Wedelolactone Ameliorates Ischemic Stroke by Inhibiting Oxidative Damage and Ferroptosis via HIF-1α/SLC7A11/GPX4 Signaling

Abstract

Purpose: Wedelolactone (Wel), a furocoumarin compound extracted from the medicinal plant Eclipta prostrata L. has been shown to exhibit significant neuroprotective effects. However, the potential of Wel to improve ischemic stroke (IS) and the underlying mechanisms remain unclear.

Methods: The middle cerebral artery occlusion and reperfusion (MCAO/R) and oxygen-glucose deprivation/reoxygenation (OGD/R) were established to evaluate the potential neuroprotective of Wel. Neurological function, brain infarct volume, brain swelling, and histopathological staining were analyzed to demonstrate the therapeutic efficacy of Wel. The occurrence of ferroptosis was confirmed by quantifying the levels of ROS and Fe²⁺, and by examining the ultrastructural features of cells. The binding affinity between Wel and HIF-1α was evaluated using Molecular docking. Immunofluorescence and Western blot analyses were conducted to explore the regulation of Wel on the HIF-1α/SLC7A11/GPX4 pathway. Finally, the expression of HIF-1α was down-regulated to verify whether Wel exerts neuroprotection by modulating HIF-1α to inhibit ferroptosis of cells.

Results: The results demonstrated that Wel effectively inhibit neuron ferroptosis after MCAO/R in a dose-time-dependent manner, thereby alleviating brain injury. Moreover, at the cellular level, Wel significantly reduced the accumulation of Fe²⁺ and ROS after OGD/R. Further investigation revealed that Wel might inhibit neuronal ferroptosis and improve IS by promoting the nuclear translocation and accumulation of HIF-1α, subsequently regulating the expression of downstream proteins SLC7A11 and GPX4. Notably, the inhibitory effect of Wel on ferroptosis was markedly attenuated by HIF- 1α siRNA in OGD/R PC12 cells.

Conclusion: Our discovery revealed that the Wel could alleviate IS by inhibiting oxidative damage and ferroptosis via HIF-1α/ SLC7A11/GPX4 signaling pathway. Wel may represent a promising active compound for anti-IS, while also providing novel insights into the elucidation of its molecular mechanisms and potential clinical implications.

Keywords: HIF-1α/SLC7A11/GPX4 pathway; Wedelolactone; ferroptosis; ischemic stroke.

Graphical abstract

Figure 1

Wel improved neurological dysfunction after MCAO/R. (A) The chemical information of Wel. (B) Flow chart of the experiment. (C) Body weight was evaluated on the 0d, 3d and 7d. The neurological scores including Bederson score (D), mNSS score (E), Right turn in corner test (%) (F) and left forelimb removal time (G) were evaluated on the 0d, 3d and 7d. Data were expressed as mean ± SD (n=10-15). **P<0.01 vs Sham; ^##P<0.01, ^#P<0.05 vs MCAO/R group. NBP: Butylphthalide.

Figure 2

Wel alleviated CI/RI after MCAO/R. (A) TTC staining representative images. (B and C) The statistical analysis of brain infarct volume and brain water content (n=6). (D and E) The quantification of LDH levels in serum and brain tissue at 3d and 7d (n=6). (F) Representative H&E and Nissl staining cerebral slices were imaged by microscopy. The black arrow indicates that the outline of the cell is irregular, the nucleus shrinks, and cracks and vacuoles appear around the cell. The red arrow indicates that the Nissl staining color became lighter and vacuoles appear. (Scale bar:50μm). (G and H) Quantification of H&E and Nissl staining. Data were expressed as mean ± SD. **P<0.01, *P<0.05 vs Sham; ^##P<0.01, ^#P<0.05 vs MCAO/R group.

Figure 3

Wel inhibited lipid peroxidation and ferroptosis after MCAO/R. (A and B) The quantification of Fe²⁺in serum and in brain tissue at 3d and 7d (n=6). (C) Representative TEM images of neurons and mitochondria in MCAO/R group and the Wel-treatment group. The red arrow indicates that the neurons with ruptured outline. The yellow arrow indicates that the mitochondria with decreased cristae (Scale bar: 2μm). (D–I) The quantification of MDA, GSH and LPO levels in serum and tissue brain at 3d and 7d (n=6). (J and K) Quantification of GPX4 fluorescence intensity (Scale bar: 20μm), the white arrows indicate the co-expression of GPX4 and DAPI. (L–M) The quantification of GPX4 in serum and brain tissue at 3d and 7d (n=6). Data were expressed as mean ± SD. **P<0.01 vs Sham; ^##P<0.01, ^#P<0.05 vs MCAO/R group.

Figure 4

Wel inhibited ferroptosis after MCAO/R through regulating the HIF-1α/SLC7A11/GPX4 signaling pathway. (A) Representative images of the infarction area on 3d and 7d after Wel treatment contained with an antibody against HIF-1α (red) and SLC7A11 (green) by immunofluorescence (Scale bar: 20 μm), the white arrows indicate the co-expression of HIF-1α, SLC7A11 and DAPI. (B and C) Quantification of HIF-1α and SLC7A11 fluorescence intensity. (D–G) Representative bands and quantitative analysis of HIF-1α, SLC7A11 and GPX4 (n=5). Data were expressed as mean ± SD. **P<0.01 vs Sham; ^##P<0.01, ^#P<0.05 vs MCAO/R group.

Figure 5

Wel attenuated OGD/R-induced neuronal injury in vitro. (A and B) Effects of Wel on viability of normal and OGD/R PC12 (n=5). (C) Effects of Wel on cell state of OGD/R PC12 (Scale bar: 40μm). (D) Quantification of LDH in PC12 cells (n=5) (E and F) Representative images and quantification of Wel on apoptosis of OGD/R PC12 (n=7, Scale bar: 200μm), the red arrows indicate apoptosis cells with nuclear shrunk. Data were expressed as mean ± SD. **P<0.01 vs Con; ^##P<0.01 vs OGD/R group.

Figure 6

Wel inhibited lipid peroxidation and ferroptosis after OGD/R in vitro. (A and B) Representative images and quantification of the level of ROS by DHE staining in PC12 cells (n=5, Scale bar: 100μm). (C) Quantification of the Fe²⁺ fluorescence intensity by Ferro-Orange staining in PC12 cells. (D) Quantification of Fe²⁺in PC12 cells (n=5). (E) Representative images of the level of the Fe²⁺ fluorescence intensity by Ferro-Orange staining in PC12 cells. (F–H) Quantification of MDA, GSH and LPO in PC12 cells (n=5). Data were expressed as mean ± SD. **P<0.01 vs Con; ^##P<0.01, ^#P<0.05 vs OGD/R group.

Figure 7

Wel protected against OGD/R-induced ferroptosis through the HIF-1α/SLC7A11/GPX4 signaling pathway. (A) Three-dimensional docking structure diagram of HIF-1α and Wel. (B–E) Representative bands and quantitative analysis of HIF-1α, SLC7A11 and GPX4 (n=5). Data were expressed as mean ± SD. **P<0.01 vs Con; ^##P<0.01, ^#P<0.05 vs OGD/R group.

Figure 8

Wel inhibits OGD/R-induced ferroptosis through knockdown of HIF-1α. (A) Efficiency of HIF-1α silence by siRNA was analyzed by qRT-PCR (n=5). (B and C) Efficiency of HIF-1αsilence by siRNA was analyzed by Western blot (n=5). (D–F) Representative bands and quantitative analysis of SLC7A11 and GPX4 (n=5). (G) The cell viability was measured by CCK-8 kits (n=5). (H) The content of Fe²⁺ was measured by assay kit (n=5). (I and J) The Hoechst 33258 staining and quantification of cell apoptosis (n=6, Scale bar: 40μm), the red arrows indicate apoptotic cells with nuclear shrunk. Data were expressed as mean ± SD. **P<0.01 vs Con; ^##P<0.01, vs OGD/R; ^&&P<0.01 vs OGD/R⁺Wel group.

Figure 9

Schematic overview of the mechanism underlying the protective effects of Wel against cerebral ischemia/reperfusion injury (CI/RI)-induce ferroptosis.