Study On The Mechanism Of Signaling Molecules Regulating Neuroinflammation In Alzheimer’s Disease And The Intervention Of Huanglian Jiedu Decoction

BackgroundObjiectiveTo clarify the cascade mechanism of intestinal microflora,microbial metabolome,and central nervous inflammation in Alzheimer’s disease(AD);It is suggested that Huanglian Jiudu decoction can improve central inflammation by reconstructing intestinal structure,regulating the metabolism/absorption/distribution/excretion of SCFAs,amino acid or neurotransmitter and improving the interaction between central immune cells and Aβ.To clarify the key active components of HLJDD to intervene in different links of the "gut-brain" axis,synergistically exert the biological effects of antiAD neuroinflammation.Method1)Male B6SJL-Tg(APPSwF1Lon,Psen1*M146L*L286V)6799Vas/J transgenic(5×FAD)mice(5-months-old)were used.Male C57BL/6 mice(5-months-old)born in the same litter were selected as WT-M mice.These transgenic mice are widely used for their ability to reproduce the pathologic features of Alzheimer’s disease,including accelerated cognitive impairment,amyloid deposition at 2 months of age,synaptic degeneration at 4 months of age,and behavioral changes at 6 months of age.2)Four different methods were selected to comprehensively evaluate the cognition and emotion of 5 ×FAD mice.The open-field test and black and white box test were used to evaluate the motion state and mood changes of rodents.The Y-maze and Moriss water maze test were used to evaluate cognitive and memory behavior in rodents.3)Immunofluorescence was used to co-locate the Aβ,IBA and GFAP proteins in the brain of 5 ×FAD mice.The size and amount of β amyloid plaques,the morphological changes and activation degree of microglia and astrocytes,and the degree of microglia or astrocyte recruitment around Aβ plaques were observed.It was used to judge the phagocytosis of microglia or astrocytes to Aβ.4)The soluble and insoluble Aβ38,Aβ40 and Aβ42 in brain homogenate of 5 ×FAD mice were detected simultaneously by MSD hypersensitive multi-factor electrochemical detection technology.5)The contents of short-chain fatty acids(GFAP)in feces,peripheral blood and brain of 5 ×FAD mice were detected by gas phase mass spectrometry.The levels of amino acids or neurotransmitters in feces,peripheral blood and brain of 5 ×FAD mice were detected by UPLC-QQQ-MS/MS technique.And polyunsaturated fatty acids or oxidized lipids in the 5 ×FAD mice were measured by UPLC-QQQ-MS/MS.6)The 16S V3-V4 region was sequenced by pyrosequencing method to search for bacteria related to AD pathology and explore the effect of drug intervention on bacterial flora.7)PPARγ,ApoE4,DRP-1,HDAC6 and other indicators in tissues were detected by RT-PCR or Western Blot.8)Related immune cells(CD3,CD4,CD8),including Th1,Th2,etc.were detected in peripheral spleen of mice by flow cytometry.Result1)Rapid accumulation of Aβ plaque deposition,and excessive activation of microglia and astrocytes were observed in Tg-M mice compared to the aged-paired wild-Type(WT-M)mice.Followed with increased levels of inflammatory factors MCP-1,IL-1α,TNF-α and lipid peroxidation.Neuroinflammation occurred in the center of 5 ×FAD mice.2)The gut bacterial richness and α-diversity in feces of 5 ×FAD mice increased,and the structure of intestinal flora unbalanced.Firmicutes,P_Campilobacterota and P_Deferribacteerota increased,while the levels of Bacteroidetes,P Proteobacteria and P_actinobacteria decreased.The bacteria associated with SCFAs and proteolytic metabolism also had significant changes,including g_Odoribacter、g_Parasutterella、f_Ruminococcacea、f_Lachnospiraceae、f_Bifidobacteriaceae(g_Lachnospiraceae_NK4A136_group、g_unclassified_f_Lachnospiraceae、g_norank_f_Lachnospiraceae、g_Lachnospiraceae_UCG-001)。3)During the 6-month observation period,the content of SCFAs in the feces of WT-M and TG-M mice increased in a senescence dependent manner.From 5 to 11 months of age,the levels of AA,PA and VA in feces of TG-M mice were always lower than those of their litter WT-M mice.The contents of BA,IB A and IVA in feces of TGM mice aged from 5 to 9 months were lower than those of WT-M mice,but the trend of the levels of these three SCFAs between TG-M and WT-M groups was reversed from 10 months of age.From 8 months of age,the level of HA in feces of mice in TGM group was higher than that in WT-M group.4)From 5 to 11 months,the levels of 5-HT,phenylalanine,alanine,methionine,glutamine,arginine,lysine,tyrosine,leucine,proline,tryptophan,serine,and threonine in feces of WT-M and Tg-M mice showed an aging dependent increase.And showed that the difference in amino acids or neurotransmitters between the two groups increased as the aging process increased.5)The level of central Aβ and the activation of immune cells in female 5 ×FAD mice were much higher than that in male 5 ×FAD mice.And the cognitive impairment of the female 5 ×FAD mice was more severe.In addition,8 out of 17 female 5 ×FAD mice were overexcited and had a high mortality rate(6 out of 17)during feeding.Compared with Tg-M mice,the fecal microbial diversity of TG-F mice was lower,accompanied by a lower level of SCFAs.The fluctuation range was unstable during the observation period,and no obvious regular changes with time were found.6)After the intervention of antibiotic ABX,the diversity of intestinal microorganisms in the feces of 5 ×FAD mice was significantly reduced and the structure of intestinal flora was reshaped.Antibiotic ABX can decrease the expression of Aβ,IB A,GFAP and anti-inflammatory factors MCP-1,IL-1α,TNF-α and lipid peroxide in 5 ×FAD mice.7)The cliff-like decrease of SCFAs in fecal of 5×FAD mice after antibiotic ABX intervention was observed,but no significant time-dependent increase or decrease.Among them,BA,VA and IBA decreased the most.8)The levels of amino acids or neurotransmitters in the feces of 5 ×FAD mice were not rapidly inhibited after the intervention of antibiotic ABX,but the contents of amino acids and neurotransmitters in the feces were increased,especially in the early stage of the action of antibiotic ABX.Phenylalanine,alanine,methionine,glutamic acid,glutamine,arginine,lysine,leucine,tryptophan,aspartic acid,histidine,and GABA in feces of 5 ×FAD mice decreased in a time-dependent manner during the 6 months of ABX intervention.Among them,phenylalanine,methionine,glutamine,arginine,lysine,tyrosine,and tryptophan in feces of 5 ×FAD mice and WT-M mice had senescence dependent increase.9)HLJDD can reduce the frequency of defecation and urination in 5 ×FAD mice in open field test.HLJDD intervention.The escape latency of 5 ×FAD mice in the water maze training experiment was reduced,and the retention time and glide distance in the target quadrant were increased.H-H performed better than H-L in the water maze test,but the frequency of defecation in the open field test was greater in the H-H group than in the H-L group.In this experiment,berberine,baicalin and the Mix group can improve the cognitive impairment and anxiety in 5 ×FAD mice.The behavioral effects of theses tress drugs were like to those of HLJDD,and no obvious advantage were shown.10)After HLJDD administration,the levels of Aβ,IBA and GFAP in brain decreased in 5 ×FAD mice.The relief effect of H-H on Aβ,IBA and GFAP was greater than H-L.RT-PCR results showed that HLJDD reduced the levels of central inflammatory factors IL-1α,TNF-α and IFN-γ in a dose-dependent manner.After administration,berberine,baicalin and the Mix group could also down-regulate the levels of central Aβ deposition,IBA and GFAP in 5 ×FAD mice,but the overall downregulation degree was not as good as H-H.Baicalin down-regulated Aβ less than berberine and combination group,mainly in insoluble Aβ40,Aβ42 and Aβ plaques in hippocampus CA3 region.However,the reduction of baicalin on GFAP was stronger than that of berberine and Mix group.Berberine,baicalin and the Mix group had similar regulatory effects on central proinflammatory factors in 5 ×FAD mice.Conclusion1)This is the first time that we have studied longitudinal changes in intestinal SCFAs,amino acids or neurotransmitters with aging based on the gut microbiome,and compared the differences in intestinal metabolites between 5 ×FAD mice and wild-type male mice at different life points.During the observation period from 5 months to 11 months of age,SCFAs and most amino acids or neurotransmitters in feces of 5 ×FAD mice showed an aging-dependent increase.This study suggests that the metabolism of our intestinal group may be a predictor of aging,and it is hoped that it can be used to predict different inflection points of Alzheimer’s disease in a non-invasive manner,to better design the best treatment plan for different diseases.2)Intestinal microbial structure disorders change the synthesis,metabolism,and absorption of SCFAs,amino acids or neurotransmitters in the intestinal tract,and then affect their signal transduction between peripheral and central in 5 ×FAD mice.Weaken the phagocytosis of microglia to Aβ,then induce central nervous inflammation.3)AD like symptoms happened in 5 ×FAD mice differ between males and females.Female 5 ×FAD mice developed the disease earlier and the degree was more serious,mainly reflected in high mortality,high temper rate,central Aβ level and the activation degree of immune cells,as well as cognitive impairment were far more than male 5×FAD mice.4)HLJDD can regulate the metabolism of intestinal microbiome,affect the signal transmission of intestinal endogenous small molecules between the"gut-brain" axis,and increase the phagocytosis of microglia and astrocytes to Aβ plaques,thus alleviating the central nervous inflammation in 5 ×FAD mice and improving cognition and mood.The effect of H-H is better than H-L.Berberine,baicalin and genipin made certain contribution to HLJDD anti-neuroinflammation,but the effect of HLJDD was better than that of single or Mix drug.Gv-971 had an anti-neuroinflammatory effect comparable to that of H-H in treating 5×FAD mice.