Materials
Hunan Slac Jingda Laboratory Animal Co., Ltd. (Changsha, China) provided 32 (age= 6 weeks) male C57BL/6 mice, weighing 22 ± 3 g. The Chinese Academy of Medical Science (Shanghai, China) provided cardiac fibroblasts (CFs) for this study. Two crucial reagents applied to this study were as follows: dl-5-methoxytryptophan (5-MTP, Sigma Co., Ltd., St. Louis, MO, USA) and lipopolysaccharide (LPS, Sigma Co., Ltd., St. Louis, MO, USA). The remaining reagents included in this investigation were standard and easily available.
Animal treatment
All mice in this study were maintained in a sterile environment (20 °C and 60% humidity in 12 h light–dark cycle) with accessibility to food and water at Nanchang University's Jiangxi Medical Animal Care Center. Mice were fed for around 2 weeks before any experimental treatments to help them adjust to their new surroundings. Then they were categorized randomly (eight in each group) into control, LPS, LPS + 5-MTP (25 mg/kg) and LPS+5-MTP (50 mg/kg) groups. (1): the mice in the control group were given phosphate-buffered saline (PBS) and no other chemicals. (2): the mice in the LPS group were injected with PBS intraperitoneally (i.p.) once per day for 7 days, and then followed by 10 mg/kg of LPS intraperitoneally (i.p.) once for another 24 h. (3): the mice in the LPS+5-MTP(25 mg/kg) group were given 5-MTP (25 mg/kg) intraperitoneally (i.p.) once every day for over 7 days, following LPS (10 mg/kg, i.p.) for another 24 h. (4): the mice in the LPS+5-MTP(50 mg/kg) group were given 5-MTP (50 mg/kg) intraperitoneally (i.p.) once every day for over 7 days, following LPS (10 mg/kg, i.p.) for another 24 h. The methods and the procedures of reagents administration applied to this study were in line with previous studies [13]. The Animal Care and Use Committee of Nanchang University's Second Affiliated Hospital authorized the current experimentations, and all experimental procedures in mice were carried out in compliance with legal requirements.
Echocardiography
The cardiac function of the mice in this study was evaluated by echocardiography and the procedures of echocardiography were as follows. 1.5% of isoflurane was applied to anesthetize the mice before being tethered to a heating pad in a reclined position. We used a Vevo770 device (Visual-Sonics, Canada) with a 30-Hz transducer for transthoracic echocardiography after the mice's body hair was removed. Left ventricle end-systolic diameter (LVEDS) and left ventricular end-diastolic diameter (LVEDS) were measured and used to calculate left ventricle ejection fraction (LVEF %) and left ventricle shortening fraction (LVSF %). Following the completion of these performances, the mice were instantly slaughtered, and for subsequent study, the heart tissues were removed.
Measurement of serum CK-MB and LDH levels
We employed pentobarbital sodium (30 mg/kg, i.p.) to anesthetize these mice after echocardiography monitoring was done, and then these mice were slaughtered. A vacuum tube was applied to collect whole blood from the right ventricle of the mice. Subsequently, the whole blood collected from the mice was used to extract the serum at a centrifugation of 4000 rpm for around 30 min, with an operating temperature of 4 °C. An automatic biochemical analyzer (Chemray240, Rayto, China) was used to detect serum levels of heart damage biomarkers CK-MB and LDH.
Measurement of serum inflammatory cytokines by ELISA (enzyme-linked immunosorbent assay)
Specific mice ELISA kits were utilized to measure inflammatory cytokines such as IL-1β, IL-6 and TNF-α levels in the blood. The ELISA kits' details were as follows: TNF-α (Catalogue No. 88-7324, Invitrogen, California, USA), IL-6 (Catalogue No. 88-7064, Invitrogen, California, USA), and IL-1β (Catalogue No. 88-7013, Invitrogen, California, USA). All experiments were conducted under manufacturers provided guidelines.
H&E (hematoxylin and Eosin) staining
Heart tissues were isolated from the mice immediately when they were sacrificed. The heart tissue was then sliced into three sections, and the central section was preserved in a 4% formaldehyde solution for roughly an hour. Following that, this piece of heart tissue was cut into 5 μm portions at several depths for an H & E staining assay. The detailed procedures in this assay were in line with standard experimental operational instructions.
Culture and treatment of mice CFs
At a density of 1 × 106 cells, the mice CFs were seeded in a 6 cm diameter cell culture dish that was filled with 3 ml cells culture medium. The cells culture medium was composed of DMEM (Dulbecco's modified Eagle's medium, Thermo Fisher, USA), 10% FBS (fetal bovine serum, Gibco, USA) and 1% streptomycin and penicillin (Hyclone). All of these cells were cultured at 37 °C with 5% CO2 in a humidified incubator. The mice CFs were pretreated for approximately 12 h with 5-MTP at low, medium, and high (5 μM, 10 μM, 50 μM, respectively) concentrations and subsequently activated with 100 ng/ml LPS for a later 24 h. These cells were randomly allocated into five groups in the current study: (1) Control group: without any other reagents treatments; (2) LPS group: stimulation of LPS (100 ng/ml); (3) LPS+5-MTP (5 μM) group: pretreatment of 5-MTP (5 μM) and stimulation of LPS (100 ng/ml); (4) LPS+5-MTP (10 μM) group: pretreatment of 5-MTP (10 μM) and stimulation of LPS (100 ng/ml); (5) LPS+5-MTP (50 μM) group: pretreatment of 5-MTP (50 μM) and stimulation of LPS (100 ng/ml). Following these treatments, the mice CFs in every group were collected for subsequent investigation of the molecular processes.
Western blot assay
Total proteins from mice cardiac tissues were isolated in the current study through a protein extraction kit purchased from Beyotime Biotechnology (Catalogue No. P0013B, Jiangsu, China). Before any performances of western blot assay, we applied a bicinchoninic-acid (BCA) protein estimation kit (Catalogue No. P0012, Beyotime Biotechnology, Jiangsu, China) to detect the protein quantities. To separate the total proteins from each sample, 10% sodium-dodecyl sulfate–polyacrylamide gel-electrophoresis (SDS-PAGE) was utilized, and the proteins were then deposited into PVDF-membranes (EMD-Millipore, MA, USA). Following that, these PVDF-membranes were treated with specific primary antibodies and incubated for approximately 12 h at 4 °C, followed by exposure to appropriate secondary antibodies for around 1 h at room temperature. In addition, we used an advanced chemiluminescence detection kit and a scanner (ThermoFisher-Scientific, MA, USA) to measure protein bands in PVDF membranes. In the present study, the detailed information of primary antibodies were as follows: anti-IL-1β (Catalogue No. ab-200478; 1:1000; Abcam, Cambridge, MA, USA), anti-IL-6 (Catalogue No. ab-6672; 1:1000; Abcam, Cambridge, MA, USA), anti-TNF-α (Catalogue No. ab-1793; 1:1000; Abcam, Cambridge, MA, USA), anti-IL-10 (Catalogue No. ab-52909; 1:1000; Abcam, Cambridge, MA, USA), anti-NLRP3 (Catalogue No. 19771-1-AP; 1:1000; Proteintech Rosemont, IL, USA), anti-phosphorylated (p)-NF-κB (Catalogue No. CST-3033S; 1:1000; Cell-Signaling-Technology, Danvers, MA, USA), anti-NF-κB (Catalogue No. CST-8242S; 1:1000; Cell-Signaling-Technology, Danvers, MA, USA), anti-cleaved-Casepase-1 (Catalogue No. CST-89332S; 1:1000; Cell-Signaling-Technology, Danvers, MA, USA), anti-Casepase-1 (Catalogue No. ab-138483; 1:1000; Abcam, Cambridge, MA, USA), anti-cleaved-Casepase-3 (Catalogue No. ab-32042; 1:1000; Abcam, Cambridge, MA, USA), anti-Casepase-3 (Catalogue No. ab-44976; 1:1000; Abcam, Cambridge, MA, USA), anti-Bax (Catalogue No. ab-32503; 1:1000; Abcam, Cambridge, MA, USA), anti-Bcl-2 (Catalogue No. ab-182858; 1:1000; Abcam, Cambridge, MA, USA), anti-GAPDH (Catalogue No. 60004-1-Ig; 1:1000; Proteintech Rosemont, IL, USA). Secondary antibodies includes goat anti-mouse-IgG (Catalogue No. 15014; 1:5000; Proteintech Rosemont, IL, USA) and goat anti-rabbit-IgG (Catalogue No. B900210; 1:5000; Proteintech Rosemont, IL, USA). At least three times in separate experiments, the western blot assay was conducted. All findings were examined using Image-Lab (version: 4.0.1) software. In the present study, the relative target protein expression levels in each group were obtained by dividing the target protein levels by the GAPDH protein level.
Statistical analyses
The results of this investigation are provided as means and standard deviations (means ± SD). For all statistical analyses, the GraphPad Prism 7.0 software (GraphPad Software Inc., San Diego, CA, USA) was utilized. To compare different groups in the current study, ANOVA (analysis of variance) was used, with a P-value of less than 0.05 considered as statistically significant, as * denote p-values < 0.05.