Fig 2: Knockdown of Circ_0030235 remitted OGD/R-induced H9c2 cell injury via regulating miR-526b.H9c2 cells were co-transfected with sh-circ/sh-NC and miR-526b inhibitor/miR-NC for 48 h, followed by 6 h of OGD exposure and 12 h of re-oxygenation treatment. (A) Measurement of ROS generation using ROS Detection Assay Kit showed that OGD/R+sh-circ+miR-526b inhibitor group has a higher ROS generation than that in OGD/R+sh-circ+miR-NC group. ELISA assays were utilized to confirm the enhancement of (B) CK-MB and (C) cTnI levels in OGD/R+sh-circ+miR-526b inhibitor group. (D) JC-1-Mitochondrial Membrane Potential assay showed that miR-526b knockdown reversed the reduction of mitochondrial membrane potential loss triggered by sh-circ in OGD/R-treated cells. (E) Mitochondrial Viability Staining revealed that miR-526b knockdown inhibited the mitochondrial viability of OGD/R-treated and sh-circ-transfected cells. The data were expressed as the mean ±S.D. from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: ATR-I protects cardiomyocytes against injury induced by H/R in vitro. Rat cardiomyocytes were divided into the following five groups: control, H/R, and H/R + ATR-I preconditioning (10, 50, and 250 µg). Enzyme-linked immunosorbent assays were performed to determine the levels of CK-MB (a), CK (b), cTnI (c), and MB (d) expression in cardiomyocytes with H/R. *P < 0.05 vs the control group; #P < 0.05 vs the H/R group; &P < 0.05 vs the H/R + ATR-I 10-µg group; $P < 0.05 vs the H/R + ATR-I 50-µg group.H/R, hypoxia/reoxygenation; ATR-I, atractylenolide I; CK-MB, creatine kinase-MB; CK, creatine kinase; cTnI, cardiac troponin I.

Fig 4: Knockdown of Circ_0030235 remitted OGD/R-induced ROS and mitochondrial injury.H9c2 cells were transfected with sh-circ and sh-NC for 48 h, followed by 6 h of OGD exposure and 12 h of re-oxygenation treatment. (A) Measurement of ROS generation using ROS Detection Assay Kit showed that OGD/R+sh-circ group has a lower ROS generation than that in OGD/R+sh-NC group. ELISA assays were utilized to confirm the inhibitory effects of sh-circ transfection on the productions of (B) CK-MB and (C) cTnI in OGD/R-treated cells. (D) JC-1-Mitochondrial Membrane Potential assay showed that silencing Circ_0030235 reversed the enhancement of mitochondrial membrane potential loss triggered by OGD/R treatment. (E) Mitochondrial Viability Staining revealed that silencing Circ_0030235 enhanced the mitochondrial viability of OGD/R-treated cells. The data were expressed as the mean ± S.D. from three independent experiments. **P < 0.01, ***P < 0.001.

Fig 5: Protective effect of ATR-I on myocardial injury in rats with I/R. Male Sprague-Dawley rats were randomly allocated to the following five groups (nine rats/group): control, I/R, and I/R + ATR-I preconditioning (10, 50, and 250 µg). (a) Staining with 2, 3, 5-triphenyltetrazolium chloride was conducted to determine the infarction size of rat hearts in the different groups. (b) Representative hematoxylin and eosin-stained histological images (× 200 magnification) of myocardial tissue. Scale bars = 50 µm. (c–f) The enzyme-linked immunosorbent assay was used to determine CK-MB, CK, cTnI, and MB expression in myocardial tissue. (g) Arrhythmia score, (h) LVSP, (i) LVDP, (j) LVEDP, (k) LVEF, and (l) LVWT. *P < 0.05 vs the control group; #P < 0.05 vs the I/R group; &P < 0.05 vs the I/R + ATR-I 10-µg group; $P < 0.05 vs the I/R + ATR-I 50-µg group.I/R, ischemia/reperfusion; ATR-I, atractylenolide I; CK-MB, creatine kinase-MB; CK, creatine kinase; cTnI, cardiac troponin I; LVSP, left ventricular systolic pressure; LVDP, left ventricular-developed pressure; LVEDP, left ventricular end-diastolic pressure; LVEF, left ventricular ejection fraction; LVWT, left ventricular wall thickness.