3.1 Effects of nerve function, body weight and visceral indexes
There was a significant decrease in body weight between M group and the sham group at 24 h, 48 h and 72 h after I/R after operation. At each time point, it showed no significant difference, but a decreasing trend can be seen (Fig. 1a). The neurological function score showed there were different degrees of improvement after 72 h of administration (Fig. 1b). Viscera index (Fig. 1c) showed that brain, lung and spleen indexes increased significantly in M group compared with the sham group, while thymus index decreased significantly.
3.2 Effects of BA-MS on cerebral infarct area and general histology
As shown in Fig. 2a, viable tissue was deep red by TTC staining, whereas area of infarction in the injured side cerebral hemisphere was white. The brain infarction area in M group was significantly increased in comparison to the sham group (P < 0.001). Compared with M group, 20:1 group showed the smallest infarction area (P < 0.001), followed by 5:1 group (P < 0.01) and H10:1 group (P < 0.01). In addition, the brains of the rats in M group exhibited an edematous morphology with neuron loss and pyknotic nuclei in H&E staining, and this condition was effectively ameliorated in each treatment group (Fig. 2b). The lungs of the rats in M group appeared pulmonary bullae with alveolar structure damaged. Alveolar capillary wall thickened and infiltrated the alveolar space and peribronchi in H&E staining, which was consistent with the development of pulmonary infection in rats after stroke [18, 19], and it was effectively ameliorated in treatment groups (Fig. 2c).
3.3 BA-MS improved biochemical indexes
The results of biochemical indexes were shown in Fig. 2d. M group showed significant increase in IL-1β, nNOS, BDNF, NGF, ET-1, PAI-1, PDGF-BB and VEGF-A levels, as well as a significant decrease in AngⅡ level in comparison with sham group. After the administration, the above indicators all tended to be normal. H5:1 group and H20:1 group were generally better than other administration groups.
As shown in Fig. 3a and Fig. 3b, the expressions of bFGF, TGF-β1 and vWF were significantly increased and TDP-43, NeuN were significantly decreased in cerebral I/R region after MCAO. Compared with M group, administration groups had obviously increased the expressions of bFGF, TGF-β1, TDP-43 and NeuN, while remarkably decreased the expressions of vWF.
3.5 Effects of BA-MS on protein expressions of NF- κ B, p-NF- κ B, TLR4, AngⅠ, AngⅡ and Tie2 in Western blot analysis
Western blotting results (Fig. 3c) showed that the expression of NF-κB, p-NF-κB and TLR4 protein in cerebral I/R region after MCAO were significantly increased, and AngⅠ, AngⅡ and Tie2 protein were significantly decreased compared with the sham group (P < 0.05). In general, the H20:1, H10:1 and H5:1 had significant effects on the expression of NF-κB, p-NF-κB, TLR4, AngⅠ, AngⅡ and Tie2, and H5:1 showed the best regulatory effect.
3.6 Metabolism analysis
The OPLS-DA score plots (Figure S1a) showed that sham and M group could be clearly separated, indicating that metabolic disorders occurred in the model group. PCA score (Figure S1b) showed that the plasma and urine samples of sham and M group had significant clustering. The administration groups deviated from M group and was closer to the sham operation group, indicating the drug administration groups could improve the metabolic disorder of I/R in MCAO rats. In the S-plot plot (Figure S1c), metabolites that deviate from the main ion cluster and VIP ≥ 1 was extracted as differential metabolites. Then, 21 potential biomarkers (Table S1, among them 9 from plasma and 12 from urine.) and 16 potential metabolism pathways (impact ≥ 0.1) were identified by HMDB (www.hmdb.ca) and Metabo Analyst (http://www.metaboanalyst.ca/) database. As shown in Figure S1d, the occurrence of I/R in MCAO rats may be associated with retinol metabolism, pentose and glucuronate interconversions, linoleic acid metabolism, glycerophospholipid metabolism, biotin metabolism, ascorbate and aldarate metabolism, phenylalanine metabolism, terpenoid backbone biosynthesis, pantothenate and CoA biosynthesis, ether lipid metabolism, sphingolipid metabolism, pyruvate metabolism, galactose metabolism, cysteine and methionine metabolism, arachidonic acid metabolism and steroid hormone biosynthesis. Among them, linoleic acid metabolism had an impact-value of 1, which suggested the administration of BA-MS could modify this metabolic pathways and be related biomarkers to exert the curative effect for I/R after MCAO rats. The results also showed BA-MS could improve metabolic disorders in I/R after MCAO by regulating metabolites. Other details see Figure S2.
3.7 Regulation of BA-MS on intestinalmicroecology
3.7.1 Gut microbiota analysis
In order to investigate whether the therapeutic effect of the bioactive parts of BA-MS in different proportions on I/R after MCAO was related to intestinal flora, we analyzed the cecum contents flora of rats after 72 h of treatment and the species diversity of the sample at OTU level.
Figure S3 showed the sample length distribution was in the range of 400 to 440 bp. Sobs index was to measure colony richness, Heip index was to measure colony evenness, and Shannon index was to evaluate species diversity of the samples at OUT level, as shown in
Figure S4a. The results showed that there were high richness, diversity and uniformity of the microflora in each group, and there was no significant difference in the diversity index. Dilution curve (
Figure S4b), showed that the current sequencing depth was enough to reflect the diversity contained in the current sample, and a large number of undiscovered new OTU cannot be detected by increasing the sequencing depth. In addition, the number of each sample at the level of phylum and genus was counted. At the phylum level
(Fig.
4a
),
firmicutes,
proteobacteria,
bacteroidetes and
verruciformis were found after the combination of species with abundance less than 0.1%, and
Firmicutes accounted for the largest proportion. At the genus level
(Fig.
4a
),
escherichia-shigella,
allobaculum, norank_f_
muribaculaceae,
akkermansia,
lactobacillus,
prevotella_9 were found after the combination of species with abundance less than 0.1%. According to the LEfSe multi-species hierarchy tree diagram
(Fig.
4b
), compared with the normal group, the intestinal flora of the model group was different in phylum, class, order, family and genus. The Linear discriminant analysis (LDA) discriminant bar graph
(Fig.
4b
) was made from phylum level to genus level. By comparing the sham group with M group and Welch's t test, significant differences were found in 3 phylums and 27 genus. It was found that H5:1 significantly increased the relative abundance of
actinobacteria and
tenericutes at the phylum level (Fig.
4c
). The administration groups could regulate 10 species of bacteria at the genus level, among which H5:1 had the best effect, followed by H10:1, H20:1, L10:1 and YXY. Further studies showed that the intestinal flora of I/R in MCAO rats were involved in 24 related functions (
Figure S5) and metabolic pathways of the top 20 abundance values (
Table S2). Among them, amino acid transport and metabolism, cell wall/membrane/envelope biogenesis, replication, recombination and repair, ribosomal structure and biogenesis, energy production and conversion have high functional abundance, and carbohydrate metabolism, amino acid metabolism, replication and repair, energy metabolism, nucleotide metabolism and lipid metabolism pathways disorder contribute directly or indirectly to the development of ischemia stroke.
3.7.2 Short-chain fatty acids analysis
In order to investigate the effects of different proportions of BA-MS bioactive ingredients on intestinal flora metabolites in I/R rats, we analyzed SCFAs in cecum contents of rats after 72 h of I/R. The chromatogram of the mixed reference substances, sham and M groups was shown in
Figure S6, and the precision, stability, repeatability, average recovery and linear relation of six SCFAs components were shown in
Table S3. It can be seen in Fig.
4d, compared with the sham group, the contents of acetic acid and propionic acid in the caecum of M group were significantly decreased, and the contents of each administration group were increased to different degrees. H5:1 had a significant effect on the contents of acetic acid and propionic acid in the contents of the I/R rats' cecum after MCAO, suggesting that H5:1 could improve the effect of ischemic stroke by increasing the content of SCFAs in the contents of the cecum of MCAO rats.
3.8 Correlation analysis
It can be seen from the heat map of correlation analysis (Fig. 5a), NeuN was positively correlated with PM5 and PM9 (r > 0.7); PDGF-BB was positively correlated with UN2 (r > 0.7); AngⅠ was correlated with UN6 (r > 0.7); p-NF-κB was negatively correlated with PM5 (r < -0.7); TDP-43 was positively correlated with PM6 (r > 0.7), VWF was negatively correlated with PM1 (r < -0.7).
Correlation analysis heat maps of the above gut microbes and SCFAs were carried out. In this experiment, the Pearson correlation coefficients r > 0.4 and r<-0.4 respectively indicate medium positive correlation and medium negative correlation. The correlation of unclassified_ks-k_dbacteria, verrucomicrobia and cyanobacteria were good at the phylum level (Fig. 5b). There were 15 good correlations at the genus level (Fig. 5b), lachnospiraceae_NK4A136_group, clostridium_sensu_stricto_1, lactobacillus, norank_f_Lachnospiraceae, bifidobacterium, streptococcus, akkermansia, turicibacter, norank_o_gastranaerophilales, proteus, parasutterella, norank_f_erysipelotrichaceae, [eubacterium]_coprostanoligenes_group, phascolarctobacterium, butyricimonas.