Electroacupuncture pretreatment attenuates myocardial ischemia/reperfusion injury-induced ferroptosis via the p53/SLC7A11/GPX4 pathway

Figures

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  Figure 1. Electroacupuncture treatment improves energy metabolism and mitigates oxidative damage in cardiomyocytes during MIRI. (A) The level of ATP in cardiomyocytes was determined through kits; (B) Intracellular MDA in cardiomyocytes was determined based on kits; (C) The ROS in cardiomyocyte was determined by flow cytometry; (D) The SOD in cardiomyocytes was determined based on kits. [Data are presented as Means ± SD of n = 6 independent biological replicates. Intergroup differences were assessed via one-way ANOVA followed by Tukey’s post-hoc multiple comparison test; **p < 0.01, ***p < 0.001; ns, not significant (p> 0.05)].

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  Figure 2. Electroacupuncture mitigates the ferroptotic biochemical phenotype and alleviates myocardial iron overload in MIRI. (A) Fe²+ levels in cardiomyocytes were measured using a specific kit; (B–D) The contents of GSH and GSSG in cardiomyocytes were detected using specific assay kit; (E) The mRNA contents of ferroptosis-related proteins (TFR1, FTH1, and ACSL4) were determined via RT-PCR; (F) The proteins contents of ferroptosis-related proteins (TFR1, FTH1, and ACSL4) were determined through Western blot. [Data are presented as Means ± SD of n = 6 independent biological replicates. Intergroup differences were assessed via one-way ANOVA followed by Tukey’s post-hoc multiple comparison test; *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant (p> 0.05)].

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  Figure 3. Electroacupuncture alleviates MIRI and preserves cardiac function by restraining the ferroptotic biochemical signature. (A–C) Serum from each group of MIRI rat was collected to detect enzymes related to myocardial injury (CK-MB, LDH and cTnI); (D, E) Apoptosis of MIRI cardiomyocytes was determined by flow cytometry using TUNNEL staining; (F) mRNA levels of proteins associated with apoptosis were identified by RT-PCR (Bax, Bcl2 and caspase3); (G) Apoptosis-related protein levels were determined by Western blot; (H) Evans Blue-TTC dual staining shows the myocardial infarct size in SD rats; (I) Histopathological HE staining of myocardial tissue in each group. [Data are presented as Means ± SD of n = 6 independent biological replicates. Intergroup differences were assessed via one-way ANOVA followed by Tukey’s post-hoc multiple comparison test; *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant (p> 0.05)].

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  Figure 4. Electroacupuncture enhances myocardial energy metabolism and alleviates oxidative damage via the p53/SLC7A11/GPX4 pathway. (A) The content of ATP in cardiomyocytes was determined; (B–D) Substances associated with oxidative damage to cardiomyocytes was measured by using kits or flow cytometry (SOD, MDA and ROS). [Data are presented as Means ± SD of n = 6 independent biological replicates. Intergroup differences were assessed via one-way ANOVA followed by Tukey’s post-hoc multiple comparison test; ***p < 0.001.]

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  Figure 5. Electroacupuncture modulates the ferroptotic biochemical phenotype through the p53/SLC7A11/GPX4 pathway. (A) Fe2+ content of cardiomyocytes was determined (EA: MIRI rat were pretreated with EA; P53+ group: cardiomyocytes of rat with MIRI were treated with p53 agonist; SLC- group: cardiomyocytes of rat with MIRI were treated with EA and then added SLC7A11 inhibitor); (B–D) The kit detected the concentrations of GSH and GSSG in cardiomyocytes; (E) RT-PCR detected the mRNA level of ferroptosis-related proteins (p53, SLC7A11, GPX4, TFR1, FTH1 and ACSL4). (F) Western blot detected protein levels of ferroptosis-related proteins (p53, SLC7A11, GPX4, TFR1, FTH1 and ACSL4).(Data are presented as Means ± SD of n = 6 independent biological replicates. Intergroup differences were assessed via one-way ANOVA followed by Tukey’s post-hoc multiple comparison test; *p < 0.05, **p < 0.01; ***p < 0.001, ns, not significant (p> 0.05)).

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  Figure 6. Electroacupuncture protects cardiomyocytes via the p53/SLC7A11/GPX4 pathway. (A–C) Myocardial cell injury markers in each group of rat were detected using ELISA kits; (D) The apoptosis of rat in each group was detected by flow cytometry; (E–G) The mRNA levels of apoptosis-related proteins in cardiomyocytes in each group were identified by RT-PCR;(H) The expression of apoptosis-related proteins in cardiomyocytes in each group was quantified by Western blot; (I) Evans Blue-TTC dual staining shows the myocardial infarct size in SD rats; (J) Histopathological HE staining of myocardial tissue in each group. [Data are presented as Means ± SD of n = 6 independent biological replicates. Intergroup differences were assessed via one-way ANOVA followed by Tukey’s post-hoc multiple comparison test; *p < 0.05, **p < 0.01, ***p < 0.001.

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  Figure 7. The SLC7A11 agonist reverses the pro-ferroptotic effects of the p53 agonist, confirming that electroacupuncture protects against MIRI via the p53/SLC7A11/GPX4 signaling pathway. (A) The Fe2+ content of cardiomyocytes in each group was quantified by the kit; (B–D) The GSH and GSSG contents of cardiomyocytes in each group were quantified; (E) The mRNA levels of proteins associated with ferroptosis in each group were identified again when the combination of p53 agonists and SLC7A11 agonists or SLC7A11 inhibitors were added. (F) The expression levels of proteins associated with ferroptosis in each group were once again identified. [Data are presented as Means ± SD of n = 6 independent biological replicates. Intergroup differences were assessed via one-way ANOVA followed by Tukey’s post-hoc multiple comparison test; *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant (p> 0.05)].