2.1 Animals, cells and main reagents
Male C57dx newborn mice (3 days old, SLAC, Shanghai, China) and male C57BL/6 mice, (8 weeks old, SLAC, Shanghai, China), were purchased from the experimental animal center of Shanghai Jiao Tong University. RPMI 1640 medium and fetal bovine serum were purchased from Gibco, USA. LPS and dimethyl sulfoxide (DMSO) were purchased from Sigma; the diquinolinic acid (BCA) protein quantitative kit was purchased from the Nanjing Nuowizan company; THBS1 siRNA and control siRNA were purchased from Santa Cruz, USA. THBS1 mouse anti-human monoclonal antibody (abl823) was purchased from Abcam. Anti-actin and sheep anti-mouse polyclonal antibodies were purchased from the Beijing Zhongshan Jinqiao Biotechnology company. All experimental procedures were carried out according with the guidelines of the Institutional Animal Care and Use Committee of Shanghai. All of the experimental procedures were performed after obtaining the approval of the Ethical Committee for Animal Experiments of Shanghai general hospital (Shanghai, China).
2.2 Cell culture and stimulation
We randomly selected 12 C57dx 3d old mice. After cervical dislocation, their hearts were extracted, the atria were cut off, the hearts were rinsed with LML collagenase, and the heart tissue was cut into approximately 1 mm3 pieces. The pieces of heart tissue were mixed with collagenase and were transferred into a centrifuge tube and were repeatedly shaken and mixed. After allowing it to stand and any solid tissue had settled, the supernatant was removed and this was repeated until the tissue mass was dissolved completely. The obtained supernatant was centrifuged at 2000 r/min for 10 min, discarded, and the precipitate was mixed with 2 ml of medium, and then inoculated into a 75 ml culture bottle. After incubating at 37℃ for 90 min, the nonmyocardial cells had adhered to the bottle, and the cell suspension, which contained the myocardial primary cells, was inoculated into another culture bottle.
2.3 Detection of THBS1 mRNA in myocardial cells was performed by real-time fluorescence quantitative PCR (qRT-PCR). Total RNA was extracted from the cardiomyocytes according to the instructions of the TRIzol reagent. The concentration of RNA was measured by a micro ultraviolet spectrophotometer and its purity was determined. The RNA was reversely transcribed into cDNA using a reverse transcription kit and amplified according to the instructions of the qRT-PCR kit. After the reaction, the amplification curve and dissolution curve were confirmed, and the results were interpreted strictly in accordance with the instructions of the kit. The 2-ΔΔCt method was used to calculate the level of THBS1 mRNA expression.
2.4 Western blotting analyses
The cells were placed in the cell lysis solution. The total protein concentration was detected using a BCA kit. The protein samples (60 µg per well) were electrophoresed on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and then transferred to a PVDF membrane. The membrane was blocked and then incubated with the indicated primary antibody at 4 ℃ overnight, washed, then incubated with the secondary antibody. The signal was detected with an ECL system using an A1600 detector and photographic exposure system. Protein expression levels were calculated using actin as the internal reference.
2.5 Effects of transfected siRNA on myocardial cell injury, oxidative stress, inflammatory response and apoptosis
2.5. 1 In the experimental group, primary cells were inoculated into a 25 cm2 culture flask and RPMI 1640 medium containing 10% fetal bovine serum by volume was added. After the cells grew well, they were digested with trypsin and inoculated into 6-well plates. Four groups were set up for the experiment: A. Control group; B. LPS group; C. THBS1 siRNA group; and D. LPS+THBS1 siRNA group. There was no intervention in the control group; in the LPS group, we used 1 mg/L LPS to stimulate the cardiomyocytes. THBS1 siRNA group: THBS1 siRNA was transfected into the cardiomyocytes with Liposome 2000. LPS+THBS1 inhibition group: THBS1 siRNA was transfected with Liposome 2000 into LPS-treated cardiomyocytes.
2.5. 2 Detection of THBS1 mRNA in cells after transfection for 48 h. The expression of THBS1 mRNA in the cells was detected by qRT-PCR, using the same method as 1.2. Each group was tested with three biological replicates twice.
2.5. 3 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 24, 48, 72 and 96 h, expression of cTNI, proBNP, ROS, IL-6, TNF-α and caspase-3 were detected by ELISA.
2.6 Sepsis model:
Healthy SPF C57BL/6 mice(n=18), male, 8 weeks old, weight approximately 20±2 g, were used. Before the experiment, the mice were given free food and water and kept at an ambient temperature of 20℃. Intraperitoneal injection of LPS 15 mg/kg was used to make the model. To judge the success of estabolishing model according to the references, It was confirmed that the sepsis modelling was successful by histopathological changes in cardiac muscle 24 h after modelling and cTNI, proBNP, Il-6, and TNF-αexpression by ELISA. Three groups(n=6 every group) have been designed in the animal experiments. A. Control group; B. LPS group; C. LPS+THBS1 Inhibition group.
2.7 H&E, TUNEL and caspase-3 staining
H&E staining and TUNEL analysis were performed to assess the histopathological changes in myocardial tissue. Solarbo (4% paraformaldehyde) was used to fix the mouse myocardium overnight at 4°C. Paraffin embedding, tissue sections and H&E staining were performed as described above. A TUNEL assay kit (Roche Diagnostics, Indianapolis, IN, USA) was used to measure the rate of apoptosis of the cardiomyocytes, according to the manufacturer's specifications. A caspase-3 fluorometry kit (Beyotime Institute of Biotechnology, Shanghai, China) was used for detection. We did not test LDH activity, which is a marker of cell injury ,Because LDH is greatly affected by Sepsis.
2.8 Collection of clinical samples. Serum was collected from patients with myocardial injury of sepsis, those without myocardial injury of sepsis and healthy people and stored at −80℃. THBS1 was detected by ELISA. The prognosis of the patients was collected during the follow-up of 28 days.
2.9 Statistical analysis. SPSS 17.0 was used to process the data. The expression levels of THBS1 mRNA, cTNI, proBNP, ROS, IL-6, TNF- α and caspase-3 in the different groups were compared by one-way ANOVA. LSD-t test was used for pairwise comparisons. Statistical significance was defined as p =0.05.