Analysis of compounds in HSD and its drug-containing serum

Figure S1 shows the total ion chromatogram for the identification of the compounds in the HSD and its drug-containing serum. The chemical composition of HSD matched 237 compounds with a composite score greater than 60 in mzCloud best match, and 30 small molecular compounds of traditional Chinese medicine were screened by comparison with the traditional Chinese medicine database (Shown in Table S1). While the chemical composition of HSD drug-containing serum full matched 61 small molecular compounds in mzCloud best match (Shown in Table S2). Among them, Wogonin identified both in HSD and in its drug-containing serum was selected as a drug in the subsequent experimental study.

HSD improves cognitive disorder in samp8 mice

The result of Morris water maze test showed that the escape latency of mice was significantly prolonged in the model group (P < 0.01) from the 4th day comparing with those in the control group. On the 5th day, the escape latency of mice in the donepezil group, HSD group and Wogonin group were significantly shortened comparing with the model group (P < 0.05, P < 0.001, P < 0.001) (Fig. 1B). On the last day of the space exploration experiment, the number of platform crossings and the time spent in the target quadrant of mice in the model group were significantly lower than those in the control group (P < 0.001, P < 0.01) (Fig. 1C, D). The donepezil group, HSD group and Wogonin group had significant increases in the time spent in the target quadrant compared with the model group (P < 0.001, P < 0.001, P < 0.001) (Fig. 1C). There was no significant difference in the number of platform crossings between the donepezil group and the model group, but the number of platform crossings in the HSD group and Wogonin group were significantly higher than that in the model group (P < 0.01, P < 0.01) (Fig. 1D). The open field results showed that there was no significant difference in the total moving distance among the groups (Fig. 1F). Compared with the control group, the time spent in the central area and the times of grooming in the model group were significantly reduced (P < 0.05, P < 0.05). Compared with the model group, the time spent in the central area and the times of grooming in the donepezil and HSD group were significantly increased (P < 0.05, P < 0.05, P < 0.01; P < 0.05, P < 0.05, P < 0.05) (Fig. 1G, H). The results of Y maze showed that there was no significant difference in the total number of arm approaches among the three groups (Fig. 1J). Compared with the control group, the model group had a significant reduction in the rate of arm entry altercountry (P < 0.001). Compared with the model group, the donepezil group and HSD group had a significant increase in the rate of arm entry altercountry (P < 0.01, P < 0.05) (Fig. 1K).

Fig. 1

Behavioral experiments. A Swimming trajectories of the Morris water maze experiment; B Evasion latency; C Time elapsed in target quadrant; D Platform crossings times; E Activity trajectories of the open field experiment; F Total distance; G Percentage of time in central zone; H Number of grooming; I Activity trajectory of the Y maze experiment; J Arm entries times; K Spontaneous alternating correct rate of Y maze in each group. The data are expressed as Mean ± SEM (n = 9). *: model group compared with the control group, #: Donepezil group compared with the model group, $: HSD group compared with the model group, &: Wogonin group compared with the model group. ns no significant difference, *,#,$,&P < 0.05, **,##,$$,&&P < 0.01, ***,###,$$$,&&&P < 0.001

HSD reduced the protein levels of pSer404-Tau and Aβ and the histopathological changes of the brain tissue in SAMP8 mice

Immunofluorescence results showed that the protein levels of pSer404-tau in the hippocampus of the model group was significantly higher than that of in the control group (P < 0.001), the levels of pSer404-tau in hippocampus of donepezil group, HSD group and Wogonin group was significantly lower than that of model group (P < 0.001, P < 0.001, P < 0.001) (Fig. 2B); Compared with the control group, the model group had a significant increase in the expression of pSer404-tau in the cortex (P < 0.001), and the donepezil, HSD and Wogonin groups had significant reduction in the expression of pSer404-tau in the cortex compared with the model group (P < 0.001, P < 0.001, P < 0.001) (Fig. 2C); Similarly, Western-blot results showed that the expression of pSer404-tau protein in the hippocampus of the model group was significantly higher than that of the control group (P < 0.01), while donepezil, HSD and Wogonin significantly reduced the levels of pSer404-tau protein in the hippocampal tissues (P < 0.001, P < 0.001, P < 0.01); The expression of pSer404-tau protein in the cortex of the model group was significantly higher than that in the control group (P < 0.001), while donepezil, HSD and Wogonin significantly reduced the expression of pSer404-tau protein in the cortical tissues (P < 0.001, P < 0.001, P < 0.001) (Fig. 2D).

Fig. 2

The expression levels of pSer404-tau and Aβ in the hippocampus and cortex and morphological structure of neurons and synapses observed by H&E staining and transmission electron microscopy. A Fluorescence expression of pSer404-tau in hippocampus (left) and cortex (right) of mice in each group. pSer404-tau protein staining (red) and nuclei re-stained (blue) (Magnification 100×, partial enlarged is Magnification 1000×). B The mean density of pSer404-tau fluorescence in hippocampus of mice in each group. C Mean density analysis of pSer404-tau fluorescence in cortex of mice in each group; D Expression of pSer404-tau protein in hippocampus and cortex of mice in each group; E Aβ deposition in hippocampus and cortex of mice in each group (Magnification 200×, partial enlarged is Magnification 1000×); F H&E staining (Magnification 40×, partial enlarged is Magnification 400×, neuronal cells were loosely arranged with karyopyknosis in model group, shown with black arrow) and transmission electron microscopy (Magnification 20,000×, red arrows indicating synaptic structure). The data are expressed as Mean ± SEM (n = 3). *: model group compared with the control group, #: Donepezil group compared with the model group, $: HSD group compared with the model group, &: Wogonin group compared with the model group. *,#,$,&P < 0.05, **,##,$$,&&P < 0.01, ***,###,$$$,&&&P < 0.001

The immunohistochemistry results showed that the Aβ deposition in the hippocampus and cortex of the model group was significantly higher than that of the control group (P < 0.01, P < 0.001). Compared with the model group, the Aβ deposition in the hippocampus and cortex of the donepezil group was significantly reduced (P < 0.05, P < 0.05). The deposition of Aβ in hippocampus and cortex was significantly reduced in HSD group (P < 0.001, P < 0.001), and the deposition of Aβ in hippocampus and cortex was also significantly reduced in Wogonin group (P < 0.05, P < 0.05) (Fig. 2E).

The results of H&E staining showed that the neurons in the hippocampus CA1, CA3, dentate gyrus and cortex of the control group were structurally intact, neatly arranged, and the nuclei were clearly stained, while the neurons in the hippocampus and cortex of the model group showed neuropathological changes such as the reduction in the number of neurons, loose arrangement, cell vacuoles, and nuclear pyknosis. The pathological changes of hippocampus and cortex in donepezil group, HSD group and Wogonin group were improved, the number of nerve cells increased, the arrangement of nerve cells was more regular, and the cell vacuolization and karyopyknosis were less (Fig. 2F). Transmission electron microscopy showed that the nucleus of the hippocampal CA1 region was atrophic and deformed, the structure of synaptic membrane was incomplete, the synaptic cleft was mostly blurred, and the vesicles were sparse in the model group. In the control group, donepezil group, HSD group and Wogonin group, the nucleus showed normal with clear synaptic structure and synaptic gap (Fig. 2F).

Proteomics analysis of hippocampal tissue of samp8 mice

A total of 56 differentially expressed proteins were obtained from the control group and the model group (more than 1.2 times as significant difference), in which 35 proteins were up-regulated with 21 proteins were down-regulated. A total of 46 differentially expressed proteins were obtained from the model group and the HSD group, of which 32 proteins were up-regulated with 14 proteins were down-regulated, while 12 proteins were differentially expressed in the three groups (Fig. 3A). Among them, 9 proteins were down-regulated and 3 proteins were up-regulated by HSD (Table S3). The volcano plot reflected the difference of the overall protein. The differentially expressed proteins were screened by FC < 1/1.2 and FC > 1.2, and P < 0.05. The red dots in the upper right corner represent up-regulated differentially expressed proteins, and the green dots in the upper left corner represent down-regulated differentially expressed proteins (Fig. 3B, C). The differential protein cluster heatmap intuitively reflected the differential expression changes of differential proteins in the two groups. Hierarchical clustering was performed on the expression patterns of differential proteins, and the clustering results were presented using a heat map. The darker the red color, the higher the protein expression. The darker the blue color, the lower the expression of the protein (Fig. 3D, E). GO functional enrichment analysis was performed on the differentially expressed proteins. A total of 453 items were enriched in Biological Process (BP) and 152 items were enriched in Cellular Component (CC) in the control group and the model group, respectively. A total of 112 items were enriched for Molecular Function (MF); In the model group and HSD group, a total of 323 items were enriched in BP, 97 items were enriched in CC, and 117 items were enriched in MF. Each subclass was annotated according to the top 20 most-enriched GO enriched terms. According to the annotation results, the GO function of the control group, model group and HSD group was mainly involved in extracellular matrix, fibrin and plasminogen, collagen, endothelial cell apoptosis, platelet regulation, etc. (Fig. 3F, G). According to the differentially expressed proteins, KEGG pathway enrichment analysis was performed, and 83 pathways were enriched in the control group and the model group, and 102 pathways were enriched in the model group and HSD group. Annotation was performed according to the top 20 most enriched KEGG pathways. The KEGG pathways of the control group, model group and HSD group were mainly involved in the Extracellular matrix (ECM) interaction of the central nervous system, complement and coagulation cascade, platelet activation, etc. (Fig. 3H, I).

Fig. 3

Proteomics analysis of hippocampus tissue of SAMP8 mice treated with HSD and the expression levels of FGB and FGG in the brain of mice in each group. A Number of differentially expressed proteins; B, C Volcano plot statistics of differentially expressed proteins in control group vs. model group and model group vs. HSD group, respectively; D, E Heat map statistics of differentially expressed proteins in control group vs. model group and model group vs. HSD group, respectively; F, G GO enrichment analysis of differentially expressed proteins in control group vs. model group and model group vs. HSD group, respectively; H, I KEGG pathway enrichment analysis of differentially expressed proteins in control group vs. model group and model group vs. HSD group, respectively; J, K FGB and FGG protein expression levels in hippocampus and cortex of mice in each group, respectively. The data are expressed as Mean ± SEM (n = 3). *: model group compared with the control group, #: Donepezil group compared with the model group, $: HSD group compared with the model group, &: Wogonin group compared with the model group. ns no significant difference, *,#,$,&P < 0.05, **,##,$$,&&P < 0.01, ***,###,$$$,&&&P < 0.001

Western-blot results showed that the model group had significant increases in the protein expression levels of Fibrinogen beta chain (FGB) and Fibrinogen γ chain (FGG) in the hippocampus compared with the control group (P < 0.001, P < 0.001), and the donepezil group had significant reductions in the protein expression levels of FGB and FGG in the hippocampus compared with the model group (P < 0.05, P < 0.001), meanwhile, the expressions of FGB and FGG in hippocampus were significantly decreased in HSD group (P < 0.001, P < 0.001) and Wogonin group (P < 0.001, P < 0.001) (Fig. 3J). Compared with the control group, the model group had significant increases in the protein expression levels of FGB and FGG in the cortex tissue (P < 0.001, P < 0.05). Compared with the model group, the donepezil group had no significant difference in the expression of FGB in the cortex tissue, and the expression of FGG was significantly decreased (P < 0.001). The expression of FGB and FGG in cortex tissue was significantly decreased in HSD group (P < 0.001, P < 0.001), while it was also significantly decreased in Wogonin group (P < 0.001, P < 0.001) (Fig. 3K).

HSD reduced the co-localization of FG and Aβ in brain of samp8 mice

The results of immunofluorescence showed that the positive expression of FGB and Aβ were increased obviously with a colocalization in the model group comparing with the control group, while the donepezil group, HSD group and Wogonin group had reduced the expression of FGB and Aβ, and the aggregation of FGB and Aβ (Fig. 4A, B). Similarly, compared with the control group, the model group had an obvious increase in the expression of FGG and Aβ with a colocalization, while the donepezil group, HSD group and Wogonin group had a reduction in the expression of FGG and Aβ, and a reduction in the aggregation of FGG and Aβ (Fig. 4C, D).

Fig. 4

Colocalization of FGB, FGG and Aβ in hippocampus and cortex of mice in each group (Magnification 100×, partial enlarged is Magnification 1000×). A, B Colocalization of FGB and Aβ in hippocampus and cortex; C, D Colocalization of FGG and Aβ in hippocampus and cortex. Blue, green and red fluorescence indicates DAPI nuclear staining, Aβ and FGB/FGG, respectively

HSD reduced the co-localization of FG and iba-1 and inhibited the activation of microglia

Figure 5 showed the positive expression of FGB and Iba-1 were increased obviously with a colocalization in the model group comparing with the control group, while the donepezil group, HSD group and Wogonin group had reduced the expression of FGB and Iba-1, and the aggregation of FGB and Iba-1 (Fig. 5A, B). Meanwhile, the model group had an obvious increase in the expression of FGG and Iba-1 with a colocalization comparing with the control group, while the donepezil group, HSD group and Wogonin group had a reduction in the expression of FGG and Iba-1, and a reduction in the aggregation of FGG and Iba-1 (Fig. 5C, D).

Fig. 5

Colocalization of FGB, FGG and Iba-1 in hippocampus and cortex of mice in each group (Magnification 100×, partial enlarged is Magnification 1000×). A, B Colocalization of FGB and Iba-1 in hippocampus and cortex; C, D Colocalization of FGG and Iba-1 in hippocampus and cortex. Blue, green and red fluorescence indicates DAPI nuclear staining, Iba-1 and FGB/FGG, respectively

HSD reduced the co-localization of FG and GFAP and inhibited the activation of astrocytes

As shown in Fig. 6, compared with the control group, the model group had an obviously increase in the expression of FGB and GFAP with a colocalization, while the donepezil group, HSD group and Wogonin group had a reduction in the expression of FGB and GFAP, and the aggregation of FGB and GFAP (Fig. 6A, B). Similarly, compared with the control group, the expression of FGG and GFAP in the model group was increased with obvious colocalization, while the expression and aggregation of FGG and GFAP in the donepezil group, HSD group and Wogonin group were reduced (Fig. 6C, D).

Fig. 6

Colocalization of FGB, FGG and GFAP in hippocampus and cortex of mice in each group (Magnification 100×, partial enlarged is Magnification 1000×). A, B Colocalization of FGB and GFAP in hippocampus and cortex; C, D Colocalization of FGG and GFAP in hippocampus and cortex. Blue, green and red fluorescence indicates DAPI nuclear staining, GFAP and FGB/FGG, respectively

HSD increased myelin generation of oligodendrocytes

The immunofluorescence results showed that the MBP positive expression in hippocampus and cortex of the model group was lower than that of the control group, while the MBP expression in the donepezil group, HSD group and Wogonin group were higher than that in the model group. It is shown that there was no colocalization of FGB and FGG with MBP (Fig. 7).

Fig. 7

FGB, FGG and MBP in hippocampus and cortex of mice in each group (Magnification 100×, partial enlarged is Magnification 1000×). A, B Positive expression of FGB and MBP in hippocampus and cortex; C, D Positive expression of FGG and MBP in hippocampus and cortex. Blue, green and red fluorescence indicates DAPI nuclear staining, MBP and FGB/FGG, respectively

HSD improved the vascular ultrastructure of samp8 mice, increased the protein levels of ZO-1 and occludin in the brain barrier, and reduced the level of bacterial DNA fragments and LPS in the brain

The morphological changes of microvessels in the brain were observed by TEM. The results showed the tight junctions between endothelial cells were blurred, the basement membrane was broken and blurred, the mitochondria of endothelial cells were swollen, the membrane was ruptured, the cristae were mostly dissolved, and the end feet of glial cells were swollen and deformed in the model group. In the donepezil group, HSD group and Wogonin group, the tight junctions between endothelial cells were clear, the basement membrane was tightly connected, and the endothelial mitochondria and glial structures tended to be in a healthy state (Fig. 8A).

Fig. 8

The ultrastructure of brain vessels, the expression levels of ZO-1 and Occludin, the relative content of bacteria or bacterial DNA fragments and LPS in the brain of mice in each group. A Ultrastructure of hippocampal blood vessels in each group (Magnification 30,000×), red scissors indicated tight junctions between endothelial cells, basement membrane, mitochondria and glial endfeet; B The mRNA expression levels of ZO-1 and Occludin in hippocampus and cortex of mice in each group; C Protein expression levels of ZO-1 and Occludin in hippocampus and cortex of mice in each group; D In situ hybridization of EUB338 fluorescent probe in hippocampus and cortex of mice in each group, blue and red fluorescence indicates DAPI nuclear staining and EUB338 positive signal, respectively (Magnification 100×, partial enlarged is Magnification 1000×); E Immunofluorescence staining of LPS in hippocampus and cortex of mice in each group, blue and green fluorescence indicates DAPI nuclear staining and LPS positive signal, respectively (Magnification 100×, partial enlarged is Magnification 1000×). The data are expressed as Mean ± SEM (n = 3). *: model group compared with the control group, #: Donepezil group compared with the model group, $: HSD group compared with the model group, &: Wogonin group compared with the model group. ns no significant difference, *,#,$,&P < 0.05, **,##,$$,&&P < 0.01, ***,###,$$$,&&&P < 0.001

The mRNA levels of ZO-1 and Occludin in brain tissue were detected by Q-PCR. The results showed that the expression of ZO-1 mRNA in hippocampus and cortex in the model group was significantly decreased comparing with the control group (P < 0.01, P < 0.05). Compared with the model group, the expression of ZO-1 mRNA in hippocampus and cortex in the donepezil group was significantly increased (P < 0.01, P < 0.01), the expression of ZO-1 mRNA in hippocampus and cortex was significantly increased in HSD group (P < 0.001, P < 0.05), and it was also significantly increased in Wogonin group (P < 0.01, P < 0.01). The expression of Occludin mRNA in the hippocampus and cortex of the model group was significantly decreased comparing with the control group (P < 0.05, P < 0.05). Compared with the model group, the expression of Occludin mRNA in the hippocampus and cortex of the donepezil group was significantly increased (P < 0.05, P < 0.001), it was significantly increased in HSD group (P < 0.05, P < 0.05) and in Wogonin group (P < 0.001, P < 0.05) (Fig. 8B). Similar to the Q-PCR results, Western-blot results showed that the expression of ZO-1 in the hippocampus and cortex of the model group was significantly decreased comparing with the control group (P < 0.01, P < 0.05). Compared with the model group, there was no significant difference in the expression of ZO-1 in the hippocampus of the donepezil group, while the expression of ZO-1 in the cortex was significantly increased (P < 0.05), it was also increased in HSD group (P < 0.05, P < 0.05) and in Wogonin group (P < 0.01, P < 0.05). Compared with the control group, the expression of Occludin in the hippocampus and cortex of the model group was significantly decreased (P < 0.01, P < 0.05) while which was significantly increased in donepezil group comparing with the model group (P < 0.001, P < 0.001), meanwhile, the expression of Occludin in the hippocampus and cortex was also significantly increased in HSD group (P < 0.05, P < 0.001) and in Wogonin group (P < 0.01, P < 0.001) (Fig. 8C).

FISH assay was used to observe the distribution and relative content of bacteria or bacterial DNA fragments in the brain of mice in each group. The results showed that the model group had significantly increased bacteria-specific fluorescence signal values in the hippocampus and cortex comparing with the control group (P < 0.001, P < 0.001). Compared with the model group, the bacteria specific fluorescence signal values in the hippocampus and cortex of the donepezil group and HSD group were both significantly decreased (P < 0.001, P < 0.001; P < 0.001, P < 0.01). In the Wogonin group, there was no significant difference in the value of bacteria-specific fluorescence signal in the hippocampus, but it was significantly reduced in cortex (P < 0.001) (Fig. 8D). Immunofluorescence staining was used to observe the distribution and relative content of LPS in the brain of mice in each group. The results showed that the model group had significantly increased LPS expression in the hippocampus and cortex comparing with the control group (P < 0.001, P < 0.01). Compared with the model group, the donepezil group had significantly reduced LPS expression in the hippocampus and cortex (P < 0.001, P < 0.01), which was also observed in HSD group (P < 0.001, P < 0.01) and Wogonin group (P < 0.01, P < 0.001) (Fig. 8E).

HSD regulates the structure of intestinal microbiota in samp8 mice

Alpha diversity showed that the Simpson and coverage values of HSD group were increased comparing with the model group with no significant difference (Fig. 9A, B). NMDS analysis of Beta diversity showed that the microbiota structure of the control group and the model group was relatively independent, while there was overlap between the control group and the HSD group, indicating that the microbiota composition of the HSD group was more similar to that of the control group (Fig. 9C). The heat map showed the genus-level composition of the intestinal bacterial clusters of mice in each group. The red blocks represented the abundance of genus was higher than others and the blue blocks represented lower (Fig. 9D). Lefse analysis showed the difference in bacterial species among groups. In the control group, the species difference bacteria included enterobacteriales, Enterobacteriaceae, MBAO8, shigella, clostridium; In the model group, the species difference bacteria included clostridia, clostridiaies, pseudomonas; The species difference bacteria in HSD group included bacteroidetes, bacteroidales, bacteroidia, bacteroidaceae, bacteroides, faecalibacterium, comamonadaceae, flavobacteriaceae, comamonas, Pseudomonadaceae, flavobacteria, flavobacteriales (Fig. 9E). The composition of intestinal microbiota showed significant differences in the abundance of ASVs/OTU sequences among different groups at the phylum, class, order, family, and genus levels. The results showed that the abundance of Firmicutes was significantly increased in the model group compared with the control group at the phylum level (P < 0.05). Compared with the model group, the HSD group had a significant reduction in the abundance of Firmicutes (P < 0.001) and a significant increase in the abundance of Bacteroidetes (P < 0.001) (Fig. 9F). At the class level, compared with the control group, the abundance of Clostridia in the model group was significantly increased (P < 0.01). Compared with the model group, the HSD group had a significant reduction in the abundance of Clostridia (P < 0.001) and a significant increase in the abundance of Bacteroidia (P < 0.001) (Fig. 9G). At the order level, the abundance of Clostridiales in the model group was significantly increased compared with the control group (P < 0.01), and the abundance of Enterobacteriales was significantly decreased (P < 0.05). Compared with the model group, the HSD group had a significantly lower abundance of Clostridiales (P < 0.001) and a significantly higher abundance of Bacteroidales (P < 0.001) (Fig. 9H). At the family level, compared with the control group, the abundance of Lachnospiraceae in the model group was significantly reduced (P < 0.05). Compared with the model group, the abundance of Enterobacteriaceae in HSD group was significantly decreased (P < 0.05) (Fig. 9I). At the genus level, compared with the model group, the abundance of unidentified_Lachnospiraceae, unidentified_Clostridiales, and unclassified_Clostridiales was significantly reduced (P < 0.05) (Fig. 9J).

Fig. 9.

16S rRNA analysis of intestinal microbiota. A Simpson value of alpha diversity index; B Coverage value of alpha diversity index; C NMDS analysis of beta diversity; D Genus-level species composition heatmap of species clustering; E Lefse analysis of bacterial species differences among groups; F Species composition and statistical analysis at phylum level; G Species composition and statistical analysis at class level; H Species composition and statistical analysis at the order level; I Species composition and statistical analysis at the family level; J Species composition and statistical analysis at the genus level. The data are expressed as Mean ± SEM (n = 5). *: model group compared with the control group, #: HSD group compared with the model group, *,#P < 0.05, **,##P < 0.01, ***,###P < 0.001.*model group compared with the control group, #HSD group compared with the model group