The Effects Of Root-securing And Brain-fortifying Method On Intestinal Flora And Neuroinflammation In AD Model Animals Based On The Theory Of "Gut-Brain" Axis

Purpose:Based on the theory of gut-brain axis,this paper discusses the effects of Chinese medicine（TCM）of Root-Securing and Brain-Fortifying（RSBF）method on intestinal flora and neuroinflammation in AD model rats.Method:1.Theoretical research:searching the database and consulting books,summarizing the modern research related to AD and the theory of Chinese medicine,exploring the relationship between AD and intestinal microbes,dementia and spleen and stomach,in order to explain the application value of gut-brain axis theory in the diagnosis and treatment of AD.2.Experimental research:Experiment 1:50 SD rats were randomly divided into normal Control group,Model group,TCM-low group,TCM-high group and Positive control group.The AD animal model was established by bilateral hippocampus injection of Aβ_25-35.On the day after the model was established,the TCM-low group（1.035 g/kg/d）and the TCM-high group（2.070 g/kg/d）were given the Chinese medicine of Root-Securing and Brain-Fortifying method,and the control group and the model group were given the same volume of normal saline by gavage for 2 months.Morris water maze was used to observe the learning and memory ability of rats,Nissl staining was used to observe hippocampal neurons,immunofluorescence was used to observe the expression of Aβin CA1 area of hippocampus,and western blot was used to observe the expression of Aβand BACE1 protein in hippocampus.Experiment 2:16S r RNA was used to detect the fecal flora of rats in experiment 1,and the expression of component of E.coli in cortex was observed by immunohistochemistry,and the expression of component of E.coli in hippocampus was observed by western blot.Experiment 3:SD rats were gavaged with low-dose Chinese medicine of Root-Securing and Brain-Fortifying method to prepare medicated serum.BV-2 cells pretreated 1%and 10%medicated serum for 2 hours,and then added 1μg/ml LPS to intervene for 24 hours.CCK-8 method was used to observe the effect of serum containing Chinese medicine of Root-Securing and Brain-Fortifying method on BV-2 cell activity.Immunofluorescence was used to observe the expression of NF-κB p65 and Iba-1 protein in CA1 region of hippocampus and p-NF-κB p65 protein in BV-2 cell.Western blot was used to observe the expression of TLR4/TRAF6/NF-κB pathway related proteins in hippocampus and BV-2 cell of experiment 1.The expression of TLR4 and TNF-αm RNA in hippocampus and IL-1βand TNF-αm RNA in BV-2 cells were observed by q RT-PCR,and the content of IL-1βin rat serum was observed by ELISA.Experiment 4:30 SD rats were randomly divided into TCM-low group,minocycline group and lipopolysaccharide（LPS）group.AD animals were established by injecting Aβ_25-35into bilateral hippocampus.From the day of modeling,rats in the TCM-low group,minocycline group and LPS group were gavaged with low-dose TCM of Root-Securing and Brain-Fortifying method（1.035 g/kg/d）.On the basis of Chinese medicine,minocycline group added 50mg/kg/d minocycline into drinking water,which started on the day of modeling and lasted for 11 days,while lipopolysaccharide group injected LPS7mg/kg/time intraperitoneally,which started after modeling and lasted for 7days.The expression of NF-κB p65 and Iba-1 protein in CA1 area of hippocampus was observed by immunofluorescence,the expression of TLR4/TRAF6/NF-κB pathway related protein in hippocampus was observed by western blot,the expression of TLR4 and TNF-αm RNA in hippocampus was detected by q RT-PCR,and the content of IL-1βin rat serum was detected by ELISA.Results:1.Results of Experiment 1（1）Morris water maze resultsCompared with the control group,the escape latency of AD rats in the model group was prolonged（P<0.01）,and the times of crossing the platform were reduced（P<0.01）.Compared with the model group,the escape latency of TCM-low group and TCM-high group decreased（P<0.05 or P<0.01）,and the times of crossing the platform in TCM-low group increased（P<0.05）.（2）Nissl staining resultsCompared with the control group,the Nissl bodies of neurons in CA1 area of hippocampus in model group decreased.Compared with the model group,the number of Nissl bodies in CA1 area in TCM-low group and TCM-high group increased and the shape was more regular.（3）Immunofluorescence resultsCompared with the control group,the expression of Aβin CA1 area of hippocampus in model group increased（P<0.01）.Compared with the model group,the expression of Aβin CA1 area of hippocampus in TCM-low group and TCM-high group decreased（P<0.01,P<0.05）.（4）western blot resultsCompared with the control group,the expression of Aβand BACE1 in hippocampus of model group increased（P<0.05 or P<0.01）.Compared with the model group,the expression of Aβand BACE1 in hippocampus of TCM-low group and TCM-high group decreased（P<0.05 or P<0.01）.2.Results of experiment 2（1）16S r RNA experimental resultsIn terms ofα diversity,compared with the control group,the Chao index and Shannon index of the model group increased significantly（P<0.01）,while the Simpson index decreased slightly（P>0.05）.Compared with the model group,the Chao index,Shannon index and Simpson index of low-dose group and high-dose group had no significant changes（P>0.05）.In terms ofβ diversity,PCA analysis showed that there was partial overlap between TCM-low group,TCM-high group and model group.In PCOA analysis,there was no obvious overlap between the TCM-low group and the model group,but there was partial overlap between the TCM-high group and the model group.Compared with the control group,there was no significant change in flora composition,Phylum level in TCM-low group,TCM-high group and positive control group（P>0.01）.Compared with the model group,the proportion of Firmicutes in the high-dose group of Chinese medicine of Root-Securing and Brain-Fortifying method increased（P<0.01）,while Firmicutes in the positive control group decreased（P<0.01）,and the proportion of Bacteroidota increased significantly（P<0.01）.At the Genus level,compared with the normal control,Alloprevotella,Lactobacillus,Ligilactobacillus,Muribaculaceae＿norank,Prevotella＿9,Prevotellaceae Ga6A1 group,Prevotellaceae UCG-001,Prevotellaceae UCG-003,and Romboutsia in the model group showed significant changes（P<0.05 or P<0.01）.Compared with the model group,the TCM-low group and TCM-high group of Chinese medicineof Root-Securing and Brain-Fortifying method showed significant changes in the Lachnospiraceae NK4A136 group,Muribaculaceae＿norank and Prevost＿9（all P<0.01）.In the combination analysis of sample cluster tree and histogram,there are obvious differences between normal control group and other groups.AD modeling groups are clustered together,among which model group is clustered with high dose group,TCM-low group and positive control group.Compared with the control group,there was no significant change in the prediction of PICRUSt2 metabolic function in the model group（P>0.05）.Compared with the model group,there was no significant change in TCM-low group and TCM-high group（P>0.05）.（2）western blot resultsCompared with the control group,the content of E.coli in hippocampus of model group increased significantly（P<0.01）.Compared with the model group,the content of E.coli components in TCM-low group,TCM-high group and positive control group decreased significantly（all P<0.01）.（3）Immunohistochemical resultsCompared with the control group,the content of E.coli in cortex of rats in model group increased significantly（P<0.01）.Compared with the control group,the contents of E.coli components in TCM-low group,TCM-high group and positive control group of Chinese medicine of oot-Securing and Brain-Fortifying method were significantly decreased（all P<0.01）.3.Results of Experiment 3（1）CCK-8 experimental resultsIn CCK-8 experiment,compared with the control group without medicated serum,the increase of RSBF method medicated serum concentration had no significant inhibitory effect on the activity of BV-2 cells（P>0.05）.（2）western blot resultsIn vivo experiments,compared with the control group,the expressions of TLR4,TRAF6,p-NF-κB p65 and IL-1βin the hippocampus of the model group were significantly increased by western blot（P<0.01）.Compared with the model group,the expression of TLR4 and p-NF-κB p65 in the TCM-low group decreased（all P<0.01）,TRAF6 and IL-1βdecreased slightly（P>0.05）,and that in the TCM-high group decreased（P<0.05）.In vitro,compared with the control group,the expression of TLR4,TRAF6,p-NF-κB p65 and IL-1βin hippocampus of model group increased significantly（P<0.05 or P<0.01）.Compared with the model group,the expressions of TLR4,TRAF6,p-NF-κB p65 and IL-1βin 1%and 10%medicated serum groups decreased（P<0.05 or P<0.01）.（3）Immunofluorescence resultsIn vivo,compared with the control group,the expression of NF-κB p65 and Iba-1 in hippocampal CA1 area of model group increased significantly（P<0.01）.Compared with the model group,the expression of NF-κB p65 and Iba-1 in the TCM-low group decreased（P<0.05 or P<0.01）.In vitro,compared with the normal control group,the expression of p-NF-κB p65 in the model group increased（P<0.01）.Compared with the model group,the expression of p-NF-κB p65 in 1%and 10%medicated serum decreased（both P<0.01）.（4）q RT-PCR resultsIn vivo,compared with the control group,the expression of TLR4 and TNF-αm RNA in the model group increased（P<0.01）.Compared with the model group,the expression of TLR4 and TNF-αm RNA in TCM-low group and TCM-high group decreased（P<0.05 or P<0.01）.In vitro experiments,compared with the control group,the expression of IL-1βand TNF-αm RNA in the model group increased（P<0.01）.Compared with the model group,the expression of IL-1βm RNA in 1%medicated serum group decreased（P<0.01）,and the expression of TNF-αm RNA in 1%and10%medicated serum groups decreased（both P<0.01）.（5）ELISA resultsIn vivo,compared with the control group,the serum IL-1βcontent in the model group increased significantly（P<0.01）.Compared with the model group,the levels of serum IL-1βin TCM-low group,TCM-high group and positive control group all decreased（all P<0.01）.4.Results of experiment 4（1）Morris water maze resultsCompared with the TCM-low group,the escape latency of minocycline group and LPS group was prolonged（P<0.05 or P<0.01）,the times of crossing the platform in minocycline group decreased（P<0.01）,and the times of crossing the platform in LPS group decreased slightly（P>0.05）.（2）Immunofluorescence resultsCompared with the TCM-low group,the expressions of Iba-1 and NF-κB p65in CA1 area of hippocampus in minocycline group and LPS group decreased（all P<0.01）,while those in CA1 area of hippocampus in lipopolysaccharide group increased（all P<0.01）.（3）western blot resultsCompared with the low-dose group,the expression of TLR4,TRAF6,p-NF-κB p65 and IL-1βin hippocampus of minocycline group decreased（P<0.05 or P<0.01）,while the expression of TLR4,TRAF6 and IL-1βin CA1area of hippocampus of LPS group increased（P<0.01）.There was no significant change in the expression ofp-NF-κB p65（P>0.05）.（4）q RT-PCR resultsCompared with the TCM-low group,the expression of TLR4 and TNF-αm RNA in hippocampus of minocycline group decreased（P<0.01）,while the expression of TLR4 m RNA in hippocampus of LPS group had no significant change（P>0.05）,but the expression of TNF-αm RNA increased（P<0.01）.（5）ELISA resultsCompared with the TCM-low group,the content of serum IL-1βin minocycline group decreased（P<0.01）,while that in LPS group increased（P<0.01）.Conclusion:1.Treating AD rats with Chinese medicine of Root-Securing and Brain-Fortifying method can improve the learning and memory function,inhibit the expression of Aβand reduce the loss of Nissl bodies of neurons;2.Chinese medicine of Root-Securing and Brain-Fortifying method has a positive regulatory effect on the intestinal flora of AD rats and reduce the content of E.coli in hippocampus and cortex;3.Chinese medicine or medicated serum of Root-Securing and Brain-Fortifying method can effectively improve neuroinflammation,and its mechanism may be related to the activation of TLR4/TRAF6/NF-κB pathway in which microglia participate,especially the regulation of the function of microglia by Chinese medicine of Root-Securing and Brain-Fortifying method.