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

Materials

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).

Preparation of SMYA

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.

High-performance liquid chromatography

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.

Animal model

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.

Echocardiography

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.

H&E staining

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).

Gut microbiota analysis

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.

Enzyme-linked immunosorbent assay

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.

Western blot analysis

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.

Statistical analysis

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.

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