| ObjectiveAlzheimer's disease?AD?is a complex neurodegenerative disease.Its clinical manifestations are mainly progressive memory impairment,emotional disorders and behavioral abnormalities.At present,there is no ideal treatment for Alzheimer's disease.As the aging population intensifies,AD has become a heavy burden for more and more families and the society.In recent years,a large number of studies have shown that gut microbiota could directly or indirectly affect the brain through vagus nerve,immune system,circulatory system and endocrine system,which is closely related to the occurrence and development of AD.LW-AFC is an innovative traditional Chinese medicine composed of LWB-b,CA-30 and LWD-b,which was further developed,followed by tracking and separating the active components in LW.Previous studies have shown that LW-AFC has the function of balancing Neuroendocrine Immunomodulation network?NIM?to improve cognitive function of AD model animals,meanwhile,it has also showed the function of regulating gut microbiota.In order to further determine whether LW-AFC could regulate gut microbiota,in the present study,the effects of LW-AFC and LW on gut microbiota were compared,and the effects of LW-AFC and its active fraction on gut microbiota were further observed and analyzed by fecal microbiota transplantation.In addition,this study also used co-housing as the meaning of intervening gut microbiota to study the role of gut microbiota in AD related "caregiver burden".Chapter I Studies on the iproved effecet of LW and LW-AFC on the anxity andlearning and memory of SAMP8 mice via modulating gut microbiotaMethod:9-months male senescence-accelerated mouse prone 8 strain?S AMP8?was used as AD model,and senescence-resistant mouse prone 1 strain?SAMR1?was used as control.LW and LW-AFC were given by intragastric administration respectively for 4 months,and memantine was used as positive control drug.Nest building,elevated plus maze,Y maze and Morris water maze were used to observe the daily activities,anxiety and spatial learning and memory ability of mice respectively.The levels of inflammatory cytokine in peripheral blood of mice were detected by Luminex multi-factor detection technique,and the species diversity and functional diversity of gut microbiota of mice were observed using 16S rRNA sequencing and metagenome sequencing techniques.Results:1.The effecet of LW and LW-AFC on the anxity and learning and memoryThe results of behavioral tests showed that,compared with SAMR1 mice,the ability of nest building in SAMP8 mice was decreased?P<0.01?.The open arm entries?P<0.05?and the total entries?P<0.01?in the elevated plus maze test was decreased.The entries of C arm in Y maze was decreased?P<0.05?.In the Morris water maze,the escape latency in lerning phase?P<0.05?and testing phase?P<0.01?were prolonged,and the number of crossing platform?P<0.01?and the time in target quadrant?P<0.01?in testing phase were reduced.These results indicated that SAMP8 has impaired daily activities and spatial learning and memory ability accompanied by elevated anxiety.Memantine,LW and LW-AFC had no effect on daily activities and spatial learning and memory ability of SAMP8,but LW could increase the number of open arm entries of SAMP8 mice in elevated plus maze test?P<0.05?,indicating that LW has the effect of improving anxiety of SAMP8 mice.2.The effecet of LW and LW-AFC on the level of inflammatory cytokine of SAMP8The detection results of nine inflammatory cytokine?IL-1?,IL-2,IL-6,IL-17,MCP-1,RANTES,M-CSF,IFN-y,TNF-??in plasma showed that,compared to SAMR1 mice,IL-6?P<0.05?,MCP-1?P<0.01?,RANTES?P<0.01?,M-CSF?P<0.01?,IFN-y?P<0.05?and TNF-a?P<0.05?in SAMP8 mice were higher.LW could reduce the levels of MCP-1?P<0.01?,RANTES?P<0.05?and IFN-??P<0.01?;LW-AFC could reduce the level of IFN-y?P<0.05?.These results showed that SAMP8 mice have immune inflammatory reaction,and LW and LW-AFC could improve it.3.The effecet of LW and LW-AFC on the bacterial community compositionResults of 16S rRNA sequencing showed that,compared to SAMRl mice,the alpha diversity of gut microbiota in SAMP8 mice decreased?P<0.05?and the microbiota structure of SAMP8 was abnormal.On the one hand,three genera did not appear in the SAMR1;On the other hand,the abundance of 16 genera has changed,of which 12 genera were up-regulated?P<0.05?and 4 genera were down-regulated?P<0.05?.LW increased alpha diversity?Chao 1,P<0.05,Shannon,P<0.001?and increased the abundance of Rikenellaceae RC9 gut group?P<0.01?in gut microbiota of SAMP8 mice;LW-AFC also increased alpha diversity?Shannon,P<0.001?and decreased the abundance of[Eubacterium]coprostanoligenesgroup in the gut microbiota of SAMP8 mice?P<0.05?.4.The effecet of LW and LW-AFC on the bacterial function compositionMetagenome sequencing results were used to obtain the function of gut microbiota in EggNOG,KEGG and CAZy databases.The results showed that,compared to SAMR1 mice,the functional structure of gut microbiota in SAMP8 mice was changed.Based on EggNOG database,20426 NOGs were found in the four groups,328 NOGs were unique to SAMR1 group,129 NOGs were unique to SAMP8 group,137 NOGs were unique to LW group,and 1359 NOGs were unique to LW-AFC group;Compared to SAMR1 mice,the abundance of 10 clusters of orthologous groups of proteins?COG?in the gut microbiota of S AMP8 mice decreased?P<0.05?and the abundance of 7 COG in the SAMP8 mice increased?P<0.05?and this 17 different COG belong to four categories of functions:function G,function C,function O and unknown function S.LW had no significont effect on gene abundance of all differentially abundant COG in gut microbiota of SAMP8 mice.However,LW-AFC significantly decreased the abundance of COG5016,COG 1720 and ENOG41123X3,and significantly increased the abundance of COG 1904,ENOG410YFAX,ENOG410XT2F,ENOG410ZNV6 and ENOG410Y1WP?P<0.05?.Based on KEGG database,4539 KOs were found in the four groups,44 KOs were unique to SAMR1,51 KOs were unique to SAMP8,27 KOs were unique to LW and 1181 KOs were unique to LW-AF C.Based on CAZy,a professional database of carbohydrate-active enzymes?CAZymes?,421 CAZymes were found in the four groups.SAMR1,SAMP8 and LW groups had no unique CAZymes,while 5 CAZymes were unique to LW-AFC.Compared to SAMR1,the abundance of 6 CAZymes including CE7,GH4312,GH1336,GT13,GH4318 and GH68 in SAMP8 was significantly decreased?P<0.05?,while the abundance of 7 CAZymes including GT39,CBM 12,CBM22,GT41,PL44,GH51 and CBM31 was significantly increased.L.W had no significantt effect on the abundance of all CAZymes genes in the gut microbiota of SAMP8 mice.However,LW-AFC significantly increased the abundance of GH4312,GH43 18 and decreased the abundance of GT39,GT41 and PL4 4 in SAMP8 mice.Conclusions:1.There were impaired ability of daily activity and spatial leranning and memory and increased anxiety-like behavior in SAMP8 mcie.LW could improve the anxiety of SAMP8 mice.2.SAMP8 had increased level of inflammatory cytokine and both LW and LW-AFC could improve it.3.The alpha diversity of gut microbiota in SAMP8 was decreased and the abudance of several genera in SAMP8 was imblanced.LW and LW-AFC could both increased the alpha diversity and balance the abundance of abnormal genera.4.The improved effect of LW on anxiety might through modulating the bacterial funcontion by its active fraction?LW-AFC?,The modulated bacterial funcontion then lead to the improved inflammation.Chapter ? The effect of LW-AFC,active fraction of LW-AFC and fecal microbiota transplantation on the learning amd memory of APP/PS1 mice.Method:8-month male APP/PS1 transgenic mice were used as AD model animals and wild type was used as control,the experiment was divided into 11 groups,that is,control group?control,deionized water was given by gavage?,model group?Model,given with deionized water?,positive drug group?Namzaric,model animals were given with Namzaric?,LW-AFC group?LW-AFC group,model animals were given with LW-AFC?,LWB-b+LWD-b group?B-b+D-b,model animals were given with two active fractions of LW-AFC,namely LWB-b and LWD-b?,CA-30 group?CA-30,model animals were given with CA-30?,and M?Control group?fecal microbiota from Conrol group was transplanted into model animals?,C?Model group?wild type was given with fecal microbiota from model group?,M?AFC group?fecal microbiota from LW-AFC group was transplanted into model animals?,M?Bb+Db group?fecal microbiota from B-b+D-b group was transplanted into model animals?,M?CA-30 group?fecal microbiota from CA-30 group was transplanted into model animals?.After 3 months of treatment,the anxiety,object recognition memory,and active avoidance response ability and spatial learning and memory ability of mice were observed by elevated plus maze,novel object recognition memory test,shuttle box and Morris water maze experiments respectively.Nissl staining and immunofluorescence staining were used to detect neuronal loss and A? deposition in mouse brain;Using 16s rRNA sequencing technology to obtain species diversity information of gut microbiota;The content of short-chain fatty acids?SCFAS?,a metabolite of gut microbiota in mice,was detected by GC-MSResults:1.The effect of LW-AFC,active fraction of LW-AFC and fecal microbiota transplantationon the learning amd memory of APP/PS1 mice.Results of behavioral tests showed that,compared to the control group,there was no statistical difference in the number of open arm entries in the elevated plus maze test.The preference index decreased?P<0.05?in the 24 hours test session of the novel object recognition memory test.The number of active avoidances?P<0.05?decreased on the 4th day of the training phase and the test phase?P<0.05?in the shuttle box test.Escape latency were prolonged?P<0.05?during the 2-5 day of the learning phase and the test phase in the Morris water maze test,the number of crossing platform?P<0.05?and the swimming distance?P<0.05?in the target quadrant was reduced in the test phase of Morris water maze test.Thses results indicated that the object recognition memory ability,active avoidance response ability and spatial learning memory ability of APP/PS1 transgenic mice were impaired.Namzaric could increase the number of active avoidances in the test session of shuttle box test?P<0.05?,shorten the escape latency?P<0.001?and increase the number of crossing platform?P<0.05?of APP/PS1 transgenic mice in the test phase of Morris water maze test;LW-AFC could improve the preference index?P<0.05?in the 24-hour test phase of novel object recognition experiment,increase the number of active avoidance?P<0.05?in the test phase of shuttle box test,and shorten the escape latency in the 3rd day of learning phase?P<0.05?and the test phase?P<0.05?of Morris watrer maze test.CA·30 could improve the preference index?P<0.01?in the 24-hour test phase of novel object recognition experiment and shorten the escape latency?P<0.01?in the test phase of Morris watrer maze test.B-b+D-b could improve the preference index?P<0.01?in the 24-hour test phase of novel object recognition experiment.These results indicated that LW-AFC,B-b+D-b and CA-30 could improve the object recognition memory ability of APP/PS1 transgenic mice,Namzaric,LW-AFC and CA-30 could improve the spatial learning and memory ability of APP/PS1 transgenic mice,and Namzaric and LW-AFC could improve the active avoidance response ability of APP/PS1 transgenic mice.Fecal microbiota from APP/PS1 transgenic mice could significantly prolong the escape latency?P<0.05?of wild type mice in the test phase of Morris water maze.Fecal microbiota from wild-type mice could significantly increase the preference index?P<0.01?of APP/PS1 transgenic mice in the 24-hour test phase of novel object recognition experiment,and significantly shorten the escape latency of APP/PS1 transgenic in the 2-4th day of learning phase and test phase in the Morris water maze test?P<0.05?.These results indicated that the gut microbiota of AD model mice could lead to the deterioration of object recognition memory and spatial learning and memory ability of wild type mice,while the gut microbiota of wild type mice could improve the spatial learning and memory ability of AD model mice.Fecal microbiota from APP/PS1 transgenic mice treatment with LW-AFC,B-b+D-b and CA-30 for three months were transplanted into APP/PS1 transgenic mice respectively.All of them could improve the preference index?P<0.01?of APP/PS1 transgenic mice in the 24-hour test phase of novel object recognition experiment and shorten the escape latency?P<0.05?of APP/PS1 transgenic mice in the test phase of Morris water maze.These results showed that,on the one hand,gut microbiota of LW-AFC,B-b+D-b,and CA-30 group could improve the object recognition memory ability and spatial learning and memory ability of APP/PS1 transgenic mice.On the other hand,LW-AFC and CA-30 improved the object recognition memory ability and spatial learning and memory of APP/PS1 transgenic mice via modulating gut microbiota,B-b+D-b improved the object recognition memory ability of APP/PS1 transgenic mice via modulating gut microbiota.2.The effect of LW-AFC,active fraction of LW-AFC and fecal microbiota transplantationon the Nissl body and A? plaque in the hippocampus of APP/PS1 mice.Nissl staining and A? immunofluorescence staining showed that compared to the control group,the number of Nissl bodies in the hippocampus of the model group mice decreased but there was no statistical difference.No positive signal of A? plaque was detected in the control group mice,while a large number of A? plaque was detected in the model group mice.APP/PS1 transgenic mice treated with LW-AFC,B-b+D-b and CA-30 have reduced A? plaque area in hippocampus?P<0.05?,and Namzaric,LW-AFC,B-b+D-b and CA-30 all increased the number of Nissl bodies?P<0.01?in hippocampus of APP/PS1 transgenic mice.Fecal microbiota from wild-type mice or APP/PS1 transgenic mice did not significantly affect the area of A? plaque and Nissl bodies in hippocampus.However,APP/PS1 transgenic mice treated with fecal microbiota from mice treated with LW-AFC,B-b+D-b or CA-30 have increased number of Nissl bodies?P<0.05?and reduced A? plaque deposition?P<0.05?in hippocampus.These results indicated that LW-AFC,B-b+D-b and CA-30 increased the number of Nissl bodies and reduced the deposition of A? plaque in hippocampus of APP/PS1 transgenic mice via modulating gut microbiota.3.The effect of LW-AFC,active fraction of LW-AFC and fecal microbiota transplantationon the gut bacterial composition.PCA analysis showed that the community structure of gut microbiota between the control group and the model group were significantly different.The fecal microbiota from wild-type mice could make the gut bacterial composition of APP/PS1 transgenic mice similar to that of wild-type mice,while the gut bacterial composition of wild-type mice transplanted with fecal microbiota from APP/PS1 transgenic mice was similar to that of APP/PS1 transgenic mice.APP/PS1 transgenic mice treated with LW-AFC,LWB-b+D-B-b or CA-30 and APP/PS1 transgenic mice transplanted with the fecal microbiota from LW-AFC,LWB-b D-B-b or CA-30 group all showed a similar bacterial composition to wild-type mice.Compared with the control group,the abundance of 9 genera decreased?P<0.05?and the abundance of 6 genera increased?P<0.05?in the model group.In the APP/PS1 transgenic mice treated with LW-AFC,LWB-b+D-B-b or CA-30,the abundance of Alloprevotella was decreased and the abundance of LachnospiraceaeNK4A136group was increased.In the APP/PS1 transgenic mice treated with LW-AFC or LWB-b+D-B-b,the abundance of unclassifiedfLachnospiraceae and norankfLachnospiraceae was increased.The abundance of LachnospiraceaeNK4A136group,unclassifiedfLachnospiraceae,norankfLachno.spiraceae,Mucispirillum and norankfRuminococccaceae were significantly increased?P<0.05?while the abundance of Faecalibaculum was significantly decreased?P<0.01?in APP/PS1 transgenic mice treated with fecal microbiota from wild-type mice.The abundance of Alloprevotella,Eubacterium]fissicatenagroup,Eubacterium]coprostanoligen was significantly increased?P<0.05?and the abundance of unclassifiedfLachnospiraceae,norankfLachnospiraceae,norankfDesul.fovibrionaceae,Ruminiclostridium9,Mucispirillum,CoulddidatusSaccharimonas,norankfRuminococcaceae,and Roseburia significantly decreased?P<0.05?in the wild-type mice treated with fecal microbiota from APP/PS1 transgenic mice.Fecal microbiota from APP/PS1 transgenic mice treated with LW-AFC decrease the abundance of Alloprevotella and increasehe abundance of Lachnospiraceae NK4A136group and norankfLachnospiraceae in APP/PS1 transgenic mice.Fecal microbiota from APP/PS1 transgenic mice treated with LWBb+Db could up-regulate the abundance of LachnospiraceaeNK4A136group?P<0.01?.Fecal microbiota from APP/PS 1 transgenic mice treated with CA-30 could reduce the abundance of Alloprevotella and increase the abundance of LachnospiraceaeNK4A136group?P<0.05?.These results suggested that unclassifiedfLachnospiraceae,norankfLachnospiraceae,Mucispirillum and norankfRuminococccaceae in gut microbiota of APP/PS1 transgenic mice and wild-type mice may be the reasons for the differences in phenotypes between the two.It was also suggested that LW-AFC up-regulated the abundance of LachnospiraceaeNK4 A13 6group and norankfLachnospiraceae and down-regulated the abundance of Alloprevotella in gut microbiota,CA-30 up-regulated the abundance of LachnospiraceaeNK4A136group and down-regulated the abundance of Alloprevotella,and B-b+D-B up-regulated the abundance of LachnospiraceaeNK4A136group,which may be the way of LW-AFC,CA-30 and B-b+D-B to improve the learning and memory ability of APP/PS 1 transgenic mice,respectively.4.The effect of LW-AFC,active fraction and fecal microbitoa transplantation on the bacterial metabolites.Targeted metabonomic analysis using GC-MS showed that compared to the control group,the contents of acetate and propionate in feces of APP/PS 1 transgenic mice did not change significantly,while the contents of butyrate increased significantly?P<0.05?,and the contents of isobutyrate,isovalerate,valerate,isocaproate and caproate decreased significantly?P<0.05?.The content of butyrate?P<0.05?was decreased in APP/PS 1 transgenic mice treated with CA-30,the content of butyrate?P<0.05?was decreased and the content of isovalerate?P<0.05?was increased in APP/PS 1 transgenic mice treated with LWB-b+D-b.The contents of butyrate,isobutyrate,valerate and caproate in APP/PS 1 transgenic mice transplanted with fecal microbiota from wild-type mice was decreased?P<0.05?,while the contents of isobutyrate,isovalerate,valerate,isocaproate and caproate in wild-type mice treated with fecal microbiota from APP/PS1 transgenic mice was decreased?P<0.05?.The content of butyric acid,valerate and caproate in APP/PS1 transgenic mice transplanted with fecal microbiota from LW-AFC group was reduced.The content of isobutyrate?P<0.05?,isovalerate?P<0.01?,valerate?P<0.05?and isocaproate?P<0.01?was increased and the contents of acetate and butyrate?P<0.05?was decreased in the APP/PS1 transgenic mice transplanted with fecal microbiota from B-b+D-B-b group.The contents of butyrate were decreased in APP/PS1 transgenic mice transplanted with fecal microbiota from CA-30 group.These results suggested that gut microbiota may affect immune inflammation through its metabolites including caproate,valerate and isobutyrate,which leads to the difference of pathology and learning and memory ability between APP/PS 1 transgenic mice and wild-type mice.Butyrate and isovalerate might be the mediators for B-b+D-b to improve learning and memory ability.Butyrate may be the mediators for CA-30 to improve learning and memory ability.Conclusions:1.The object recognition memory ability,active avoidence and spatilal learing and memory ability of APP/PS1 mice were impaired;the fecal microbiota from APP/PS1 mice could result in impairement in object recognition memory ability and spatilal learing and memory ability of C57 mice while the fecal microbiota from C57 mice could improve the spatilal learing and memory ability of APP/PS1 mice.2.LW-AFC,B-b+D-b and CA-30 could improve object recognition memory ability of APP/PS1 mice;Namzaric,LW-AFC and CA-30 could improve the spatilal learing and memory ability of APP/PS1 mice;Namzaric and LW-AFC could improve active avoidance of APP/PS1 mice.The improved effect of LW-AFC,CA-30 and B-b+D-b on the learing and memory ability,Nissl body and A? plaque were through modulating gut microbiota.3.The gut bacterial community composition was significantly different between APP/PS1 and C57 mice;LW-AFC,LWB-b+D-B-b,CA-30 and the fecal microbiota from mice treated with LW-AFC,LWB-b+D-B-b or CA-30 could modulate the bacterial composition of APP/PS1 mice similar to that of C57 mice.Gut microbitoa may induce the pathologic phenomenon and impaired learning and memory in APP/PS1 mice by the bacterial metabolites.Chapter ? The effect of co-housing on the anxiety and learning and memory of SAM mice.Method:Six-month-old male SAMP8 mice were used as model animals and SAMR1 mice were used as control animals.Co-housed SAMP8 with SAMR1 mice for 6 months.S pontaneous locomotor activity test,nest building test,grading score for evaluation of the degree of senescence,open field test,elevated plus maze test and Morris water maze test were used to evaluate the spontaneous locomotor activity,daily activity,aging degree,anxiety and spatial learning and memory ability of mice respectively.16S rRNA sequencing technology was used to observe the species diversity of gut microbiota in mice.Results:1.The effect of co-housing on the anxiety and learning and memory of SAM mice.Compared to SAMR1 mice,the nesting ability of SAMP8 mice was decreased and the aging degee was increased.The central time and central distance in open field test and the open arm entries in elevated plus maze of SAMP8 were reduced.In the morris water maze,the escape latency in learning phase and test phase of SAMP 8 were both prolonged,and the platform entries were also reduced.These results showed that the daily activity and spatial learning and memory ability of SAMP8 mice were impaired,and SAMP8 mice had accelerated aging process and increased anxiety.Co-housing had no effect on daily activities,aging process,anxity and spatial learning and memory ability of the SAMP8 mice,while increase the aging degree?P<0.01?,decreased the central time?P<0.01?and central distance?P<0.001?in the SAMR1 mice.Besides,co-housing prolonged the escape latency of SAMR1 mice in 2nd and 5th day of learning phase and test phase while also decreased the platform entries?P<0.01?of SAMR1 mice.These results indicated co-housing induced impaired spatial learning and memory,accelerated senescence and anxiety in SAMR1 mice.2.The effect of co-housing on gut microbitoa16S rRNA sequencing showed that the bacterial community composition between SAMR1 and SAMP8 mice was significantly different.There were 24 differentially abundant genera between SAMR1 and SAMP8 mice.Compared with SAMR1,the abundance of Rikenella,norankfRs-E47termitegroup,RuminococcaceaeUCG-010 and norankcCyanobacteria in SAMP8 was significantly reduced?P<0.05?.After co-housing,the abundance of Rikenella,norankfRs-E47termitegroup,RuminococcaceaeUCG-010 and norankcCyanobacteria was significantly reduced in SAMR1 mice.Furthermore,the correlation analysis showed that the abundance of four genera,that is,Rikenella,norankfRs-E47termitegroup,RuminococcaceaeUCG-010 and norankcCyanobacteria were significantly correlated with the central time?P<0.05?and the central distance?P<0.05?of mice in the open field test.Conclusion:There were significantt differences in gut bacterical community and abundance of severa specific genera between SAMR1 and SAMP8 mice.Co-hosuing might induced accelerated senescense,anxiety and impaired spatial learning andmemory via chaning the gut microbiota in SAMR1 mice. |
PDF Full Text Request