Male C57dx newborn mice and male C57BL/6 mice were pur-chased from the Experimental Animal Center of Shanghai Jiao Tong University (Shanghai, China). All of the experimental procedures were performed after obtaining the approval of the Ethical Committee for Animal Experiments of Shanghai General Hospital (Shanghai, China; 2020AWS006); all experimental procedures were carried out according with the guidelines of the Institutional Animal Care and Use Committee of Shanghai General Hospital. RPMI-1640 medium and fetal bovine serum were purchased from Gibco (Thermo Fisher Scientific, Inc.). Lipopolysaccharide (LPS). BCA protein quantitative kit was purchased from Vazyme Biotech Co., Ltd. siTHBS1 and control siRNA were purchased from Santa Cruz Biotechnology, Inc. Mouse anti-human THBS1 monoclonal antibody (cat. no. ab1823) was purchased from Abcam. Anti-β-actin (cat. no. ab8226) was purchased from Abcam and goat anti-rabbit polyclonal antibodies (HRP-conjugated) (cat. no. 31460; Thermo Fisher Scientific, Inc.).

A total of 12 male C57dx mice (age, 3 days; weight 1.5–2 g) were randomly selected. After cervical dislocation, the hearts were extracted and the atria were cut off; the ventricles were rinsed with 1 ml collagenase, and the heart tissue was cut into ~1 mm³ pieces. The pieces of heart tissue were mixed with collagenase and transferred into a 15-ml centrifuge tube and were repeated-ly shaken and mixed at 37°C for 1 min. After allowing the tubes to stand and for the solid tissue to settle, the supernatant was removed, and this was repeated until the tissue mass was dissolved completely. The obtained supernatant was centrifuged at 2,000 × g for 10 min (37°C), the supernatant was discarded and the precipitate was mixed with 2 ml of medium, which was inoculated into a 75 ml culture bottle. After incubating at 37°C for 90 min, the non-myocardial cells had adhered to the bottle, and the cell suspension (5×10⁵−6×10⁵ cells/ml), which contained the myocardial primary cells, was subsequently inoculated into another culture bottle and incubated at 37°C for 24 h.

Total RNA was extracted from the cardiomyocytes (5×10⁵−6×10⁵ cells/ml) using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The concentration of RNA was measured by a NanoDrop 8000 spectrophotometer (Thermo Fisher Scientific, Inc.) and its purity was determined. Total RNA was reverse transcribed into cDNA using a TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) (Agrisera) for 30 min at 42°C, then 5 sec at 85°C. qPCR was subsequently performed using Unique Aptamer qPCR SYBR Green Master Mix (MedChemExpress), according to the manufacturer's protocol. After the reaction, the amplification and dissolution curves were verified, and the results were interpreted strictly in accordance with the instructions of the kit. The 2^−ΔΔCq method (15) was used to calculate the level of THBS1 mRNA expression. The primer sequences were as follows: THBS1, forward 5′-GGAAAGAUUUCACUGCAUATT-3′, reverse 5′-UAUGCAGUGAAAUCUUUCCAG-3′; and GAPDH, forward 5′-GTCAAGGCTGAGAACGGGAA-3′, reverse, 5′-AAATGAGCCCCAGCCTTCTC-3′. The thermocycling conditions used for qPCR were as follows: Initial denaturation at 94°C for 5 min; followed by 30 cycles of 5 sec at 94°C and annealing and extension at 65°C for 30 sec.

The cells (5×10⁵−6×10⁵ cells/ml) were placed in RIPA lysis buffer (Beyotime Institute of Biotechnology). The total protein concentration was detected using a BCA kit. The protein samples (60 µg/well) were separated by 8% SDS-PAGE and then transferred to a PVDF membrane. The membrane was blocked with 5% skimmed milk powder (37°C for 1 h) and then incubated with the indicated primary antibodies, anti-THBS1 (1:200; cat. no. ab1823) or β-actin (1:200; cat. no. ab8226) at 4°C for 12 h. Following the primary antibody incubation, the cells were washed with TBS-Tween 20 and incubated with the secondary antibody (1:2,000) at 37°C for 1 h. Protein bands were visualized with an ECL system (Abcam) using a western blot gel imager (ChemiDoc; Bio-Rad Laboratories, Inc.). Protein expression levels were calculated using ImageJ V1.8.0.112 software (National Institutes of Health) using actin as the internal reference.

Primary cells (5×10⁵−6×10⁵ cells/ml) were inoculated into a 25 cm² culture flask containing RPMI-1640 medium supplemented with 10% fetal bovine serum by volume. Then, cells were digested with trypsin and inoculated into 6-well plates. Four experimental groups were prepared: i) Control group; ii) LPS group; iii) siTHBS1 group; and iv) siTHBS1 + LPS group. There was no intervention in the Control group. Cells were treated at 37°C for 24 h with 1 mg/l LPS to stimulate the cardiomyocytes. siTHBS1 was transfected into the cardiomyocytes using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C at 24 h with or without previously stimulating with LPS, aforementioned.

The expression of THBS1 mRNA in the cells was detected by RT-qPCR, aforementioned. Each group was tested with three biological replicates twice.

Cell injury, oxidative stress, inflammatory response and apoptosis were detected 48 h after transfection. The cells were inoculated into a 96-well plate. After routine culture for 48 h, expression of cardiac troponin I (cTNI) (mouse TNNI3/cTn-I ELISA kit; cat. no. D721149-0048; Sangon Biotech Co., Ltd.), pro-brain natriuretic peptide (proBNP; mouse NT-proBNP ELISA kit; cat. no. E-EL-M0834; Elabscience Biotechnology, Inc.), reactive oxygen species (ROS; ROS ELISA kit; cat. no. ZK-M5156; ZIKER), IL-6 (IL-6 ELISA kit; cat. no. ab46100; Abcam), TNF-α (TNF-α ELISA kit; cat. no. SEKM-0034; Beijing Solarbio Science & Technology Co., Ltd.) and caspase-3 (caspase-3 ELISA kit; cat. no. BH-E0764; Thermo Fisher Scientific, Inc.) were detected by ELISA. Supernatant was used in the ELISA, which was obtained by centrifugation of the cell culture at 4°C at 6,000 × g for 5–10 min.

Healthy specific pathogen free male C57BL/6 mice (n=18; age, 8 weeks; weight ~20±2 g) were used. Before the experiment, the mice were provided free access to food and water, and maintained at an ambient temperature of 20°C and 40–60% humidity under a 14/10-h light/dark cycle. Intraperitoneal injection of LPS 15 mg/kg was used to make the model, and successful model establishment was determined based on a previous study (16); sepsis modelling was confirmed successful by histopathological changes in cardiac muscle 24 h after modelling, as well as determination of cTNI, proBNP, Il-6, and TNF-α levels by ELISA using serum which was collected following euthanasia. Three groups (n=6 mice/group) were used in the in vivo experiments: i) Control group; ii) LPS group; and iii) siTHBS1 + LPS group (15 nmol/20 g weight siTHBS1 was injected through the caudal vein). Euthanasia was performed in a CO₂ chamber with a flow rate of 50% volume displacement/min. The means used to verify mouse death were whether the heart stopped completely and whether the pupils were dilated.

Upright fluorescence Metallurgical Microscope was NIKON ECLIPSE C1 and inverted fluorescence microscope was NIKON ECLIPSE TI-SR (magnifications, ×200 and 400). H&E staining and TUNEL analysis were performed to assess the histopathological changes in myocardial tissue. Mouse myocardial tissues were fixed with 4% paraformaldehyde (Beijing Solarbio Science & Technology Co., Ltd.) for 12 h at 4°C. The nuclei were counterstained in the TUNEL and caspase-3 staining assay with DAPI for 10 min at 37°C. Paraffin embedding, tissue sections and H&E staining were performed (the width of the sections used was 0.5-mm and H&E staining was performed at 37°C for 30 min). A TUNEL assay kit (Roche Diagnostics; cat. no. 11684817910) was used to measure the rate of apoptosis of the cardiomyocytes, according to the manufacturer's specifications. A caspase-3 fluorometry kit (Wuhan Servicebio Technology Co., Ltd.) was used for detection of caspase-3 levels. LDH activity, which is a marker of cell injury, was not examined because LDH is greatly affected by sepsis (17).

A total of 2 ml serum was collected from patients with sepsis-induced myocardial injury (n=84), those without sepsis-induced myocardial injury (n=84) and healthy individuals (n=10) and stored at −80°C. The inclusion criteria included patients who were diagnosed with sepsis and were 18–80 years old. The exclusion criteria included the presence of malignant tumors and post-transplant patients. THBS1 was detected by ELISA, aforementioned. The prognosis of the patients was recorded during the 28-day follow-up. Ethics approval was obtained for the use of humans/human tissues prior to the start of the study from the ethics committee based at Shanghai General Hospital (approval no. 2020sq049). Informed consent was obtained from each patient prior to participation. Basic clinicopatholgical features, including age and sex, of the patients with sepsis-induced myocardial injury (n=84) are shown in Table I.

SPSS v17.0 (SPSS, Inc.) was used to process the data. The expression levels of THBS1 mRNA and cTNI, proBNP, ROS, IL-6, TNF- α and caspase-3 levels in the different groups were compared by one-way ANOVA; Fisher's LSD test was used for pairwise comparisons. Kaplan-Meier survival curves were analyzed by log-rank; the cut-off value used to separate high vs. low THBS1 was the mean value of expression 271.179 ng/ml. The optimal cutoff values were determined by the highest values of sensitivity and specificity indicated in the area under the receiver operatic characteristic (ROC) curve (AUC) analysis. P≤0.05 was used to indicate a statistically significant difference.

Successful knockdown of THBS1 by siRNA is shown in Fig. 1. THBS1 expression was reduced in the in vivo model mice injected with siTHBS1 compared with the control group, as determined using western blotting and RT-qPCR (Fig. 1A and B). Similarly, THBS1 expression was reduced in the in vitro primary myocardial cell cultures following siTHBS1 transfection compared with the control group, as determined using western blotting and RT-qPCR (Fig. 1C and D).

In the LPS-stimulated primary cardiomyocytes, RT-qPCR analysis demonstrated a significant increase in THBS1 mRNA expression compared with the Control group (Fig. 2B); a similar increase was observed for THBS1 protein expression detected by western blotting (Fig. 2A).

In LPS-stimulated primary cardiomyocytes, ELISA demonstrated a significant increase in caspase-3 and ROS levels in the LPS group compared with the control group. Caspase-3 was significantly decreased compared with the LPS group, indicating that myocardial cell apoptosis was decreased (Fig. 2C), and ROS levels were significantly decreased compared with the LPS group, indicating that the oxidative stress response was decreased (Fig. 2D).

In the LPS-stimulated primary myocardial cells, ELISA results demonstrated that the cTNI, proBNP, Il-6 and TNF-α levels were significantly increased compared with the control group (Fig. 3A-D, respectively). cTNI, proBNP, IL-6 and TNF-α levels were significantly reduced in the siTHBS1 + LPS group compared with the LPS group.

In the mouse cardiac pathology sections from mice intraperitoneally injected with LPS (15 mg/kg), myocardial histopathological changes were observed 24 h after mouse modeling under a light microscope. In the Control group (Fig. 4A and D), the myocardial fibers were arranged neatly with clear horizontal stripes; the cardiomyocytes displayed normal morphology, the cytoplasm was pink, the nuclear membrane was intact, the chromatin was dense and deeply stained, and there were no significant changes in the myocardial interstitium. In the LPS group (Fig. 4B and E) the myocardial fibers were irregular; there was interstitial edema, hemorrhage, and inflammatory cell infiltration. In the siTHBS1 + LPS group (Fig. 4C and F), there was interstitial edema, but inflammatory cell infiltration was reduced compared with the control. Successful knockdown of THBS1 by siRNA is shown in Fig. 1A and B.

TUNEL staining was used to observe the changes of the myocardial cells in mice 24 h after modeling. LPS (15 mg/kg) treatment (Fig. 5B) led to increased apoptosis compared with the Control group (Fig. 5A), whereas the siTHBS1 + LPS group (Fig. 5C) showed decreased apoptosis compared with the LPS group.

Caspase-3 immunostaining was used to observe the changes of myocardial cells in mice 24 h after modeling. The LPS group (Fig. 5E) displayed increased caspase-3 expression compared with the Control group (Fig. 5D), and there was decreased caspase-3 expression in the siTHBS1 + LPS group compared with the LPS group (Fig. 5F).

THBS1 serum levels were significantly increased in patients with sepsis accompanied by myocardial injury compared with the control group (Fig. 6A), and THBS1 was also higher in deceased patients compared with those who survived (Fig. 6B). The survival rate of patients with high THBS1 was lower compared with that of patients with low THBS1 over the 28-day follow-up (Fig. 6C). THBS1 expression can be used to predict myocardial injury, the cutoff value of THBS1 is 271.2 ng/ml. When THBS1>271.2 ng/ml, myocardial injury may happen (AUC =0.7906; P<0.001; Fig. 6D).