Aspirin in combination with gastrodin protects cardiac function and mitigates gastric mucosal injury in response to myocardial ischemia/reperfusion

Abstract

Myocardial ischemia/reperfusion (I/R) injury after percutaneous coronary intervention (PCI) is common in acute myocardial infarction. Aspirin is commonly prescribed as anti-thrombotic therapy with coronary heart disease (CHD). However, long-term use of aspirin causes severe gastric mucosal damage. Gastrodin is a Chinese natural medicine with anti-inflammatory and anti-oxidative properties. In this study, we investigated the effects of combined therapy with aspirin and gastrodin on the myocardial and gastric mucosal injury in response to myocardial I/R injury and underlying mechanisms using the Sprague-Dawley (SD) rat model. Our results demonstrated that myocardial I/R caused significant cardiac dysfunction and gastric mucosal damage. Administration of aspirin led to significantly reduce myocardial infarction size and myocardial enzyme release, as well as significantly improved cardiac function through exerting anti-inflammatory effects. However, aspirin exacerbated gastric mucosal damage by increasing the levels of inflammatory mediators and endothelin (ET) while reducing prostaglandin E2 (PGE2) levels. The combined treatment with aspirin and gastrodin not only significantly protected gastric mucosa by normalizing the expression levels of the inflammatory factors, ET and PGE2, but also significantly reduced myocardial infarction size and improved cardiac function by inhibiting inflammation in response to I/R. The combination therapy also dramatically down-regulated the levels of pyroptosis-related proteins in the myocardium and gastric mucosa. The combination therapy showed obviously reduced level of thromboxane B2 (TXB2), which was simultaneously accompanied with increased levels of the tissue plasminogen activator (t-PA). This suggested that gastrodin did not inhibit the anti-thrombotic function of aspirin. Accordingly, aspirin in combination with gasrtodin protected the structural and functional integrity of the heart and stomach by suppressing pyroptosis and inflammation. Therefore, combination of aspirin and gastrodin is a promising treatment for cardiac dysfunction and gastric mucosa injury after myocardial I/R.

Keywords: Aspirin; Gastrodin; gastric mucosal injury; myocardial ischemia/reperfusion injury; pyroptosis.

FIGURE 1

Aspirin inhibits myocardial inflammation and alleviates cardiac dysfunction in response to I/R injury. (A) Representative electrocardiogram shows the successful establishment of myocardial I/R injury model (n = 5 per group). The red arrows represent changes in the ST segment. (B) Representative images show TTC staining of the myocardium from rats (n = 5 per group) belonging to the sham, I/R, and I/R + 50 mg/kg aspirin groups. The infarct size quantification of the 3 groups of rats is also shown. (C) ELISA assay results show the serum levels of creatine kinase-MB (CK-MB) and cardiac troponin T (cTnT) in the sham, I/R, and I/R + aspirin group rats (n = 5 per group). (D) Laggendorff in vitro cardiac perfusion results show the left ventricular developed pressure (LVDP), maximum and minimum rates of pressure development (max dP/dt and min dP/dt), and heart rate (HR) in the sham, I/R, and I/R + aspirin group rats (n = 5 per group). (E) Representative H&E staining images (×200 and ×400) show the histological details of the myocardium from the sham, I/R, and I/R + aspirin group rats (n = 5 per group). (F,G) Representative immunoblots and quantification of the expression levels of COX-1 and COX-2 in the sham, I/R, and I/R + aspirin group rats (n = 5 per group). (H) ELISA results show the tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) level in the myocardial tissues of the sham, I/R, and I/R + aspirin group rats (n = 5 per group). The tissues were harvested 2 h post-reperfusion. The data are represented as means ± SD; *p < 0.05 vs. the sham group; ^# p < 0.05 vs. the I/R group.

FIGURE 2

Aspirin aggravates gastric mucosal injury by inducing inflammation. (A) Representative images of the harvested stomach specimens from the sham, I/R, and I/R + 50 mg/kg aspirin group rats (n = 5 per group). (B) Byron gastric mucosal injury scores in the sham, I/R, and I/R + aspirin group rats (n = 5 per group). The scoring criteria are shown in Table 1. (C) Representative images (×100 and ×200) of the H&E-staining gastric mucosal tissue sections from the sham, I/R, and I/R + aspirin group rats (n = 5 per group). (D) ELISA assay results show the levels of prostaglandin E2 (PGE2) and endothelin (ET) in the gastric tissues of the sham, I/R, and I/R + aspirin group rats (n = 5). (E,F) Representative immunoblots and quantification analysis of COX-1 and COX-2 protein levels in the gastric tissues of the sham, I/R, and I/R + aspirin group rats (n = 5). (G) ELISA assay results show the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the gastric tissues of the sham, I/R, and I/R + aspirin group rats (n = 5). The data are represented as means ± SD; * p < 0.05 vs. the sham group; ^# p < 0.05 vs. the I/R group.

FIGURE 3

Gasrtodin alleviates dual gastric mucosal injury by suppressing inflammation. (A) Representative images of the harvest stomach specimens from the sham, I/R, I/R + aspirin, I/R + 50 mg/kg aspirin + 200 mg/kg gastrodin group, and I/R + 200 mg/kg gastrodin group rats (n = 5 per group). (B) Byron gastric mucosa injury scores of the sham, I/R, I/R + aspirin, I/R + aspirin + gastrodin group, and I/R + gastrodin group rats (n = 5 per group). The scoring criteria are shown in Table 1. (C) Representative images (×100 and ×200) of the H&E-staining gastric tissue sections from the sham, I/R, I/R + aspirin, I/R + aspirin + gastrodin group, and I/R + gastrodin group rats (n = 5 per group). (D) Representative immunofluorescence staining images (×200) show the COX-1 and COX-2 expression levels in the gastric tissues of the sham, I/R, I/R + aspirin, I/R + aspirin + gastrodin group, and I/R + gastrodin group rats (n = 5 per group). The green denotes COX-1, red denotes COX-2, and blue denotes the nucleus. (E–G) ELISA assay results show the levels of cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), prostaglandin E2 (PGE2) and endothelin (ET) in the gastric tissues of the sham, I/R, I/R + aspirin, I/R + aspirin + gastrodin group,and I/R + gastrodin group rats (n = 5 per group). The data are represented as means ± SD; * p < 0.05 vs. the sham group; ^# p < 0.05 vs. the I/R group; ^ p < 0.05 vs. the I/R + aspirin group; ^& p < 0.05 vs. the I/R + aspirin + gastrodin group.

FIGURE 4

Combined therapy with aspirin and gastrodin alleviates cardiac dysfunction induced by myocardial I/R. (A,B) Representative TTC staining images of the myocardium from the sham, I/R, I/R + aspirin, I/R + aspirin + gastrodin group, and I/R + gastrodin group rats (n = 5 per group). Quantification of the infarct size based on TTC staining of the myocardium from the sham, I/R, I/R + aspirin, I/R + aspirin + gastrodin group, and I/R + gastrodin group rats (n = 5 per group) is also shown. (C) Laggendorff in vitro cardiac perfusion measurements show the left ventricular developed pressure (LVDP), maximum and minimum rates of pressure development (dP/dt max and dP/dt min) and heart rate (HR) in the sham, I/R, I/R + aspirin, I/R + aspirin + gastrodin group, and I/R + gastrodin group rats (n = 5 per group). (D,E) ELISA assay results show the serum creatine kinase-MB (CK-MB) and cardiac troponin T (cTnT) levels in the sham, I/R, I/R + aspirin, and I/R + aspirin + gastrodin group rats (n = 5 per group). (F) Representative H&E-staining images (×200 and ×400) show the myocardial histology and morphological characteristics in the sham, I/R, I/R + aspirin, and I/R + aspirin + gastrodin group rats (n = 5 per group). (G) ELISA assay results show the tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels in the myocardial tissues of the sham, I/R, I/R + aspirin, and I/R + aspirin + gastrodin group rats (n = 5 per group). The samples for ELISA were harvested 2 h post-reperfusion. (H) Representative immunofluorescence staining images (×400) show the COX-1 and COX-2 expression levels in the heart tissue of the sham, I/R, I/R + aspirin, and I/R + aspirin + gastrodin group rats (n = 5 per group). Green represents COX-1; red represents COX-2; blue represents the nucleus. The data are represented as means ± SD; * p < 0.05 vs. the sham group, ^# p < 0.05 vs. the I/R group, ^ p < 0.05 vs. the I/R + aspirin + gastrodin group.

FIGURE 5

Combined treatment with aspirin and gastrodin protects against cardiac and gastric mucosal injury by suppressing pyroptosis. (A,B) Representative immunoblots and quantification analysis of the NLRP3, ASC, caspase-1, and GSDMD protein levels in the myocardium of the sham, I/R, I/R + aspirin, and I/R + aspirin + gastrodin group rats (n = 5 per group). (C,D) Representative immunoblots and the quantification analysis of the NLRP3, ASC, caspase-1, and GSDMD protein expression levels in the gastric tissues of the sham, I/R, I/R + aspirin, and I/R + aspirin + gastrodin group rats (n = 5 per group). The data are represented as means ± SD; * p < 0.05 vs. the sham group; ^# p < 0.05 vs. the I/R group; ^ p < 0.05 vs. the I/R + aspirin group.

FIGURE 6

Combined treatment with aspirin and gastrodin shows effective antithrombotic effects. (A,B) ELISA assay results show the serum levels of thromboxane B2 (TXB2) and tissue-type plasminogen activator (t-PA) in the sham, I/R, I/R + aspirin, and I/R + aspirin + gastrodin group rats (n = 5 per group). The samples were obtained 2 h post-reperfusion. The data are represented as means ± SD; * p < 0.05 vs. the sham group; ^# p < 0.05 vs. the I/R group; ^ p < 0.05 vs. the I/R + aspirin group.

FIGURE 7

Combined treatment with aspirin and gastrodin protects against cardiac and gastric mucosal injury in response to myocardial ischemia/reperfusion. Myocardial I/R causes both myocardial and gastric mucosal damage. However, combined treatment with aspirin and gastrodin exerts a dual protective effect on the myocardium and gastric mucosa by inhibiting pyroptosis. In the heart, the combined treatment of aspirin and gastrodin reduced the myocardial infarct size and myocardial cell apoptosis, down-regulated the release of CK-MB and cTnT into the blood, and restored cardiac function indicators such as LVDP, ±dp/dt. In the stomach, the combined treatment of aspirin and gastrodin inhibited the inflammatory response, reduced the gastric mucosal edema, necrosis and exfoliation, and restored the near normal expression levels of PGE2 and ET.