Guhong injection mitigates myocardial ischemia/reperfusion injury by activating GST P to inhibit ASK1-JNK/p38 pathway

Background: Guhong injection (GHI), a novel compound preparation that is composed of a chemical drug, namely aceglutamide, and the aqueous extract of safflower (Carthamus tinctorius L.), exhibits extreme antioxidative, antiapoptotic, anti-inflammatory, and neuroprotective effects. Since oxidative stress, apoptosis, and inflammatory response are all the dominant mechanisms of myocardial ischemia/reperfusion (MI/R) injury, we probe into the protective mechanism of GHI on MI/R injury for the first time. Methods: In this research, we first employed molecular docking to determine whether three active ingredients in GHI, acetylglutamine (NAG), hydroxysafflor yellow A (HSYA), and syringin, possessed the potential activity to modulate the protein, glutathione S-transferase P (GST P). We further identified the protective effect of GHI on myocardial tissue with TTC staining, HE staining, TUNEL staining, and ELISA, and on H9c2 with flow cytometry and ELISA. We next explored whether the cardioprotective effect of GHI on left anterior descending ligation-reperfusion in rats and hypoxia/reoxygenation (H/R) in H9c2 cells was related to activate GST P to inhibit ASK1-JNK/p38 pathway via approaches of qRT-PCR and Western blot. Results: Results of molecular docking indicated that all three compounds spontaneously docked to GST P, among them the binding affinities of both HSYA and syringin to GST P were higher than NAG. In vivo, GHI reduced myocardial infarction size and mitigated myocardial pathological injury. In vitro, GHI enhanced cell viability and extenuated depolarization of mitochondrial membrane potential. In addition, the results of in vivo and in vitro studies demonstrated that the cardioprotection of GHI was associated with improving the mRNA and protein expression levels of GST P to modulate oxidative stress, and inhibiting the levels of mRNA expression and protein phosphorylation of ASK1, JNK, and p38. However, the suppressed effect of GHI on ASK1-JNK/p38 pathway was reversed by ethacrynic acid (EA, a GST inhibitor), indicating that the regulation of GHI on ASK1-JNK/p38 was related to the activity of GST P. Besides, the in vitro results of qRT-PCR and western-blot also certified that the inhibited JNK and p38 further reduced Bax expression and elevated Bcl-2 expression to reduce the expression of caspase-3 to exert anti-apoptosis effects. Conclusion: Taken together, the cardioprotection of GHI mainly incarnated in activating GST P to relieve oxidation properties, thereby inhibiting ASK1-JNK/p38 pathway to suppress apoptosis.