Alprostadil Injection Attenuates Coronary Microembolization-Induced Myocardial Injury Through GSK-3β/Nrf2/HO-1 Signaling-Mediated Apoptosis Inhibition

Figure 1. Alprostadil injection ameliorated myocardial morphological alterations of CME rats (magnification ×200, scale bar=50 μm) (n=6, per group). The black arrow indicates microspheres, red arrow represents inflammatory cell infiltration, and white arrow shows microinfarction focus.

Figure 2. Cardiac function-based indices of rats were measured by echocardiography (n=6 per group).

Figure 3. Myocardial mitochondrial morphology observed by transmission electron microscopy (magnification ×20,000, scale bar=500 nm). The black arrow represents the typical vacuolated degeneration and enlarged mitochondria, and white arrow indicates the normal mitochondrial morphology (n=3 per group).

Figure 4. Alprostadil injection suppressed CME-induced myocardial apoptosis. (A) Representative images of TUNEL staining, in which apoptotic nuclei were yellow-brown stained, while normal nuclei were blue stained (magnification ×400, scale bar=20 μm). The black arrow indicates apoptotic cells. (B) Relative TUNEL-positive nuclei in each group (n=6 per group). *P<0.05, compared with Sham group, #P<0.05, compared with CME group, aP<0.05, compared with High group.

Figure 5. Alprostadil injection reduced expressions of pro-apoptotic proteins and improved anti-apoptotic protein expression levels. (A) The bands of Bax, Bcl-2, cytosol-Cyt-c, cleaved-caspase-9, and cleaved-caspase-3. (B–F) Quantification of expression levels of Bax, Bcl-2, cytosol-Cyt-c, cleaved-caspase-3, and cleaved-caspase-9. GAPDH served as an internal control (n=4 or 6, per group). *P<0.05, compared with Sham group; #P<0.05, compared with CME group; aP<0.05, compared with High group.

Figure 6. Alprostadil injection reduced myocardium micro-infarct size following CME (magnification ×200, scale bar=50 μm). (A) Representative images of HBFP staining; (B) The percentage of micro-infarct area in each group. The black arrow indicates micro-infarct focus. (n=6 per group). *P<0.05, compared with Sham group, #P<0.05, compared with CME group, aP<0.05, compared with High group.

Figure 7. Alprostadil injection increased mRNA levels of Nrf2 and HO-1. (A) Nrf2; (B) HO-1 (n=6, per group). The results are presented as the mean±standard deviation (SD) of three independent experiments. *P<0.05, compared with the Sham group; #P<0.05, compared with CME group; aP<0.05, compared with High group.

Figure 8. Effects of alprostadil injection on GSK-3β/Nrf2/HO-1 signaling pathway in CME-induced myocardial injury. (A) The expressions of GSK-3β, p-GSK-3β (Ser9), Nrf2, and HO-1 determined by Western blot. (B) Alprostadil injection increased the ratio of p-GSK-3β (Ser-9)/GSK-3β. (C) Quantification of Nrf2 expression levels. (D) Quantification of HO-1 expression levels (n=4 or 6, per group). *P<0.05, compared with the Sham group; #P<0.05, compared with the CME group; aP<0.05, compared with the High group.

Figure 9. Double immunofluorescence staining of Nrf2 and HO-1 in the heart sections (magnification ×400, scale bar=25 μm) (n=6, per group). The red color indicates Nrf2, green color represents HO-1, and blue color shows nuclear.