showed that compared with control group,the mRNA relative expression levels of iNOS and IL-1? in model group were significantly increased(P<0.05);Compared with model group,iNOS and IL-1? in moxibustion group were significantly decreased(P<0.05);Compared with the control group,the mRNA content of Arg-1 in the model group was significantly increased(P<0.05).Compared with the model group,the mRNA expression of Arg-1 in the moxibustion group was significantly increased.Compared with control group,the mRNA content of CD206 in moxibustion group was significantly increased(P<0.05).3.The expression of NLRP3 inflammasomeWestern blot results:The protein expressions of NLRP3,ASC and Caspase-1 in the hippocampus of mice in each group were compared.The model group was significantly increased compared with the control group(P<0.05),while the moxibustion group significantly decreased compared with the model group(P<0.05).Compared with the control group,TLR4 in the model group was significantly increased(P<0.05),but there was no significant difference between the moxibustion group and the model group(P>0.05).Compared with the control group,the contents of IL-1? and c-fos in the hippocampus of mice in each group were significantly increased in the model group(P<0.05).Compared with the model group,the histone content of mice in the moxibustion group was significantly decreased(P<0.05).Immunohistochemical results:The integrated optical density(IOD)of NLRP3,ASC,caspase-1,TLR4,IL-1? and c-fos expressions in each group were compared.Compared with control group,the IOD value in model group was significantly increased(P<0.05).Compared with the model group,the IOD value of moxibustion group was significantly decreased(P<0.05).4.Comparison of the effects of moxa smoke and cigarette?The Congo red staining in the hippocampus of mice in each group showed that there were a lot of orange A? precipitated particles in the model group and the cigarette group.The cells in the cigarette group were scattered and irregular,with more gaps and serious apoptosis and necrosis.In the group of MCP,A? particles were scattered,the cells were arranged neatly,and the gaps caused by the absence of neurons were occasionally seen.?Comparison of western blot results showed that from high to low,the cigarette group>model group>MCP group>control group.Compared with the model group,NLRP3 and TLR4 in the cigarette group were significantly increased(P<0.05),while there were no significant differences in caspase-1 and IL-1?(P>0.05).Compared with model group and cigarette group,NLRP3,Caspase-1,TLR4 and IL-1? were significantly decreased in MCP group(P<0.05).?Comparison of immunohistochemical IOD values showed that from high to low,cigarette group>model group>MCP group>control group.Compared with control group,NLRP3,Caspase-1,TLR4 and IL-1? in model group and cigarette group were significantly increased(P<0.05);Compared with model group and cigarette group,the IOD value of mice in MCP group was significantly decreased(P<0.05).?ELISA method showed that TNF-?,IL-8 and CRP in model group and cigarette group were significantly increased compared with control group(P<0.05),and TNF-?,IL-8 and CRP in MCP group were significantly decreased compared with cigarette group(P<0.05).Conclusion:1.Moxibustion can effectively improve the learning and memory ability and emotional abnormalities of AD model mice,reduce the deposition of ? amyloid protein,inhibit the activation of microglia,and the polarization of M2 microglia with anti-inflammatory and neuroprotective effect is obvious.Moxibustion can inhibit the neuroinflammation in brain and regulate the polarization of microglia.2.Moxibustion can protect nerve and control chronic neuroinflammation by inhibiting the expression of NLRP3 inflammasome related protein,regulating the upstream inflammatory factors and inhibiting the inflammatory cascade.3.Compared with cigarette smoke,moxa smoke did not aggravate the pathological changes of AD,reduced the A? protein deposition pathology in hippocampus of APP/PS1 mice,inhibited the expression of NLRP3 inflammasome related protein,and decreased the expression levels of serum inflammatory markers TNF-?,IL-8 and CRP. |
PDF Full Text Request