Treating Vascular Dementia With Shenmayizhi Decoction By Targeting The Interaction Between Microglia And Blood Brain Barrier

Objective: To progressively observe the pathological features of brain tissue in rats with bilateral common carotid artery ligation(2-VO),and to explore the protection of Shenmayizhi decoction on blood-brain barrier(BBB)by intervening microglial phenotype transformation.Methods: In this study,2-VO rats were used as the main research model for transcriptome sequencing.First,data quality control,transcriptome library quality evaluation,and comparison with the specified reference genome were performed.According to the comparison results,the transcripts were assembled,the expression levels of genes in different samples were calculated,and the gene expression profiles at different time points were constructed.Secondly,the DESeq algorithm was used to screen differential expression genes(DEGs)in different sample groups with |log2(FC)|>1,P<0.05 as the threshold,and the DEGs were clustered and displayed on a volcano plot.GO and KEGG enrichment analysis was carried out for the screened DEGs at different time points,and the protein-protein interaction(PPI)network was constructed to screen the key genes.At the same time,immunohistochemistry was used to evaluate the changes of microglia and astrocytes at different time points.Combined with the previous sequencing results,traditional Chinese medicine intervention was carried out.They were divided into 6 groups: Sham group,Model group,DNPQ group,and SMYZ-L,M and H dose group.Morris water maze was used to evaluate the spatial learning and memory function of 2-VO rats,Nissl staining was used to evaluate the survival of neurons,immunofluorescence was used to evaluate the effect of 5 and 14 days after administration on the timely spatial distribution of phenotypic transformation of astrocytes and microglia,real-time quantitative PCR was used to detect the genes related to blood-brain barrier tight junction protein,and verify the key genes of transcriptome sequencing.Results: 1.The transcriptome expression profiles of 2-VO rat hippocampus at different time points were constructed,and the DEGs were screened.There were 288,464,437,391 and 361 DEGs in 2-VO 1d,2-VO 3d,2-VO 5d,2-VO 7d and 2-VO14 d,respectively.GO and KEGG enrichment analysis of the screened DEGs was performed.According to P<0.05,there were 599,833,936,634,611 GO terms,30,38,37,10,24 KEGG terms,respectively.And a PPI network was constructed,and the genes of top30 at different time points were obtained by ranking the degree value.We further performed enrichment analysis on top30 genes,and found that the genes at different time points were mostly related to extracellular space and extracellular matrix.Therefore,we crossed the top30 genes in the PPI network with the genes enriched in extracellular space and extracellular matrix,and a total of 14 key genes(Apob,Lgals3,Lcn2,Timp1,Mmp14,Crispld2,Vwf,Itga5,Mmp2,Mmp13,Plau,Ttr,Sppl,Sv2c)with high expression levels were screened out.Iba-1 and GFAP at different time points after modeling were observed by immunohistochemical staining.Typical resting branched microglia could be observed in the Sham group.On the 5th day after modeling,the number of microglia increased the cell protrusions retracted,and the cell body became round and enlarged compared with Sham group(P<0.05).On the 7th day after modeling,the number of microglia increased significantly,the cell body enlarged,and the branches decreased compared with Sham group(P<0.05).On the 14 th day after modeling,compared with the 7th day after modeling,the number of cells decreased significantly,and the cell body was smaller than before,but compared with the Sham group,the number was still increased(P<0.05).Astrocytes were labeled with GFAP.Compared with Sham group,astrocytes began to proliferate after modeling,which showed that the number of cells increased,the volume became larger,and the protrusions increased and intertwined into a network.On the 1st day after modeling,the number of astrocytes was increased than that in Sham group(P<0.05).On the 3rd day after modeling,the number of astrocytes was significantly increased compared with the Sham group(P<0.05).However,the number decreased on 5th days after modeling,but it was still more than that in Sham group,with no significant statistical difference(P>0.05).The number increased slightly at 7th days after modeling compared with 5th days,with statistical difference compared with Sham group(P<0.05).And the number decreased at 14 th days after modeling,and had a statistical difference compared with 3th days after modeling(P<0.05).Compared with Sham group,the number was still higher,but there was no statistical difference.2.Effects of SMYZ on the behavior and nerve cells of 2-VO ratsLocation navigation experiment: Compared with Sham group,the escape latency of Model group was significantly prolonged(P<0.01).Compared with Model group,the latency time of the SMYZ-M and SMYZ-H groups was significantly shortened(P<0.05),and the DNPQ and SMYZ-L groups were also shortened to varying degrees,but there was no statistical difference.Space exploration experiment:Compared with the Sham group,the distance ratio and time ratio of the target quadrant of Model group were significantly reduced,with a statistical difference(P<0.01),and the number of crossing platforms was reduced,but there was no statistical difference.Compared with Model group,the distance ratio in SMYZ-H group increased with a statistical significance(P<0.05),but there was no difference in the time ratio.The results of Nissl staining showed that after 5 days of intragastric administration of SMYZ,the number and layers of neurons in the hippocampal CA1 area of the Sham group were more,arranged orderly and dense,the cytoplasm was stained darker,and the number of Nissl corpuscles was higher and dark blue.Compared with the Sham group,the Model group showed that the number and layers of neurons in the CA1 area were less,arranged disorderly,the number of Nissl bodies was decreased or even lost,and the degree of blue staining was lighter.Compared with the Model group,the number of neurons in the DNPQ and SMYZ-L groups increased slightly,and the loss of Nissl bodies was less,but the arrangement was still relatively disordered.The number of neurons and Nissl bodies in the SMYZ-M and SMYZ-H groups increased in varying degrees,the cell level did not decrease significantly,and the arrangement is relatively close and orderly.After 14 days of intragastric administration,it can be seen that the number and layers of neurons in the Sham group are numerous,neatly and densely arranged,and the Nissl corpuscles are dark blue with a large number.Compared with the Sham group,in the Model group,the number of neurons and layers in the CA1 area were less,the arrangement was neater than that in 5 days,the number of Nissl bodies was decreased or even lost,and the degree of blue staining was lighter.Compared with the Model group,the number of neurons and Nissl bodies in the DNPQ,SMYZ-L,SMYZ-M,and SMYZ-H groups increased to varying degrees.Compared with 5 days of intragastric administration,the arrangement was close and orderly,the cell layers was not significantly reduced,and the loss of Nissl bodies was less.3.The effect of SMYZ on GFAP after 5d and 14 d of intragastric administration The results of immunofluorescence showed that after 5 days of intragastric administration,compared with the Sham group,the model group had enlarged cell bodies,thickened protrusions,and a highly activated state,and the number of GFAP positive cells increased,but there was no statistical difference between the two groups(P>0.05).Compared with the Model group,the protrusions retracted and the number decreased after DNPQ and SMYZ treatment,but there was no statistical difference(P>0.05).And compared with the Model group,especially the SMZY-H group,the number of GFAP positive cells decreased more,but the difference was not significant(P>0.05).After 14 days of intragastric administration,compared with the Sham group,the astrocytes in the Model group expanded,had more protrusions and became thicker,and the number of GFAP positive cells increased.There was a significant difference between the two groups(P<0.01).Compared with the Model group,the number of GFAP positive cells in the DNPQ and SMYZ-L,SMYZ-M group decreased slightly after treatment,but there was no statistical difference(P>0.05).And compared with the Model group,especially in the SMYZ-H group,the morphology of the cell body tended to the Sham group,and the number of GFAP positive cells decreased more with a significant difference(P<0.05).4.Effects of SMYZ on Microglia Phenotypic Transformation and the time and space distributionAfter 5 days of intragastric administration with SMYZ,the number of Iba/CD31 and Iba/Claudin5 double-stained positive cells in the Model group decreased compared with the Sham group.The number of Iba/CD31 and Iba/Claudin5 double-stained positive cells in the DNPQ,SMYZ-L,SMYZ-M and SMYZ-H group increased,and the increase was significant in SMYZ-H group.After 14 days of intragastric administration,compared with the Sham group,the number of Iba/CD31 doublestained positive cells in the Model group decreased.The number of Iba/CD31 and Iba/Claudin5 double-stained positive cells increased in the DNPQ,SMYZ-L,SMYZM and SMYZ-H group,and the increase was significant in SMYZ-H group.5.Effects of SMYZ on blood-brain barrier-related gene expression and damaging factors IL-1,TNF-α mRNA and protective factors TGF-β,VEGF mRNA Compared with the Sham group,the expression of ZO-1,Occludin and Claudin5 mRNA in the hippocampus of the Model group were significantly decreased(P<0.05).The expressions of ZO-1,Occludin and Claudin5 mRNA in DNPQ and SMYZ-L,SMYZ-M,SMYZ-H group were increased compared with those in the Model group(P<0.05).Compared with the Sham group,the expressions of TNF-αand IL-1 in the Model group increased(P<0.05).After the intervention of DNPQ and SMYZ,the expression of TNF-α mRNA decreased.In the SMYZ-H group,the expression of TNF-α decreased with a statistical difference(P<0.05).The expression of IL-1 decreased in DNPQ and SMYZ-L,SMYZ-M,SMYZ-H group with a statistical difference(P<0.05).The mRNA expressions of VEGF and TGF-βdecreased in Model group(P<0.05).After intervention of DNPQ and SMYZ,the expressions of VEGF and TGF-β mRNA increased with a statistical difference(P<0.05).6.Effects of SMYZ on key targets of transcriptome sequencingCompared with the Sham group,Plau,Ttr,Mmp13 and Spp1 mRNA were downregulated in the Model group(P<0.05).After the intervention of SMYZ and DNPQ,the expressions of Plau,Ttr and Mmp13 mRNA were increased to varying degrees.There was a statistical difference in the SMYZ-H group(P<0.05).While the expression of SPP1 mRNA did not change significantly.The expressions of Apob,Lcn2,Timp1,Mmp14,Mmp2,Crispld2,Sv2 c,Itga5,Lgals3,and Vwf increased in the Model group(P<0.05).After the intervention of SMYZ and DNPQ,the expressions of Apob,Sv2 c,Lgals3 and Vwf mRNA decreased to varying degrees(P<0.05),while Lcn2,Timp1,Mmp14,Mmp2,Itga5 mRNA expression were in a dose-dependent,and decreased significantly in the SMYZ-H group(P<0.05).However,there was no statistical difference in Crispld2 mRNA expression(P>0.05).Conclusion:(1)We constructed the transcriptome expression profile of 2-VO rat hippocampus at different time points,screened the differential expression genes,and found that these DEGs were mainly related to extracellular space and extracellular matrix;(2)SMYZ can improve the learning and memory ability of 2-VO rats,reduce the activation of glial cells,and protect hippocampal neurons,thereby improving the cognitive function of 2-VO rats,which may be through the interaction between microglia and blood-brain barrier and modulating the expression of key targets.