Pre-treatment with a combination of Shenmai and Danshen injection protects cardiomyocytes against hypoxia/reoxygenation- and H2O2-induced injury by inhibiting mitochondrial permeability transition pore opening

Lin Li; Zhengmei Sha; Yanyan Wang; Dongli Yang; Jinghao Li; Zhenzhen Duan; Hongbo Wang; Yuhong Li

doi:10.3892/etm.2019.7462

Pre-treatment with a combination of Shenmai and Danshen injection protects cardiomyocytes against hypoxia/reoxygenation- and H₂O₂-induced injury by inhibiting mitochondrial permeability transition pore opening

Exp Ther Med. 2019 Jun;17(6):4643-4652. doi: 10.3892/etm.2019.7462. Epub 2019 Apr 2.

Authors

Lin Li^{1

2

3

4}, Zhengmei Sha⁴, Yanyan Wang^{1

2

3

4}, Dongli Yang⁴, Jinghao Li⁴, Zhenzhen Duan⁴, Hongbo Wang⁴, Yuhong Li^{1

2

3

4}

Affiliations

¹ Key Laboratory of Pharmacology of Traditional Chinese Medical Formulae, Ministry of Education, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, P.R. China.
² Key Research Laboratory Prescription Compatibility among Components, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, P.R. China.
³ Tianjin Key Laboratory of Chinese Medicine Pharmacology;, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, P.R. China.
⁴ Institute of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, P.R. China.

Abstract

Increasing evidence has indicated that opening of the mitochondrial permeability transition pore (mPTP) has a vital role in myocardial ischemia/reperfusion (I/R) injury. Shenmai injection (SMI) plus Danshen injection (DSI) combination, termed Yiqi Yangyin Huoxue (YYH) therapy is used in the clinic to treat cardiovascular diseases, including myocardial I/R injury. Previous studies by our group have demonstrated the protective effect of pretreatment with YYH against myocardial I/R injury in isolated rat hearts. The present study aimed to examine the protective effect of YYH against hypoxia/reoxygenation (H/R)- and H₂O₂-induced cardiomyocyte injury, and to determine whether this effect is produced by inhibition of mPTP opening. Primary cardiomyocytes isolated from neonatal rats were cultured and randomly grouped into a control group, injury group and pretreatment group, with six duplicated wells in each group during each assay. Cardiomyocytes in the injury group were subjected to H/R to simulate I/R or exposed to H₂O₂ for 2 h to induce oxidative injury. Cellular injury was assessed via release of creatine kinase (CK) and lactate dehydrogenase (LDH), and cell viability was measured by an MTT assay. The mitochondrial membrane potential (ΔΨm) and cytosolic reactive oxygen species (ROS) were detected using the fluorescent probes rhodamine123 (Rh123) and chloromethyl-2,7-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA), respectively. Intracellular Ca²⁺, mitochondrial Ca²⁺ and mPTP opening were measured using fluo-4 acetoxymethyl (Fluo-4/AM), rhodamine-2 acetoxymethyl (Rhod-2/AM) and calcein acetoxymethyl (Calcein/AM) probes, respectively. The results indicated that pretreatment with YYH enhanced cell viability, increased ΔΨm, reduced CK and LDH release, and decreased intracellular ROS and Ca²⁺, thus reducing cardiomyocyte injury induced by H/R or H₂O₂. LY294002, a specific phosphoinositide 3-kinase (PI3K) inhibitor, and PD98059, a specific inhibitor of the extracellular signal-regulated kinase 1/2 (Erk1/2) pathway, eliminated the protective effects of the combination therapy on cell viability and the change in the ΔΨm in cardiomyocytes. In conclusion, pre-treatment with YYH has cardioprotective effects against H/R injury and oxidative stress via activation of the PI3K/Akt and Erk1/2 signaling pathways, which reduces mPTP opening, overproduction of ROS and calcium overload.

Keywords: Danshen injection; H2O2 injury; Shenmai injection; hypoxia/reoxygenation injury; mitochondrial permeability transition pore opening; neonatal rat cardiomyocytes.