OBJECTIVE To investigate the protective effects of Qihuang Zhuyu Formula (QHZYF) against myocardial ischemia/reperfusion (I/R) injury in rats and the underlying mechanisms.

METHODS Thirty-six male Sprague–Dawley rats were randomly divided into groups: a control group, a sham group, an I/R group, a low-dose QHZYF group (I/R + QHZYF-L), a middle-dose QHZYF group (I/R + QHZYF-M), and a high-dose QHZYF group (I/R + QHZYF-H), with six rats in each group. After 2 weeks of continuous intragastric administration of QHZYF, the left anterior descending (LAD) coronary artery was ligated for 45 min followed by 4 h of reperfusion to establish the I/R model. Electrocardiographic changes before and after surgery were recorded. Left ventricular function was evaluated by echocardiography. 2,3,5-Triphenyltetrazolium chloride (TTC) staining was performed to evaluate changes in infarct size. Masson’s trichrome staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining were performed to evaluate myocardial fibrosis and cardiomyocyte apoptosis, respectively. Serum lactate dehydrogenase (LDH) activity was determined using biochemical assays. Enzyme-linked immunosorbent assay (ELISA) was used to measure the serum levels of creatine kinase-MB (CK-MB), cardiac troponin I (cTnI), interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). In addition, immunohistochemistry and Western blot were employed to detect the expression levels of WW domain-containing E3 ubiquitin protein ligase 2 (WWP2), Krüppel-like factor 5 (KLF5), and TNF-related apoptosis-inducing ligand (TRAIL) in myocardial tissue. The interaction between WWP2 and KLF5 was further analyzed by co-immunoprecipitation (Co-IP).

RESULTS Compared with the control and sham groups, rats in the model group showed a significantly increased myocardial infarct size, markedly elevated levels of myocardial injury markers, including LDH, CK-MB, and cTnI, as well as inflammatory factors, including IL-1β, IL-6, and TNF-α (P<0.001); early myocardial fibrotic changes were aggravated (P<0.001), cardiomyocyte apoptosis was increased (P<0.001), left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were significantly decreased (P<0.001), and left ventricular end-systolic dimension (LVDs) was significantly increased (P<0.001). The protein expression levels of WWP2, KLF5, and TRAIL in myocardial tissue were significantly up-regulated (P<0.01,P<0.001,P<0.000 1). Compared with the model group, treatment with QHZYF at different doses significantly reduced the myocardial infarct area and markedly decreased the levels of myocardial injury markers, including LDH, CK-MB, and cTnI (P<0.001). Inflammatory factors, including IL-1β, IL-6, and TNF-α, were also significantly reduced, with the medium- and high-dose groups showing particularly pronounced effects (P<0.001). In addition, QHZYF improved myocardial tissue structure, attenuated fibrosis (P<0.001), and reduced cardiomyocyte apoptosis (P<0.001). Echocardiographic results showed that LVEF, LVFS, and LVDs were improved to varying degrees in all QHZYF-treated groups. Among them, LVEF was significantly increased in the middle- and high-dose groups (P<0.05, P<0.01), while LVFS was significantly increased and LVDs was significantly decreased in the high-dose group (P<0.05). Furthermore, the protein expression levels of WWP2, KLF5, and TRAIL in myocardial tissue were markedly downregulated (P<0.05，P<0.01，P<0.001, P<0.000 1). Co-IP results showed that WWP2 interacted with KLF5 in myocardial tissue; this interaction was enhanced following I/R injury but was attenuated after QHZYF treatment.

CONCLUSION QHZYF may exert cardioprotective effects by regulating the WWP2/KLF5/TRAIL-related signaling axis; however, the direct upstream-downstream causal relationship among these three molecules remains to be further clarified by functional studies.