RIPC improves myocardial injury by promoting mitochondrial protection via the PGC-1α/Nrf2 pathway

Figure 1. PGC-1α and Nrf2 were highly expressed in the serum of patients with MI treated with RIPC.A, expression of PGC-1α and Nrf2 was assessed by qRT-PCR in the serum samples of 20 patients with MI and 20 patients with MI treated with RIPC. B, Pearson correlation analysis evaluated the correlation between PGC-1α and Nrf2 expression in patients with MI treated with RIPC. n = 20; ∗∗p < 0.01; ∗∗∗p < 0.001.

Figure 2. Constructing an MI model through hypoxia in vitro and RIPC treatment through intermittent hypoxia/reoxygenation.A, hypoxia caused vacuolization, the disappearance of mitochondrial cristae, and ultrastructural damage in AC16 mitochondria. After RIPC treatment, the damage to mitochondrial ultrastructure was reduced while the tendency for mitochondrial fission increased due to hypoxia; the therapeutic effect of RIPC disappeared after inhibiting PGC-1α. The arrow refers to mitochondria. Scale bar = 500 nm. B, following cellular hypoxia, mitochondrial ROS levels significantly increased, and after receiving RIPC treatment, mitochondrial ROS levels significantly decreased. After silencing PGC-1α, the effect of RIPC disappeared. C, RIPC treatment significantly increased the mitochondrial membrane potential in hypoxic AC16 cells, but this effect was negated by silencing PGC-1α. At high membrane potential, JC-1 forms red-fluorescent J-aggregates, while at low membrane potential, it remains in a green-fluorescent monomeric form. Scale bar = 100 μm. D, CCK8 cell viability test results showed that after receiving RIPC treatment, cell viability increased significantly, and the effect…

Figure 3. Silencing PGC-1α by specific shRNA abolished the upregulation of RIPC on Nrf2 expression in AC16 cells.A, Hypoxia reduces Nrf2 levels while RIPC treatment boosts Nrf2 expression, but this effect vanishes with PGC-1α inhibition. Scale bar = 25 μm. B, Nrf2 mRNA level drops with hypoxia, rise with RIPC, and decrease again when PGC-1α is silenced. C, the expression of Nrf2 protein was markedly reduced following hypoxic, elevated subsequent to RIPC treatment, and notably diminished upon PGC-1α silencing. n = 3. ∗∗∗p < 0.001.

Figure 4. In vivo silencing of PGC-1α reversed the protective effects of RIPC against hypoxia-induced cardiac injury.A, as identified by TTC staining, treatment of RIPC attenuated MI-induced cardiac infarct size, while the reversal effect disappears following PGC-1α silencing. B, mouse heart tissue was collected for qRT-PCR analysis. In MI mice with PGC-1α silencing, the RIPC-induced elevation in Nrf2 expression was effectively counteracted. C, doppler echocardiography showed that RIPC significantly enhanced EF and FS in MI mice, an effect not observed in the absence of PGC-1α. D, mouse heart tissue was harvested for WB analysis. In PGC-1α-silenced MI mice, the RIPC-induced increase in Nrf2 expression was significantly diminished. E, pathological changes of mice cardiac tissues in HE staining, scale bar = 100 μm. F, representative mitochondrial electron microscopy images from the heart tissue of mice, scale bar = 1 μm. n = 5. ∗∗p < 0.01; ∗∗∗p < 0.001.