| Objective:Alzheimer's disease(Alzheimer's disease,AD)is a common multi-factor central nervous system degenerative disease.At present,it has been proved that a variety of new anti-AD drugs aimed at a single target are ineffective,and traditional Chinese medicine has the advantage of multi-target pharmacological activity,so it is of great significance to seek effective TCM treatment and classical prescriptions for the prevention and treatment of AD.Based on this study,RSBFL was used to interfere with APP/PS1 double transgenic AD model mice,and the effects of RSBFL on intestinal flora,intestinal permeability and related indexes of hippocampus were observed.Then the germ-free mouse model was established,and the fecal bacteria of mice in RSBFL intervention group were transplanted into germ-free mouse model to observe whether RSBFL affected the deposition of A? by improving intestinal flora,and to further explore the mechanism of RSBFL in the treatment of AD,in order to provide new ideas for clinical prevention and treatment of AD.Methods:1.Theoretical discussion: conduct literature research,sort out the theory of TCM understanding of AD from Spleen-Stomach Theory and the correlation between Spleen-Stomach Theory and intestinal flora,on this basis,systematically explain the therapeutic mechanism of RSBFL on AD.2.Experimental study: experiment 1: 50 6-month-old APP/PS1 mice were randomly divided into 5 groups: model group(Model),high dose of RSBFL(RSBFL-H),medium(RSBFL-M)and low(RSBFL-L)groups,and equal-month-old gene negative C57/BL6 mice were used as blank control group(Control),(n = 10).The corresponding RSBFL was given to each drug intervention group at the dose of 2ml/100g/d,and the same volume of pure water was given to the Control group and the Model group for 12 weeks.Morris water maze test was used to evaluate the learning and memory ability of mice,HE staining was used to observe the morphology of colon tissue and hippocampal neurons,immunohistochemistry was used to observe the expression of Occludin and Zo-1 in colon,and the expression of GDNF and GFR ?-1 in hippocampal DG region of mice in each group.The expression of Zo-1and occludin in colon and the expression of ? APP,A ? 1-42,BDNF and Tr KB in hippocampus of mice in each group were detected by Western Blot,the expression of GDNF and GFR ?-1m RNA in hippocampus of mice in each group was detected by Real Time-PCR,and the feces of mice in each group were sequenced by 16 Sr RNA technique to detect the difference of intestinal flora in each group.Experiment 2: 30 6-month-old APP/PS1 mice were fed with aseptic water containing four antibiotics(ampicillin 1g/L,neomycin sulfate 1g/L,metronidazole 1g/L,vancomycin 500mg/L)for one week to establish a germ-free mouse model.10 C57/BL6 mice of the same month in the same litter were used as blank control group,and then the feces of each group were sequenced by 16 Sr RNA technique to evaluate the effect of modeling.Experiment 3: the(FMT)experiment was carried out.Normal mouse feces(FMT-control),APP/PS1 mouse feces(FMT-model)and RSBFL high-dose mouse feces(FMT-RSBFL-H)were transplanted into germ-free mice respectively.and untreated blank control group(Control),were transplanted once a week for 8 weeks.Morris water maze test was used to evaluate the learning and memory ability of mice,HE staining was used to observe the morphology of colon tissue and hippocampal neurons,immunohistochemistry was used to observe the expression of Occludin and Zo-1 in colon,and the expression of GDNF and GFR ?-1 in hippocampal DG region of mice in each group.The expression of Zo-1 and occludin in colon and the expression of?APP,A?1-42,BDNF and Tr KB in hippocampus of mice in each group were detected by Western Blot,the expression of GDNF and GFR?-1m RNA in hippocampus of mice in each group was detected by Real Time-PCR,and the feces of mice in each group were sequenced by 16 Sr RNA technique to detect the difference of intestinal flora in each group after fecal bacteria transplantation.Result:1.Results of experiment 1(1)results of Morris water maze: compared with Control group,the escape latency and total swimming distance of mice in Model group were significantly increased,the times of crossing the original platform and the time of staying in the target quadrant were significantly decreased,and the time of arriving at the platform for the first time was significantly prolonged(P<0.01);Compared with Model group,the escape latency and total swimming distance of RSBFL-H group,RSBFL-M group and RSBFL-L group decreased in different degrees,while the times of crossing the original platform and the time of staying in the target quadrant were significantly increased,and the time of arriving at the platform for the first time was significantly decreased(P<0.01,P<0.05).(2)the results of HE staining: compared with the Control group,the number of neurons in the DG and CA3 regions of the hippocampus in the Model group decreased significantly(P<0.01),slight edema of the colon,infiltration of inflammatory cells in the intestinal epithelium and lamina propria,and mild inflammatory reaction(P< 0.01).Compared with Model group,the number of neurons in hippocampal DG region and CA3 area in RSBFL-H group,RSBFL-M group and RSBFL-L group increased in different degrees(P<0.01,P<0.05),the degree of colonic swelling decreased significantly,the morphology of villi was complete and neatly arranged,and the intestinal epithelium was only slightly exfoliated.The integrity of intestinal mucosa in RSBFL-H group was better than that in RSBFL-M group and RSBFL-L group(P<0.01).(3)Immunohistochemical results: compared with Control group,the expression of GDNF and GFR ?-1 in hippocampal DG region,Zo-1 and occludin expression in colon decreased significantly in Model group(P<0.01);Compared with Model group,the expression of GDNF and GFR ?-1 in hippocampal DG region increased significantly in RSBFL-H group and RSBFL-M group(P<0.05),the expression of GDNF and GFR ?-1 in DG area did not change significantly in RSBFL-L group(P>0.05),but the expression of Zo-1 and occludin in colon increased in different degrees in RSBFL-H group,RSBFL-M group and RSBFL-L group(P<0.01,P<0.05).(4)the results of Western Blot experiment: compared with Control group,the expression of ?APP and A?1-42 protein in hippocampus of Model group was significantly increased,while the expression of BDNF and Tr KB protein was significantly decreased,and the expression of Zo-1 and occludin protein in colon was significantly decreased in Model group(P<0.01).Compared with Model group,the expression of ?APP and A?1-42 protein in hippocampus of RSBFL-H group,RSBFL-M group and RSBFL-L group decreased in different degrees,while the expression of BDNF and Tr KB protein increased in different degrees,and the expression of Zo-1 and occludin protein in colon increased in different degrees(P<0.01,P<0.05).(5)the results of Real Time-PCR test: compared with Control group,the expression of GDNF and GFR-A1 m RNA in hippocampus of Model group decreased significantly(P<0.01),while the expression of GDNF and GFR ?-1m RNA in RSBFL-H group,RSBFL-M group and RSBFL-L group increased in different degrees compared with Model group(P<0.01,P<0.05).(6)16Sr RNA test results: compared with Control group,Simpson index,Shannon index and Chao1 index in Model group were lower than those in Control group(P<0.05),and intestinal Simpson index,Shannon index and Chao1 index in RSBFL-H group,RSBFL-M group and RSBFL-L group were higher than those in Model group(P<0.05).Compared with Control group,the intestinal microflora of Model group was down-regulated in bacteroidetes phylum and up-regulated in Proteobacteria(P<0.05),while that of RSBFL-H group,RSBFL-M group and RSBFL-L group has up-regulated in bacteroidetes and down-regulated in Proteobacteria(P<0.05).In the classification level of families and genera,compared with Control group,Desulfoviridae was up-regulated and Lactobacillus(Lactobacillus)was down-regulated in Model group(P<0.05);Compared with Model group,Desulfoviridae in RSBFL-H group,RSBFL-M group and RSBFL-L group were down-regulated in different degrees,and Lactobacillaceae were all up-regulated in different degrees(P<0.05).2.The results of experiment 2showed that after drinking antibiotic water for one week,the Simpson index,Shannon index and Chao1 index of GF group were lower than those of Control group,and there was significant difference between GF group and Control group(P<0.05);and the diversity and richness of intestinal flora in GF group decreased significantly at the five classification levels of phylum,class,order,family and genus(P<0.05).3.Results of experiment 3(1)results of Morris water maze: compared with Control group,the escape latency and total swimming distance of mice in FMT-Model group were significantly increased(P<0.01),the times of crossing the original platform and the time of staying in the target quadrant were significantly decreased(P<0.01),and the time of arriving at the platform for the first time was significantly prolonged(P< 0.01).Compared with FMT-Model group,the escape latency and total swimming distance of mice in FMT-Control group and FMT-RSBFL-H group were decreased(P<0.05),the times of crossing the original platform and the time of staying in the target quadrant were significantly increased(P <0.05),and the time of arriving at the platform for the first time was significantly decreased(P<0.05).(2)HE staining results: compared with Control group,the number of neurons in DG and CA3 region of hippocampus in FMT-Model group decreased significantly(P<0.05),while in FMT-Model group,the colon showed slight edema and inflammatory cell infiltration in intestinal epithelium and lamina propria showed mild inflammatory reaction(P<0.05);Compared with FMT-Model group,the number of neurons in hippocampal DG region and CA3 area in FMT-Control group and FMT-RSBFL-H group was significantly increased(P<0.05),the degree of colon swelling was significantly decreased,the intestinal epithelium was only slightly exfoliated,and the morphology of villi was more intact(P< 0.05).(3)Immunohistochemical results: compared with Control group,the expression of GDNF and GFR ?-1 in hippocampus and the expression of Zo-1 and occludin in colon tissue of FMT-Model group decreased significantly,and the distribution was sparse(P<0.05).Compared with FMT-Model group,the expression of GDNF and GFR ?-1 in hippocampal tissue of FMT-Control group and FMT-RSBFL-H group was significantly higher,while the expression of Zo-1 and occludin in colon tissue was significantly higher and denser(P<0.05).(4)the results of Western Blot experiment: compared with Control group,the expression of ?APP and A?1-42 protein in hippocampus of FMT-Model group was significantly increased,the expression of BDNF and Tr KB protein in hippocampus was significantly decreased(P<0.01),and the expression of Zo-1 and occludin protein in colon was significantly decreased in FMT-Model group(P< 0.01).Compared with FMT-Model group,the expression of ?APP and A?1-42 protein in hippocampus of FMT-Control group and FMT-RSBFL-H group decreased,while the expression of BDNF and Tr KB protein increased in different degree(P<0.01,P<0.05).The expression of Zo-1and occludin protein in colon increased in different degree(P<0.01,P<0.05).(5)the results of Real Time-PCR experiment: the expression of GDNF and GFR ?-1m RNA in hippocampus of FMT-Model group was significantly lower than that of Control group(P<0.01),while the expression of GDNF and GFR ?-1m RNA of FMT-Control group and FMT-RSBFL-H group was higher than that of FMT-Model group(P<0 05).(6)16Sr RNA test results: the results showed that the Simpson index,Shannon index and Chao1 index in FMT-Model group were lower than those in Control group,and there was significant difference between FMT-Model group and Control group(P<0.05).Compared with FMT-Model group,intestinal Simpson index,Shannon index and Chao1 index in FMT-Control group and FMT-RSBFL-H group increased in different degrees(P<0.05).Compared with Control group,the taxonomic level of intestinal flora in FMT-Model group has bacteroidetes down-regulated,Proteobacteria up-regulated,Desulfuricaceae up-regulated,Lactobacilli down-regulated,Helicobacter pylori up-regulated,Lactobacillus and Bacteroides down-regulated(P<0.05).Compared with FMT-Model group,the up-regulation of Bacteroides and Proteobacteria down-regulated,Desulfuricaceae,Helicobacter pylori down-regulated;Lactobacillus and Bacteroides in FMT-Control and FMT-RSBFL-H groups were up-regulated in different degrees(P<0.05).Conclusion:(1)the pathogenesis of AD is closely related to the acquired spleen and stomach,and the spleen and stomach and intestinal flora influence each other,so the treatment of AD can start from the spleen and stomach.(2)RSBFL can change the abundance and diversity of intestinal microflora in APP/PS1 mice.(3)RSBFL can improve the learning and memory ability of APP/PS1 mice,reverse the apoptosis of hippocampal neurons in AD model mice,reduce A ? deposition,improve the increase of colonic permeability in dementia model mice,and improve intestinal inflammation.(4)through the FMT experiment,it was found that the feces of mice in the high dose group could successfully replicate the intestinal flora structure of the high dose group(RSBFL-H group),and the fecal bacteria transplantation could also improve the learning and memory ability of APP/PS1 mice,reverse the apoptosis of hippocampal neurons,reduce A? deposition and improve intestinal inflammation in dementia model mice.It is proved that RSBFL can improve A? deposition and inhibit neuronal apoptosis so as to reverse the pathogenesis of AD by improving intestinal flora and regulating "brain-gut axis". |
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