Estrogen Alleviates Myocardial Ischemia–Reperfusion Injury by Inhibiting NLRP3 Inflammasome‐Mediated Pyroptosis

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  Figure 1. Experimental timeline of the in vivo animal study. C57BL/6 female mice underwent bilateral ovariectomy (OVX) at Week 0, followed by a 4 week recovery period. From Week 4 to Week 8, animals received daily subcutaneous injections of either 17β-estradiol (E2; 5 μg/mL in sesame oil) or vehicle control (sesame oil). At Week 8, myocardial ischemia/reperfusion (I/R) surgery was performed via transient ligation of the left anterior descending coronary artery (30 min ischemia/24 h reperfusion), with sham-operated controls undergoing identical procedures without ligation. Terminal analysis occurred at Week 8 + 1 day, encompassing infarct size quantification (TTC/Evans blue staining), serum biomarker assessment (LDH, CK-MB), and pyroptosis evaluation (TUNEL, Western blot for TXNIP/NLRP3 pathway proteins). Experimental groups included OVX + Sham (vehicle), OVX + I/R (vehicle), and OVX + E2+I/R (n = 14/group).

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  Figure 2. Comparison of plasma E2 level, infarct size, pyroptosis and pyroptosis-related proteins in mice (n = 10 in each group). (a) Plasma E2 level alteration induced by OVX and estrogen replacement. No significant difference of plasma E2 was observed after OVX between Sham and MI/R groups. E2 dramatically increased as a result of estrogen replacement therapy for 4 weeks. The results shown are mean ± SD from three independent experiments. ∗∗p < 0.01 compared with OVX + MI/R group; (b) representative TTC–Evans Blue stained sections of hearts from OVX + Sham, OVX + MI/R, and OVX + MI/R + E2 group. The area stained with Evans blue represented the non-I/R myocardium, whereas the unstained area was the I/R myocardium or AAR. Within the AAR, the brick red-stained area represented viable myocardium, whereas the unstained (white) area represented infarcted myocardium; (c) the ratio of IS/AAR. The results shown are mean ± SD from three independent experiments. ∗∗p < 0.01 compared with OVX + MI/R group; (d) comparison of TUNEL-positive cells in each group. TUNEL-positive cells significantly increased following MI/R. E2 replacement therapy significantly reduced TUNEL-positive cells. The results…

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  Figure 3. Comparison of cell injury TUNEL-positive cells in each group in primary cardiomyocytes (n = 6 in each group). (a) Comparison of cell viability in each group. Cell viability decreased significantly after H/R procedure. E2 pretreatment significantly preserved cell viability, which could be partly reversed by ER antagonist ICI 182780. Compared with the H/R + E2 + ICI 182780 group, pretreatment with either Bay11-7082 or AC-YVAD-CMK could significantly preserve cell viability. The results shown are mean ± SD from three independent experiments. ∗∗p < 0.01 compared with the H/R group; ##p < 0.01 compared with the H/R + E2+ICI 182780 group. (b) Comparison of LDH in each group. LDH increased significantly after H/R procedure. E2 pretreatment significantly reduced LDH, which could be partly reversed by ER antagonist ICI 182780. Compared with the H/R + E2+ICI 182780 group, pretreatment with either Bay11-7082 or AC-YVAD-CMK could significantly reduce LDH. The results shown are mean ± SD from three independent experiments. ∗∗p < 0.01 compared with H/R group; ##p < 0.01 compared with the H/R + E2+ICI 182780 group. (c) Comparison of TUNEL-positive cells in each group. TUNEL-positive cells…

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  Figure 4. Comparison of Caspase-1 activity, TXNIP, IL-1β, and IL-18 in each group in primary cardiomyocytes (n = 6 in each group). (a) Comparison of Caspase-1 activity in each group. Caspase-1 activity increased significantly after H/R procedure. E2 pretreatment significantly reduces Caspase-1 activity, which could be partly reversed by ER antagonist ICI 182780. Compared with the H/R + E2+ICI 182780 group, pretreatment with either Bay11-7082 or AC-YVAD-CMK could significantly reduce Caspase-1 activity. The results shown are mean ± SD from three independent experiments. ∗∗p < 0.01 compared with the H/R group; ##p < 0.01 compared with the H/R + E2+ICI 182780 group. (b) Comparison of TXNIP in each group. TXNIP level increased significantly after H/R procedure. E2 pretreatment significantly reduces the TXNIP level, which could be partly reversed by ER antagonist ICI 182780. Compared with the H/R + E2 + ICI 182780 group, pretreatment with either Bay11-7082 or AC-YVAD-CMK could significantly reduce the TXNIP level. The results shown are mean ± SD from three independent experiments. ∗∗p < 0.01 compared with the H/R group; ##p < 0.01 compared with the H/R + E2 + ICI 182780 group. (c) Comparison of…

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  Figure 5. Comparison of pyroptosis-related proteins in each group in primary cardiomyocytes (n = 6 in each group). (a) Representative blots were displayed. (b) Comparison of pyroptosis-related proteins in each group. These proteins increased significantly after the H/R procedure. E2 pretreatment significantly reduces the expression of these proteins, which could be partly reversed by ER antagonist ICI 182780. Compared with the H/R + E2 + ICI 182780 group, pretreatment with either Bay11-7082 or AC-YVAD-CMK could significantly reduce the expression of these proteins. The results shown are mean ± SD from three independent experiments. ∗∗p < 0.01 compared with the H/R group; ##p < 0.01 compared with the H/R + E2 + ICI 182780 group.

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  Figure 6. Estrogen alleviates myocardial I/R injury by inhibiting NLRP3 inflammasome-mediated pyroptosis. E2 alleviated myocardial I/R injury via its receptor ER to downregulate TXNIP–NLRP3 and further inhibit Caspase-1, thereby suppressing ASC, and IL-18, IL-1β, and GSDMD.