Study On The Mechanism Of Cognitive Impairment In Mice With SAD Neuroinflammation By Improving Cerebral Lymphatic Function Of Meningeal Lymphatic Drainage With Danggui-Shaoyao-San

Sporadic Alzheimer’s disease(sAD)is an urgent public health problem in an aging population and there is still a lack of effective therapeutic drugs.sAD neuroinflammation hypothesis suggests that neuroinflammation is the hallmark pathological event of sAD and is the driver of sAD progression.In 2015,the discovery of the structure and function of human meningeal lymphatic vessels(MLVs)updated the understanding of the immune privilege of the central nervous system.MLVs are now recognized as important structures that regulate neuroinflammation.sAD belongs to the category of dementia in traditional Chinese medicine.Neuroinflammation is closely related to the pathological nature of phlegm-stasis pathogenesis in sAD.Danggui-Shaoyao-San(DSS)can nourish blood,invigorate blood,strengthen the spleen and relieve dampness,and has significant efficacy in the treatment of sAD.Improving neuroinflammation is one of the important mechanisms of action of DSS in the treatment of sAD.Increasing evidence suggests that DSS is likely to exert anti-sAD neuroinflammatory effects by improving the function of MLVs.Objective:To establish a mouse model of sAD neuroinflammation with MLVs dysfunction,and to elucidate the mechanism of sAD neuroinflammation exacerbated by MLVs draining brain lymphatic dysfunction.And to use DSS to intervene in the above disease model to explore the mechanism of action and pharmacodynamic material basis of this formula for treating sAD neuroinflammation.Methods:Part Ⅰ.Study on the mechanism of cognitive impairment in mice with sAD neuroinflammation exacerbated by draining cerebral lymphatic dysfunction of MLVs1.Mice were divided into Control group,Saline group,LPS group,LPS+vehicle+photoconversion group,LPS+veterpofin group,LPS+veterpofin+photoconversion group,and LPS-induced sAD neuroinflammation model was established according to the group,and 10 days later,MLVs ablation surgery was performed with sham surgery group as the control.Model testing was performed on postoperative day 7 using the Evans Blue tracer injection method in the cerebellar medullary pool to determine successful modeling.The mouse cortex,hippocampus and midbrain were taken on postoperative day 7,and the degree of activation of Iba1-marked microglia in each brain region was examined by immunofluorescence to determine the brain regions most sensitive to neuroinflammation caused by dysfunction of MLVs.2.Using sensitive brain regions as the study subjects,the expression levels of proinflammatory factor TNF-α and anti-inflammatory factor YM-1 at the same level in sensitive brain regions and the degree of neuronal damage marked by MAP2 were detected by immunofluorescence to assess the degree of neuroinflammation in sensitive brain regions exacerbated by dysfunction of MLVs.The expression of MAP2-marked neurons in other nonsensitive brain regions was also examined using immunofluorescence to verify the consistency with the results of microglia activation marked by Iba1.Novel object recognition was performed on postoperative days 6 and 7 to test cognitive function in mice to further clarify the dysfunction of sensitive brain regions.3.To further clarify the mechanism of cognitive impairment in sAD neuroinflammatory mice exacerbated by dysfunction of MLVs,according to the mechanism of MLVs regulating neuroinflammation by draining cerebral lymph to maintain cerebral perfusion function,scavenging neurotoxic protein function and draining immune cell function,the cerebral perfusion impairment in sensitive brain regions was detected using the cerebellar medullary pool injection of Evans blue tracer,and the immunofluorescence method was used to detect deep cervical lymph nodes for CD43+leukocytes and CD68+monocytes/macrophages priming,and hippocampal Aβ and tau levels were measured by Elisa to clarify the role of dysfunctional clearance of neurotoxic proteins by MLVs.To identify the specific dysfunction of MLVs as the mechanism exacerbating cognitive impairment in sAD neuroinflamed mice.4.To take sensitive brain regions for transcriptomic assays to further predict the specific molecular mechanisms of MLVs dysfunction that exacerbate neuroinflammation at the genetic level.Part Ⅱ Study on the mechanism of cognitive impairment in sAD neuroinflammatory mice by different DSS extracted components to improve the draining brain lymphatic function of MLVs1.To establish a model of LPS-induced neuroinflammation in sAD with MLVs impairment,saline,aqueous DSS and alcoholic DSS were administered to the model mice for 6 days using a novel object recognition assay to assess the cognitive function of the hippocampus,and immunofluorescence was used to detect the degree of activation of hippocampal Iba1 marker microglia.The expression levels of hippocampal pro-inflammatory factor TNF-α and anti-inflammatory factor CD 163 were detected using immunofluorescence assay.2.To detect MLVs draining cerebral lymphatic injury at the same level in the hippocampal region using the Evans Blue tracer injection method in the cerebellar medullary pool.3.Transcriptomic assays were performed on hippocampal tissues to clarify the specific molecular mechanisms by which different DSS-extracted components improve the draining cerebral lymphatic function of MLVs.Part Ⅲ:Screening and validation of active ingredients of aqueous DSS to improve the draining cerebral lymphatic function of MLVs for the treatment of cognitive impairment in sAD neuroinflammatory mice1.Combining the transcriptomic results and protein interaction network analysis in PartⅡ,we identified the key proteins of aqueous DSS to improve the brain lymphatic function of draining MLVs for the treatment of sAD neuroinflammation.2.Using molecular docking technology,we docked the key protein of the action of aqueous DSS with the main active ingredient of this formula to screen the key components of aqueous DSS to improve the draining cerebral lymphatic function of MLVs for the treatment of sAD neuroinflammation.3.Determine the dose of albiflorin administration based on the content determination of the pharmacodynamic components of aqueous DSS in the previous study.A mouse model of LPS-induced sAD neuroinflammation with MLVs disorder was established,and saline and screening monomers were administered to the model mice for 6 days,using the shamoperated group as control.Immunofluorescence was used to verify whether the effect of albiflorin on the expression of key proteins was consistent with aqueous DSS.The cognitive function of each group of mice was examined using novel object recognition assay,and the degree of hippocampal Iba1 marker microglia activation and TNF-a/CD163 marker neuronal damage were detected using immunofluorescence,and the screening monomer was tested for potency.Results:Part Ⅰ.Study on the mechanism of cognitive impairment in mice with sAD neuroinflammation exacerbated by MLVs drainage brain lymphatic dysfunction1.The results of Evans blue tracer experiment showed that the fluorescent signal of Evans blue in deep cervical lymph nodes was weaker and occupied a smaller percentage of the distribution area in the LPS+veterpofin+photoconversion group compared with the control group(P<0.05,P<0.01 vs.LPS+veterpofin+photoconversion group),indicating dysfunctional drainage of MLVs is successful modeling.2.Immunofluorescence results showed the most significant activation of hippocampal microglia in the LPS+veterpofin+photoconversion group compared with the control group(P<0.01 vs LPS group),while no significant changes were seen in the cortex and midbrain(P>0.05 vs LPS group).Using the hippocampus as the study object,immunofluorescence results showed that a significant increase in TNF-α expression,a significant decrease in YM1 expression,and a decrease in MAP2 expression(P<0.05,P<0.001 vs LPS group)were found in the hippocampus of the LPS+veterpofin+photoconversion group compared with the control group.In the novel object recognition experimental test session,the blank and saline groups showed a higher percentage of time spent exploring new objects(P<0.01,P<0.001),the LPS group,the LPS+veterpofin+photoconversion group,and the LPS+veterpofin group did not show a preference for old and new objects,while the LPS+veterpofin+photoconversion group showed a higher percentage of time spent exploring old objects(P<0.001).The recognition index was lower in the LPS+veterpofin+photoconversion group compared to the LPS group(P<0.001).3.Injection of Evans Blue tracer into the medullary pool of the cerebellum showed higher Evans Blue content in the hippocampus of the LPS+veterpofin+photoconversion group compared with the LPS group(P<0.01).Immunofluorescence results showed no difference in the expression levels of CD4+leukocytes and CD68+monocytes/macrophages in deep cervical lymph nodes between groups(P>0.05 vs LPS+veterpofin+photoconversion group).Elisa results showed no difference in Aβ1-40 and tau levels between hippocampal groups(P>0.05 vs LPS+veterpofin+photoconversion group).4.The results of GO functional enrichment analysis of hippocampal transcriptomics indicated that the hippocampal vascular circulatory system was extensively affected.Among the processes associated with the dysfunction of MLVs in draining cerebral lymph to maintain cerebral perfusion include blood circulation,circulatory system process,regulation of blood pressure,regulation of blood circulation,and vascular process in circulatory system,etc.Part Ⅱ Study on the mechanism of cognitive impairment in mice with sAD neuroinflammation treated with different DSS extract components to improve the drainage brain lymphatic function of MLVs1.In the novel object recognition experiment,the Sham and Model groups showed a higher percentage of time spent exploring old objects(P<0.001),while the water-extracted DSS and alcohol-extracted DSS groups showed a higher percentage of time spent exploring new objects(P<0.001).Compared to Model,the aqueous DSS group had a higher recognition index than the alcoholic DSS group(P<0.001),but there was no pharmacodynamic difference between the two(P>0.05).Immunofluorescence showed that the expression of Ibal and TNF-α in hippocampus was significantly decreased(P<0.01,P<0.001 vs Model group)after the intervention in the aqueous DSS and alcoholic DSS groups.However,there was no difference between the two in any of the above indicators(P>0.05).2.In the Evans blue tracer experiment,the hippocampal Evans blue content was significantly decreased in the aqueous DSS and alcoholic DSS groups compared with the Model group(P<0.05).3.Transcriptomics suggested that aqueous DSS ameliorated the mechanism of sAD neuroinflammation exacerbated by MLV impairment mainly related to interferon beta,including response to interferon-beta and cellular response to interferon-beta.In contrast,the mechanism of sAD neuroinflammation exacerbated by alcoholic DSS ameliorating MLV disorder was mainly related to TNF signaling pathway and IL-17 signaling pathway.Part Ⅲ Screening and validation study of active ingredients of aqueous DSS via interferon regulatory factor 7 to improve the drainage brain lymphatic function of MLVs to treat cognitive impairment in mice with sAD neuroinflammation1.Based on the transcriptomic differential gene results in part Ⅱ,protein interaction analysis revealed that the key protein of aqueous DSS to improve MLVs draining brain lymphatic dysfunction exacerbating sAD neuroinflammation was interferon regulatory factor 7.Differential gene FPKM index suggested that aqueous DSS inhibited the expression of interferon regulatory factor 7 in the hippocampus of model mice(P<0.01).2.Molecular docking results with interferon regulatory factor 7 showed that albiflorin was a key component of aqueous DSS via interferon regulatory factor 7 to ameliorate MLVs draining brain lymphatic dysfunction for sAD neuroinflammation.3.Immunofluorescence suggested that,consistent with the pharmacological effect of aqueous DSS,albiflorin showed the same inhibitory effect on interferon regulatory factor 7(P<0.05).In the novel object recognition assay,the Model group showed a higher percentage of time spent exploring old objects(P<0.001),while the albiflorin group showed a higher percentage of time spent exploring novel objects(P<0.001).The recognition index was higher in the albiflorin group compared to the Model group(P<0.001).Immunofluorescence showed a decrease in Iba1 expression(P<0.01 vs Model group)in the hippocampus after the albiflorin intervention.Conclusion:1.The hippocampus is more sensitive to neuroinflammation in sAD exacerbated by dysfunction of MLVs compared to the cortex and midbrain.dysfunction of MLVs draining cerebral lymphatic maintenance of brain perfusion,but not dysfunction of draining immune cells or dysfunction of draining neurotoxic proteins,exacerbates hippocampal neuroinflammation and its cognitive impairment in mice.The alteration of cerebrovascular circulatory system may be the main mechanism of neuroinflammation caused by the dysfunction of drainage of cerebral lymphatic maintenance of brain perfusion by MLVs.2.Aqueous DSS and alcoholic DSS could improve the drainage of cerebral lymphatic maintenance of cerebral perfusion by MLVs to suppress hippocampal neuroinflammation,thus exerting the effect of improving cognitive function in mice with sAD neuroinflammation.Notably,there was no significant difference in the pharmacological effects of aqueous DSS and alcoholic DSS in improving cognitive function,hippocampal neuroinflammation and hippocampal cerebral lymphatic drainage in mice.The mechanisms by which aqueous DSS and alcoholic DSS improved brain lymphatic dysfunction of MLVs drainage to inhibit hippocampal neuroinflammation in sAD were related to response to interferon β and TNF signaling pathway,respectively.The different mechanisms by which aqueous DSS and alcoholic DSS exert their pharmacological effects may be related to the differences in the composition of pharmacological substances of DSS and their contents under different extraction methods.3.Albiflorin may be the key pharmacodynamic substance of aqueous DSS in regulating interferon regulatory factor 7 to improve the drainage brain lymphatic function of MLVs to inhibit sAD neuroinflammation.