Ws20051129

The TF was performed at the middle of the right tibia of Col2al‐ICAT transgenic mice and C57BL/6J mice. After injections with 2% nembutal (WS20051129, Sinopharm Chemical Reagent Co., Ltd, Shanghai, China) for anesthetization, the mouse right tibia was exposed. After transection of the middle of the tibia, a bone nail (0.45 mm in diameter) was used to fix it and then the wound was closed. The following day, X‐ray examination was carried out to verify successful model establishment. A total of 21 C57BL/6J mice were assigned into the TF group [after establishment of TF, C57BL/6J mice were injected with 10 μL of phosphate buffer saline (PBS) at the site of the fracture], the siRNA group [after establishment of TF, C57BL/6 J mice were injected with 10 μL of MEG3 siRNA (4 mg/kg, MEG3 siRNA was synthesized by Sangon Biotech (Shanghai, China) at the site of the fracture] and the scramble group (after establishment of TF, C57BL/6J mice were injected with 10 μL of scramble siRNA at the site of the fracture). A total of 14 Col2al‐ICAT transgenic mice were assigned into the Col2a1‐ICAT group (after establishment of TF, Col2al‐ICAT transgenic mice were injected with 10 μL of PBS at the site of the fracture) and the Col2a1‐ICAT +siRNA group (after establishment of TF, Col2al‐ICAT transgenic mice were injected with 10 μL of MEG3 siRNA [4 mg/kg] at the site of the fracture).

The mice received 2% pentobarbital sodium (WS20051129, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) intraperitoneal injections and underwent transthoracic echocardiography analyses at T₀ (30 min prior to ischemia), T₁ (30 min of ischemia) and T₂ (120 min of reperfusion). High frequency ultrasonoscope Acuson sequoia 512 (Acuson Corp., Silicon Valley, CA, U.S.A.) was employed at a probe frequency of 8.5 mHz and a scanning speed at 100 mm/s. The mice were anesthetized using 2% pentobarbital sodium and subsequently fixed on the experiment table. The M-curve was measured at the long axis of papillary muscle and left ventricle section level. The following variables were measured and averaged during the three consecutive cardiac cycles: left ventricular end-systolic diameter (LVSD), left ventricular end-diastolic diameter (LVDD), left ventricular end-systolic volume (LVSV), and left ventricular end-diastolic volume (LVDV). The left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) values were converted by the Simpson method using the following formula: LVEF = (LVDV – LVSV)/LVDV × 100%; LVFS = (LVDD – LVSD)/LVDD × 100%. LVEF and LVFS were used as parameters indicating cardiac function. The experiment was conducted three times and the mean value was obtained.

On P21, rats were randomized to fasting in 30% oxygen (O₂)/70% nitrogen (N₂) or anesthetized with 3% sevoflurane (about 1.5 MAC, Maruishi Pharmaceutical Co., Ltd., Japan, catalog: 95071) plus 30% O₂/70% N₂ for 4 h in control or anesthesia chambers. Inspired anesthetic and oxygen concentrations were monitored using a gas analyzer (RGM 5250; Datex-Ohmeda, Louisville, CO, United States). The temperature in the chambers was maintained via a heating pool at 37 ± 0.5°C. Rats rectal temperature were maintained at 37 ± 0.5°C. Rats for harvest at 2 h, 24 h, and 7 days time points post-treatment were assigned randomly into two study groups: (1) control groups and (2) anesthesia groups. Rats for harvest at the 4 days post-treatment time point were assigned randomly into eight study groups: (1) non-anesthesia + vehicle; (2) non-anesthesia + inhibitor of caspase-3; (3) non-anesthesia + inhibitor of LC3B; (4) non-anesthesia + inhibitor of NF-κB; (5) anesthesia + vehicle; (6) anesthesia + inhibitor of caspase-3; (7) anesthesia + inhibitor of LC3B; (8) anesthesia + inhibitor of NF-κB (n = 6 each group). Rats were sacrificed by intraperitoneal injection of 100 mg/kg pentobarbital (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China, catalog: WS20051129).