Effects of Si-Miao-Yong-An decoction on myocardial I/R rats by regulating gut microbiota to inhibit LPS-induced TLR4/NF-κB signaling pathway

For hematoxylin–eosin (H&E) staining, the G1120 kit from Solarbio Technology (Beijing, China) was used. The CK-MB (RS2011) and LDH (RS4044) reagents were obtained from InTec PRODUCTS, INC (Xiamen, China). ELISA kits for serum and colon tissue homogenate assays were procured from the North Institute of Biotechnology (Beijing, China) for TNF-α (R2784/1), IL-6 (R3021/1), IL-1β (R3001/1), IL-17A (R4421/1), and TLR4 (R3566/1). LPS (EC80545S) assay CETAL kits were obtained from Xiamen Bioendo Technology Co., Ltd (Xiamen, China). Primary antibodies for TLR4 (66350–1-Ig), MyD88 (23230–1-AP), P65 (10745–1-AP), Occludin (27260–1-AP), β-actin (66009–1-1 g), and Lamin B1(12987–1-AP) were purchased from Proteintech (UT, United States). Antibodies for p-P65 (3033S) and ZO-1 (13663) were acquired from Cell Signaling Technology (MA, USA). HRP-conjugated goat anti-mouse IgG (H + L) (A0216) and cell lysis buffer for Western blot and IP (P0013) were used as secondary antibodies and supplied by Beyotime Biotechnology (Shanghai, China). Protein marker (180–6003) and ECL substrate solution (high sensitivity) (180–501) were procured from Tanon (Shanghai, China). Aspirin (acetylsalicylic acid) (S17061) was acquired from Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai, China).

The herbs used in the study were procured from the Hefei HuarunSanjiu Pharmaceutical Co., Ltd. (Hefei, China). L. japonicaeflos was sourced from Shandong, China (production batch No. 20200903); S. ningpoensisHemsl. was sourced from Hubei, China (production batch No. 200313); A. sinensis Radix was sourced from Gansu, China (production batch No. 201028); and Glycyrrhizae Radix et Rhizoma was sourced from Neimenggu, China (production batch No. 20160068). About 30 g L. japonicaeflos, 30 g S. ningpoensisHemsl., 18 g A. sinensis Radix, and 18 g Glycyrrhizae Radix were diluted in distilled water at a 1:5 volume ratio of the total grams of the drug. The mixture was decocted for 50 min, and then distilled water was added in thrice the volume and decocted for 50 min again. After blending the two decoctions, their concentration was adjusted to a final value of 4 g/mL, equivalent to 4 g of SMYA decoction per mL of liquid. The drug was concentrated, filtered, and stored at –20 °C. Based on Clinical Medicine and the Laboratory of Pharmacology, a gavage dose of 8.64 g/kg/d was administered to the rats.

The SMYA decoction that was prepared was converted into a dry powder using spin-drying. A quantity of 4 g of the SMYA powder was weighed and dissolved in 12 mL of distilled water. This solution was mixed with 28 mL of anhydrous ethanol in a volume of 40 mL and sonicated until it was fully dissolved. Standard compounds, including chlorogenic acid, neochlorogenic acid, isochlorogenic acid A, cryptochlorogenic acid, harpagoside, harpagide, imperatorin, liquiritin, isoliquiritin, glycyrrhizic acid, and others, were dissolved in methyl alcohol. The supernatant obtained after centrifugation (10000 rpm, 45 min) was used for analysis. The HPLC analysis was carried out using Waters Acquity I-Class UPLC-Waters SYNAPT G2-Si Q-TOF with Acquity UPLC HSS T3 (100 × 2.1 mm, 1.8 µm). The mass spectrum parameters were as follows: acquisition mode of Negative MSE, collection scope of 100–1500 Da, capillary voltage of 3.0 kV, ion source temperature of 120 °C, MS/MS energy range of 25–45. Chromatographic separation was achieved using a gradient elution solvent of methanol and formic acid, with a gradient elution of 0–20 min, B% 2%-35%; 20–30 min, B% 35%-95%. The flow velocity was set to 0.4 mL/min, and the column temperature was set at 45 °C. An injection volume of 2 µL was used for the determination of compound content in SMYA based on the retention times of standards.

The Animal Management Committee of the Institute of Basic Theory of Chinese Medicine (Beijing, China) approved the study (Permit Number: IBTTCMCAMS21–2104–04). Male Sprague Dawley rats weighing approximately 180 ± 20 g were purchased from Charles River Laboratories(Beijing, China) and housed in a room with a 12-h light/dark cycle, five rats per cage, at a temperature of 20–22 °C and relative humidity of 55–65%. Following a one-week adaptation period, 40 rats were randomly assigned to Control group, I/R group, I/R + SMYA group, and I/R + Aspirin group, with 10 rats in each group. For the I/R group, I/R + SMYA group, and I/R + Aspirin group rats, cardiac ischemia was induced under anesthesia by inhalation of 5% isoflurane followed by 2% isoflurane maintenance, and the rats were placed on the operating table. The laboratory animal ventilator was connected, and the surgical area was disinfected with iodophor. The rats were positioned at the 3rd/4th left rib space for heart exposure. Cardiac ischemia was simulated in the animals using a surgical suture (6.0 size) to ligate the proximal portion of the left anterior descending artery. The ligation was considered successful when the ligated part of the myocardium became clearly white. The ligature wire was loosened after 60 min to simulate reperfusion. After the closure of the chest, the rats were placed in clean enclosures and given appropriate post-surgical care. The rats were allowed to recover for one day before treatment. The rats in the I/R + SMYA group were orally administered 8.64 g/kg/d SMYA, while those in the I/R + Aspirin group received 50 mg/kg/d Aspirin. The rats in the other groups received an equivalent volume of saline. The experimental protocols ensured the well-being of the rats.

On day 28, echocardiography was performed on all rats in each group to evaluate cardiac function. A high-resolution ultrasound imager, Vevo 3100 (VisualSonics, Canada) MX250 probe, was used to examine the cardiac function of the rats at 21 Hz. The rats were placed on thermal blankets at 37 °C after shaving their chest hair and were anesthetized by inhaling 2% isoflurane. To obtain images of the papillary muscle in a horizontal short-axis ultrasound and M-ultrasound, the probe was rotated 90°. Images were taken to determine the values of left ventricular fractional shortening (LVFS), left ventricular ejection fraction (LVEF), left ventricular end-systolic diameter (LVEDs), and left ventricular end-diastolic diameter (LVEDd). Three consecutive cardiac cycles were measured, and the average of the three measurements was taken.

To evaluate tissue damage in the colon and myocardium, H&E staining was conducted. Following deep anesthesia with 5% isoflurane, tissues were harvested after euthanasia of the rats. Myocardial tissues were collected from the I/R region, while colonic tissues were washed in phosphate-buffered saline (PBS). Tissues were fixed in 4% paraformaldehyde for 48 h, embedded in paraffin, and sliced into 4 µm sections. These sections were then dewaxed and rehydrated by immersing them in xylene and gradient ethanol, and subsequently stained with hematoxylin for 5 min and eosin for 1 min. Finally, neutral gum was used to seal the sections, and the tissue samples were observed under an optical microscope (Olympus, Tokyo, Japan).

In this study, fresh fecal samples were obtained from rats’ colons, transferred into sterile cryotubes, and frozen in liquid nitrogen. The 16S rDNA sequencing was carried out to study the microbial diversity of the fecal samples using Illumina-based deep sequencing, universal primers 338F: 5'-ACTCCTACGGGAGGCAGCA-3’ and 806R: 5'-GGACTACHVGGGTWTCTAAT-3 and the V3-V4 region of the 16S rRNA gene was amplified. The polymerase chain reaction (PCR) was conducted in a 20 µL reaction volume, consisting of 13.25 µL of H₂O, 2.0 µL of 10 × PCR ExTaq Buffer, 2.0 µL dNTP, 1.0 µL of each primer at a concentration 10 mmol/L, 0.5 µL of 100 ng/mL template DNA, and 0.25 µL of 5 U/mL ExTaq. The PCR cycling conditions were 95 °C for 5 min, 30 cycles of 95 °C for 30 s, 58 °C for 20 s, and 72 °C for 6 s, with the final extension step at 72 °C for 7 min. This was followed by purification and recovery of the amplified products through 1% agarose gel electrophoresis. High-quality sequences were clustered/denoised using USEARCH at a similarity threshold of 97%. The species annotation at the genus and phylum levels was performed using RDP Classifier. The microbial diversity was assessed using α-diversity indices, including Chao1, ACE, Simpson’s and Shannon indices, and rarefaction curve with Motherv.1.30. The β-diversity analysis was performed using NMDS and PCoA at corresponding distances based on the distance matrix in R. Finally, statistically significant biomarkers were identified using LefSe analysis based on the LDA scores between groups.

In this research, after 28 days of treatment, rats were fasted for 8 h and anesthetized with 5% isoflurane inhalation to collect 5 mL of abdominal aorta blood. The collected blood was centrifuged at 1000 rpm and 4 °C for 10 min to separate the serum supernatant. A sample of colon tissue weighing 0.5 g was collected into a centrifuge tube and mixed with 1 mL of normal saline. The tissue was then homogenized using magnetic beads and a homogenizer, followed by centrifugation at 2500 rpm and 4 °C for 10 min. The supernatant of the tissue homogenate was collected for further analysis. An ELISA kit was used to assess the concentration of TNF-α, IL-6, IL-1β, IL-17A, and TLR4 in both the serum and colon tissue of the rats. The concentrations of LPS, CK-MB, and LDH in the serum were also measured using the same ELISA kit as per the manufacturer’s instructions.

In this study, the BCA assay kit (Beyotime) was utilized to quantify the protein levels in the colon and heart tissues. To this end, the tissue samples were lysed using a lysis solution and homogenized on ice for 45 min. After centrifugation at 1000 rpm and 4 °C for 10 min, the supernatant was collected for further analysis. SDS-PAGE was performed using a 12% separating gel and a 5% stacking gel. Each well was loaded with 60 µg of total protein, electrophoresed, and transferred to a PVDF membrane. The membrane was incubated with TLR4 (1:1000), MyD88 (1:1000), P65 (1:1000), Occludin (1:1000), β-actin (1:20,000), p-P65 (1:1000), ZO-1 (1:1000), or Lamin B1 (1:5000) antibody after being blocked with 5% skimmed milk powder. The PVDF membrane was incubated with the antibody incubation solution for 12 h at 4 °C, washed, and then subjected to an HRP-conjugated goat anti-mouse secondary antibody (1:1000 dilutions) for 2 h at room temperature on a shaker. After adequate washing, images were captured using a Tanon automated chemiluminescence analyzer, and the Image J software was used to calculate the grayscale values. The internal reference proteins used were β-actin and Lamin B1. The grayscale values of the target protein and the internal reference protein were normalized, and the final results were presented using the normalized ratio.

The data was processed using GraphPad Prism 7.0 and presented as mean ± SD. Student’s t-test was used for comparison between two groups, and One-way ANOVA was used for multiple group comparisons. The association of differential microflora with inflammatory biomarkers was evaluated using Spearman’s correlation test. Benjamini-Hochberg (BH) method was followed for calibration. *P < 0.05, **P < 0.01 vs. Control group, #P < 0.05, ##P < 0.01 vs. I/R group.

HPLC analysis was used to identify the main components of SMYA. Fifteen chromatographic peaks were obtained based on the retention time consistency, as shown in Fig. 1. The identified compounds in SMYA are listed in Table 1.

To evaluate the efficacy of SMYA against myocardial damage, the serum levels of lactate dehydrogenase (LDH) and creatine kinase isoenzyme (CK-MB) were measured. Compared to the control group, the I/R group had significantly higher levels of CK-MB and LDH, while the administration of SMYA and Aspirin resulted in a significant decrease in these serum indices in I/R rats (Fig. 2A). Therefore, SMYA could effectively reduce serum CK-MB and LDH levels, thereby protecting the myocardium and reducing myocardial injury. Echocardiography was used to assess cardiac function. The treatment with SMYA led to a reduction in LVEDs and LVEDd values and an increase in LVEF and LVES values, although the difference was not statistically significant (Fig. 2B). The I/R group exhibited enlarged ventricles and thinner ventricular walls than the control group. Moreover, the heart tissue of the I/R group showed evident structural damage, while the administration of SMYA and Aspirin resulted in improved ventricular and ventricular wall structures in myocardial I/R rats (Fig. 2C). The myocardial cells’ structural changes were examined by analyzing H&E-stained cross-sections of the heart. The I/R rats displayed disordered myocardial filaments and varying degrees of necrosis. However, the administration of SMYA and Aspirin resulted in a significant reduction in myocardial tissue damage (Fig. 2D).

To investigate the effectiveness of SMYA in combating serum inflammation, various inflammatory markers were examined. The I/R group showed significantly higher levels of LPS compared to the control group, while the levels were reduced in the I/R + SMYA and I/R + Aspirin groups (Fig. 3A). The I/R group also exhibited a significant increase in downstream inflammatory factors (TNF-α, IL-6, IL-1β, and IL-17A). However, SMYA treatment led to a statistical reduction in these inflammatory cytokines (Fig. 3B-G). The I/R model demonstrated a notable elevation in serum LPS levels, which could enter the systemic circulation through the intestinal barrier, activate TLR4 signaling, and release downstream inflammatory factors. SMYA administration could effectively reduce LPS levels, the secretion of downstream inflammatory cytokines, and the level of inflammation in the serum.

To investigate the mechanism of SMYA in reducing inflammation in an in vivo I/R model, the myocardial proteins in the TLR4/NF-κB pathway were analyzed (Fig. 4A). The discussion mainly focused on the SMYA mechanism; thus, the I/R + Aspirin group was no longer included. MyD88 and TLR4 proteins were significantly upregulated in the I/R group and downregulated after SMYA administration (Fig. 4B, C). In the I/R group, myocardial tissue p-P65 protein and p65 nuclear protein expression were upregulated, while in the I/R + SMYA group, the protein expression was considerably downregulated (Fig. 4D, E). Conversely, p65 cytoplasmic protein expression was downregulated in the I/R group and upregulated in the I/R + SMYA group (Fig. 4F). Therefore, SMYA can inhibit the expression of proteins in the TLR4/NF-κB pathway, reducing the release of inflammatory factors.

In this study, 16S rDNA sequencing was performed on the colon feces of rats. The Rarefaction Curve analysis indicated that the sequencing data were adequate for data analysis, as the curves for the three groups were flat. However, the I/R group showed a higher alpha-diversity index compared to the other groups, which was also supported by the Rank Abundance Curve (Fig. 5A, B). Although there was no significant difference in the Shannon index among the three groups, Simpson’s index showed a significant difference between the I/R and control rats, indicating a higher diversity in the I/R group. The species abundance and evenness were also higher in the I/R group rats, indicating a higher diversity. Additionally, the Ace and Chao1 indices showed a more significant number of species in the I/R rats compared to the control rats (Fig. 5C-F). The alpha diversity of the I/R + SMYA group decreased, although the difference was not statistically significant when compared to the I/R group, and the trend was similar to that observed in the normal group. For beta diversity, similarity analysis among the three groups was conducted using principal coordinate analysis (PCoA) and non-metric multi-dimensional scaling (NMDS). PCoA classified the samples based on their main elements and structure, assuming linear relationships in the data, whereas NMDS differentiated the samples based on the non-linear structure of ecological data. Both methods could distinguish the three groups, with an R-value of 0.91 and a p-value of 0.001, and a Stress value of 0.0034 in NMDS, indicating better clustering and separation of the groups (Fig. 5G, H). These results suggest a significant change in gut microbiota in the I/R and I/R + SMYA groups.

The analysis of gut microbiota in the I/R rats revealed an increase in Tenericutes and Patescibacteria at the species level and a decrease in Cyanobacteria at the phylum level. However, in the I/R + SMYA rats, these changes were reversed, and there was an increase in Bacteroidetes (Fig. 6A). Previous studies have reported an increase in Firmicutes and a decrease in Bacteroidetes in patients with CAD. Although the I/R rats did not show such a trend, the administration of SMYA resulted in a significant reduction in Firmicutes and an increase in Bacteroidetes. In the I/R group, there was an elevation at the genus level for Ruminococcaceae_UCG-014, Ruminococcaceae_UCG-005, and Lachnospiraceae_NK4A136. However, the administration of SMYA reduced this elevation and increased Phascolarctobacterium and Alloprevotella (Fig. 6B). Linear Discriminant Analysis Effect Size (LefSe) assessment of the differential bacteria in the three groups showed significantly enriched LDA scores (LDA > 4), with an increase in Prevotellaceae_Ga6A1_group (genus, species) and Ruminococcaceae_UCG_005 (genus, species) in the I/R group. However, SMYA inhibited these bacteria by increasing Bacteroidetes (phylum, class, order), f__Prevotellaceae (family, genus, species), Prevotellaceae_UCG_001 (genus, species), g__Prevotellaceae_NK3B31_group (genus, species), g__Alloprevotella (genus, species), s__uncultured_bacterium_g_Phascolarctobacterium (genus) (Fig. 6C, D). In conclusion, SMYA could regulate gut microbiota structure in myocardial I/R rats.

The study analyzed the correlation between the identified differential microbiota and serum inflammatory factors. After removing evolutionary duplicate microbiota, eleven differential microbiota were analyzed using hierarchical clustering, Spearman’s correlation analysis, and subsequent correction using the BH method. The analysis revealed two correlation clusters between the serum LPS and inflammatory factors (Fig. 7). Bacteroidetes were found to be positively correlated with Prevotellaceae, Prevotellaceae_UCG_001, and Prevotellaceae_NK3B31 (adjusted P < 0.01). On the other hand, Firmicutes showed a positive correlation with Ruminococcus and Gram-positive bacteria and a negative correlation with most other differential bacteria, especially Bacteroidetes, Prevotellaceae, and Prevotellaceae_UCG_001 (adjusted P < 0.01). The serum LPS was positively correlated with Prevotellaceae_Ga6A1 and negatively correlated with Bacteroidetes, Prevotellaceae_UCG_001 (adjusted P < 0.05), and Prevotellaceae_NK3B31 (adjusted P < 0.01). Additionally, serum IL-17A was positively correlated with Prevotellaceae_Ga6A1 and negatively correlated with Prevotellaceae_NK3B31. Interestingly, the increase in individual microbiota in the I/R + SMYA group showed a negative correlation with the inflammatory factors, whereas the increased microbiota in the I/R group showed a more positive correlation. These findings suggest a potential association between gut microbiota, LPS, and inflammatory factors.

To validate the effect of SMYA on the gut mucosa, H&E staining was performed. The I/R rats showed mucosal damage, altered villi structure, loss of crypt cells, and inflammatory cell infiltration that could extend up to the mucosal muscle layer, in contrast to the control rats (Fig. 8A). However, the I/R + SMYA group exhibited improved colonic mucosal damage, reduced inflammatory infiltration, and a better villi structure than the I/R group. The I/R rats also showed significantly decreased expression of ZO-1 and occludin tight junction proteins compared to the control rats, while the I/R + SMYA group exhibited higher levels of these proteins and a weakened intestinal permeability (Fig. 8B-D). These results suggest that SMYA can reduce intestinal mucosal damage, maintain the intestinal barrier, and decrease intestinal permeability. Moreover, the study explored the effect of SMYA on colonic inflammation by examining the colonic tissue homogenate levels of TLR4 and its downstream inflammatory factors (IL-6, IL-1β, TNF-α, and IL-17) biomarkers. The I/R rats showed significantly higher levels of these markers than the control rats, whereas the I/R + SMYA group exhibited lower cytokine expressions. These findings indicate that SMYA has an inhibitory effect on TLR4 levels and its downstream inflammatory factors in colonic tissues (Fig. 8E-I).

All methods were carried out in accordance with the relevant guidelines and regulations. Plant material complied with IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora. This study was performed in line with the principles of the National Standard of People’s Republic of China (GB/T 35892–2018) and the guidelines 2.0 of the ARRIVE. Approval was granted by the Animal Management Committee of the Institute of Basic Theory of Chinese Medicine (Beijing, China) approved the animal study (Permit Number: IBTTCMCAMS21-2104–04).

Not applicable.

The authors declare no competing interests.

Data are available from the corresponding author on reasonable request. The sequencing data used in this study are stored in NCBI SRA database (PRJNA940314). https://www.ncbi.nlm.nih.gov/sra/PRJNA940314↗.