Animal, Sepsis model and treatment
Wild-type (WT) C57BL/6 male mice (8-10weeks old, 20-25g) were purchased from Slac Laboratory (Shanghai, China) and were fed a standard rodent diet with free access to food and tap water. All experimental procedures were in accordance with the guidelines for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (8th Edition, 2011), and all protocols were approved by the Institute’s Animal Ethics Committee of Shanghai Jiao Tong University. All the mice were kept at constant temperature (21 ± 2°C) in a light and dark cycle with relative humidity of 50±5%, and free access to food and water. The experimental mice were randomly divided into 4 groups (10 mice per group): The control group, the JuA treatment group (JuA), the LPS + normal saline group (LPS+NS) and the LPS + JuA group (LPS+JuA). Lipopolysaccharide (LPS) from Escherichia coli O55:B5 (Sigma, Saint Louis, MO, USA) was dissolved in PBS and JuA was purchased from MedChemExpress (HY-N0659, Monmouth Junction, NJ, USA).
To investigate the potential effects, mice were given JuA (20mg/kg, 0.2ml, p.o.) or the equal volume of saline (0.2ml, p.o.) for consecutive 7 days. On the last day, all the mice were given a single intraperitoneal injection of LPS (20mg/kg) or the same volume of saline to mimic sepsis-induced cardiomyopathy. After 12 hours, cardiac function was detected by M-mode echocardiography and all the mice were sacrificed to harvest hearts.
The survival rate was observed using other 20 mice in each group. These LPS/saline-induced mice were returned to their cages after JuA/NS-treatment and were closely monitored for up to 7 days, as well as given ad libitum access to food and water.
Cardiac function and structure were assessed 12 hours after injecting LPS using echocardiography (Vevo 2100, VisualSonics, Toronto, Canada) with a 25MHz imaging transducer. Mice were anesthetized by inhalation of 2% isoflurane. Cardiac function parameters such as left ventricular ejection fraction (LVEF), left ventricular fraction shortening (LVFS), left ventricular end‑diastolic diameter (LVEDD), left ventricular end‑systolic diameter (LVESD), left ventricular end‑diastolic volume (LVEDV), left ventricular end‑systolic volume (LVESV) were calculated using computer algorithms.
Cell Lines and Cell Culture
The cardiomyoblast cell line H9c2 was purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Gibco, CA, USA) and antibiotics (100 U/mL penicillin and 100 U/mL streptomycin) and maintained at 37°C in 5% CO2. And the cells were passaged every 2-3 days to maintain growth. For in vitro treatment, the cells were pretreated with JuA (0 µ M and 20 µ M) or an equal volume of PBS for 1 h, and then were treated with LPS (20µg/mL) for 24 h.
TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)
TUNEL staining was performed using the In Situ Cell Death Detection Kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions to assess apoptosis in heart tissues. Diphenyl phenylindole (DAPI) staining was used to stain the cell nucleus. The apoptosis index was defined as the number of TUNEL-positive myocytes/the total number of myocytes stained with DAPI. Images were obtained using the Leica laser fluorescence microscope at a magnification of ×200 and analyzed with ImageJ software (ImageJ version 1.43r; NIH).
Transmission electron microscopy (TEM)
The heart tissues were harvested to determine the quantification of autophagosomes. The heart tissues were fixed with 2.5% glutaraldehyde and stored at 4°C. According to the manufacturer’s instructions, each specimen was performed by a series of manipulation, including fixation, dehydration, embedding, curing, biopsy, and dyeing. The autophagosomes were observed by a transmission electron microscope (JEOL, Tokyo). Random sections were imaged and analyzed by two technicians blinded to the experiment. The mean number (and standard deviation) of autophagosomes per field was calculated.
At the predetermined time points, these mice were anesthetized by isoflurane, the plasma samples were obtained from blood after centrifugation for 15 minutes at 3000g at 4°C, and stored at -80°C until use in various biochemical assays. The concentrations of TNF-α, IL-6, IL-1β, IL-18 were measured by ELISA kits according to the manufacturer’s instructions (Sangon Biotech, Shanghai, China). OD450 was calculated by subtracting the background, and standard curves were plotted. Values are expressed as pg/ml of total protein.
Flow cytometry was used to determine the apoptosis rate in LPS-induced H9c2 cells. Apoptotic cells were differentiated from viable or necrotic cells by the combined application of annexin V (AV)–fluorescein isothiocyanate (FITC) and propidium iodide (PI) according to the manufacture of the Annexin V-FITC cell apoptosis kit (C1062, Beyotime Biotechnology). Cells were washed twice and adjusted to a concentration of 106 cells/ml. AV–FITC (5 µl) and PI (10 µl) were added to 195 µl of binding buffer in each sample and incubated for 20 min at room temperature in the dark. Then without washing, samples were analyzed by using flow cytometry. Each experiment was performed at least in three independent experiments.
Western blot analysis
H9c2 Cells and the heart tissue were lysed by RIPA lysis buffer with protease inhibitors and phosphatase inhibitors according to the manufacturer’s protocol (Sigma Aldrich, St. Louis, MO, USA). Protease inhibitor cocktail was from Sigma Chemicals (St. Louis, MO, USA). The protein concentration was determined with the BCA assay kit (Thermo Fisher Scientific, Inc.). Total protein from myocardium tissues and cardiomyocytes was separated by SDA-PAGE gels and were electro-blotted onto PVDF membranes. The membranes were blocked with 5% non-fat milk for 1 h at room temperature, and then incubated overnight at 4°C with primary antibodies against Bax (1:1000, ab32503, Abcam), Bcl-2 (1:500, sc-7382, Santa Cruz Biotechnology), β-actin (1:500, Santa Cruz Biotechnology); GAPDH (1:1000, ab9485, Abcam); LC3B (1:1000, AL221, Beyotime Biotechnology); p62 (1:1000, A19700, ABclonal Technology); Beclin1(1:1000, AF5123, Beyotime Biotechnology); caspase-3 (1:1000, 9662, Cell Signaling Technology ); iNOS (1:1000, ab178945, Abcam); gp91phox (1:1000, sc-130543, Santa Cruz Biotechnology) antibodies. After incubation with the primary antibodies, blots were incubated with horseradish peroxidase-linked secondary antibodies (1:5000) for 60 min at room temperature. Blots were again washed three times with TBST and immunoreactive bands were detected using an ECL chemiluminescence. Band intensities were quantified using Gel-Pro Analyzer software version 4.0 (Media Cybernetics, Silver Spring, MD, USA).
Quantitative reverse transcription PCR (q-PCR) analysis
Total RNA of myocardial tissues was isolated using TRIzol Reagent (Invitrogen). The Concentration of mRNA was determined using absorbance at 260 and 280 nm. The sample RNA was reversely transcribed to cDNA with reverse transcription reagent kit (Takara BIO) according to the manufacturer’s instructions. q-PCR was performed using a SYBR® Premix Ex TaqTM Perfect Real Time Kit (Takara BIO) in the 7300 System SDS Software (Roche Applied Science). The primer sequences utilized for real-time PCR are presented in Table S2. Results from 7300 System SDS Software are presented as Ct values, normalized against GAPDH, and shown as 2−∆∆Ct .
Cell viability assay
Cell Counting Kit-8 (CCK8) was applied to assess the number of viable cells. Cells were seeded in a 96-well plate at a concentration of 1 × 105 cells/mL, and were allowed to acclimatize overnight. and then cultured in complete medium in the presence or absence of LPS and JuA (20µM). The cells were pre-treated with various concentrations of the JuA (0 µM to 20 µM) for 1 h, and then treated with LPS 20 µg/mL for 24 h. The OD450 was tested at 3h, 6h, 12h, 24 h using a microtiter plate reader (SkanIt Software 5.0, RE, ver. 22.214.171.124). The results were calculated using the following formula: Cell viability = (Treatment Group OD − Blank Group OD)/ (Control Group OD – Blank Group OD).
All data are presented as mean ± standard deviation at least three experiments. Statistical analysis was performed using SPSS software (SPSS Inc., Chicago, IL). Unpaired t-tests were used to determine the statistical differences between 2 groups in each analysis. A one-way analysis of variance (ANOVA) was used for multiple comparisons. Survival was analyzed with Kaplan–Meier survival curves and compared with the log-rank test. A p value of less than 0.05 was considered significant. P<0.05 was considered to be statistically significant.