Production of recombinant rSj-Cys
DNA coding for Sj-Cys was cloned in-frame into pET28a and the sequencing confirmed recombinant plasmid DNA with correct insert was transformed into E. coli BL21 by calcium transfection method. The recombinant Sj-Cys (rSj-Cys) with His-tag at N-terminus was induced with 1 mM isopropylthio-β-galactoside (IPTG, Sigma-Aldrich, Steinheim, Germany) at 37℃ for 5 hours, and purified from the induced bacteria soluble fraction using a HisPur™ Ni-NTA Spin Column (Thermo Fisher Scientific Inc., USA). The contaminated endotoxin was removed from the purified recombinant protein using a ToxOut™ High Capacity Endotoxin Removal Kit (BioVision, Palo Alto, California, USA) and the residual endotoxin level was measured by ToxinSensor™ Chromogenic Limulus Amebocyte Lysate (LAL) Endotoxin Assay Kit (GenScript Biotechnology, Nanjing, China) following the manufacturer’s protocol. The concentration of rSj-Cys was measured using a Bicinchoninic Acid Protein Assay Kit (Beyotime Biotechnology, Shanghai, China) and the recombinant protein stored at -80 °C until use.
Male BALB/c mice with 6-8 weeks old (body weight: 20~22 g) and specific pathogen free (SPF) were purchased from Anhui Medical University Experimental Animal Facility (approval no: AMU26-08061). The mice were housed in a climate-controlled facility maintained at 23 ± 1° C and 55 ± 5% humidity with a 12 h light/dark cycle and free access to food and water ad libitum.
The mice were subjected to CLP surgery according to the method described previously . Briefly, mice were fasted for 12 h with drinking water only and then anesthetized by intraperitoneal injection of 0.2 ml/20 g of 4% chloral hydrate. Following a 1-2 cm midline laparotomy incision, 66% of the cecum was ligated with a 4-0 silk tie (Syneture, Norwalk, CT). A through-and-through puncture was made on the anti-mesenteric side with an 18-gauge needle and a small amount of feces was extruded through the puncture holes to ensure perforation. The cecum was placed back in its original location and the abdomen was closed in two layers with 4-0 silk. Following CLP, sterile normal saline (300 µl) was injected subdermally for fluid resuscitation. Sham mice underwent the above process except for CLP.
Treatment of sepsis-induced cardiomyopathy with rSj-Cys
Total 6 CLP-operated mice were treated intraperitoneally with 10 µg of rSj-Cys in a total volume of 200 μl 30 min after surgery. The same number of CLP-operated mice were given with 200 µl of PBS only as control. As normal controls, 12 mice with sham surgery were divided into two groups and 6 received the same amount of rSj-Cys and 6 received PBS only. Twelve hours later, all mice were measured for echocardiography. Blood was collected from each mouse under anesthesia and sera were separated by centrifuged and stored at -80°C until use. All mice were euthanized and hearts were collected for histopathological staining and measurement.
After the mice were treated in different ways, echocardiographic evaluation was performed using a high-resolution echocardiograph (Vevo 2100, VisualSonics, Canada). Briefly, a mixture of 1% isoflurane and oxygen was inhaled via a nose cone, and each mouse was carefully kept under mild anesthesia and then subjected to M-mode and Doppler echocardiography according to the method described . The ejection fraction (EF%) and fractional shortening (FS%) of left ventricle were calculated from M-mode tracing to reflect left systolic function. Peak early-diastolic transmitral velocities (E wave) and peak late-diastolic transmitral velocities (A wave) across mitral valve inflow were examined on Doppler flow tracings and were used to calculate E/A ratios, a commonly used parameter of left ventricular diastolic function. All echocardiographic procedures are performed by the same skilled operator and the data are averaged form at least three consecutive cardiac cycles.
Histological examination of myocardium
Mouse hearts collected from different experimental groups were fixed in 4% buffered paraformaldehyde for 12 hours. Fixed heart left ventricles were sectioned and stained with hematoxylin and eosin (H&E) stain. H&E stained sections were observed under light microscopy (200×) (Nikon, Tokyo, Japan) for pathological changes.
The heart-released myoglobin (Mb), cardiac troponin I (cTnI) and N-terminal pro-Brain Natriuretic peptide (NT-proBNP) in sera and myeloperoxidase (MPO) in heart tissue were measured as biochemical markers for heart injury. The levels of cTnI and NT-proBNP in sera were detected using enzyme-linked immunosorbent assay (ELISA) kit (Elabscience Biotechnology Co., Ltd, Wuhan, China). The concentration of Mb was measured in the mouse sera by a Fully Automated Biochemistry Analyzer (Beckman Coulter, Brea, California, USA). The heart tissue was weighed and homogenized, the MPO activity in the homogenate was determined using a MPO test kit (Bioenginering Institute, Nanjing, China).
Detection of IL-6, TNF-α, TGF-β and IL-10 in sera and cell supernatants
The concentration of pro-inflammatory (TNF-α, IL-6) and regulatory (IL-10, TGF-β) cytokines in cell culture supernatants and experimental mouse sera were detected by ELISA in accordance with the manufacturer’s instructions (ABclonal Biotechnology Co., Ltd. Wuhan, China).
Detection of cardiac TNF-α, IL-6, IL-10, TGF-β, iNOS and Arg-1 mRNA expression by quantitative real time PCR (qRT-PCR)
Total RNA from left ventricular myocardium was extracted with QIAzol reagent (Ambion, USA). Then cDNAs were reverse-transcribed from 2 μg total RNA with reverse transcription kit (Thermo Electron, Waltham, MA, USA). The cDNA was used as templates for qRT-PCR using SYBR Green Super mix kit (Takara Bio，Inc., Japan). All samples were duplicated and the qRT-PCR signal of the target transcript in the treated group was compared with the control housekeeper gene (GAPDH) signal by relative quantification. The 2–ΔΔCT method was used to analyze the relative changes in gene expression. The primers (GAPDH, TNF-α and IL-6) were designed and synthesized by Sangon Biotech (Shanghai, China). The forward and reverse primers of target genes are listed in Additional file 1: Table S1.
Cell culture and treatment
H9C2 rat embryo cardiomyocytes were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’ s modified Eagle’ s medium (DMEM) containing 10% fetal bovine serum (Biowest S.A.S, USA) and 1% penicillin/streptomycin (Gibco, Grand Island, NY) at 37℃, 5% CO2. Cultured H9C2 cells were treated with rSj-Cys (0.5 ug/ml) for 0.5 h, and then exposed to 1 µg/mL of LPS (Solaibao, Beijing, China) for 24 h. Cells incubated with LPS without rSj-Cys treatment, or cells incubated with rSj-Cys or medium only were used for controls. After 24 h incubation, the culture was centrifuged at 1,000 rpm for 15 minutes at 4°C, the supernatants were stored at −80°C until use, and the cells were used for flow cytometry assays.
Detection of myocardial cell apoptosis by flow cytometry (FCM)
The LPS-induced myocardial cell apotosis was measured by annexin v-fitc and propidium iodide (pi) staining in accordance with the manufacturer’s instructions (Invitrogen，Thermo Fisher Scientific, USA). Flow cytometric analysis was performed on a CYTEK DxP AthenaTM Analyzer (CyTeK Biosciences, USA). The results were analyzed with FlowJo V7.6.5 software.
Detection of MyD88 by Western blotting
MyD88 expression level in treated H9C2 cells and myocardial tissue was determined by Western blotting. Briefly, cells or left ventricular myocardium were collected and homogenized in ice-cold RIPA buffer containing 0.1% phenylmethylsulfonyl fluoride. The homogenates werecentrifuged at 12,000 rpm for 15 min at 4℃. Supernatants were collected and protein concentration was quantified using BCA assay kit (Pierce, Rockford, IL, United States). Equal amounts of cell extracts or heart homogenates were separated by 12% SDS-PAGE and electroblotted onto PVDF membranes. After blocking with 5% non-fat milk for 2 h at room temperature, the membranes were incubated with rabbit anti-MyD88 antibody (1:800) (Cell Signaling Technology, Danvers, Massachusetts, USA) overnight at 4°C, followed by HRP-conjugated goat anti-rabbit IgG (1:4,000) (Merck Millipore, Basilica, Massachusetts, USA) for 1 h at 37°C. Immunoreactive protein bands were visualized using a Tanon 5,200 Chemiluminescence imaging system (Tanon, Shanghai, China).
All data are expressed as the mean ± SEM (standard error of the mean), and the statistical analyses were performed by using GraphPad Prism 5.0 software (GraphPad Inc., La Jolla, CA, USA). One-way ANOVA followed by the Student-Newman-Keuls test was used for multigroup comparisons. P < 0.05 was considered as statistically significant.