The alkaloids of Corydalis hendersonii Hemsl. contribute to the cardioprotective effect against ischemic injury in mice by attenuating cardiomyocyte apoptosis via p38 MAPK signaling pathway
Authors: Ge Fuxing , Gao Xiaoli , Zhou Xiaochun , Li Junjun , Ma Xiaojing , Huang Meiwen , Wuken Sana , Tu Pengfei , An Chao , Chai Xingyun 
Published: March 2023

Abstract
Background
There is a characteristic Tibetan folk medicine in China named Corydalis hendersonii Hemsl. (CH) has been used for treatment of cardiovascular related diseases, called “plethora” in Tibetan medicine. Previous studies demonstrated that ethanol extract of CH showed anti-acute myocardial infarction (AMI) effect through inhibiting fibrosis and inflammation. Rich alkaloids fraction (RAF) is isolated from CH, but whether RAF possessing an equivalent effect with the CH ethanol extract and by which mechanism it protects against AMI has not yet reported. The paper aimed to study the potential role of RAF on myocardial injured mice and its underlying mechanism.

Materials and methods
Liquid chromatography mass spectrometry-ion trap-time of flight (LCMS-IT-TOF) was used to analyze the chemical profile and isolate pure compounds. The ligation of left anterior descending (LAD) of coronary artery in mice was used to evaluate the in vivo anti-AMI effect, by dividing into eight groups: Sham, Model, Fosinopril (10 mg/kg, i.g.), total extract (TE, 400 mg/kg, i.g.), poor alkaloids fraction (PAF, 300 mg/kg, i.g.), and RAF (25, 50, and 100 mg/kg, respectively, i.g.) groups. Echocardiography was used to evaluate mice heart function through the index of left ventricular end-systolic diameter (LVEDs), left ventricular end-diastolic diameter (LVEDd), fractional shortening (FS) and ejection fraction (EF). We detected the lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) in the serum and the plasma level of angiotensin II (AngII). The apoptosis of mice myocardial tissue was verified by TUNEL assay. The expression of p38 mitogen-activated protein kinases (p38 MAPK), Bcl-2 and Bcl-2-associated X protein (Bax) were detected through immunofluorescence staining, qRT-PCR and western blot in mice heart tissue and H9c2 cells.

Results
Echocardiography data indicated that the values of LVEDd and LVEDs were reduced and the values of FS and EF were improved by TE and RAF significantly. RAF also decreased the levels of LDH, CK-MB and AngII and significantly inhibited inflammatory cells in the marginal zone of myocardial infarction. The TUNEL assay results showed that RAF significantly attenuated cell apoptosis. Immunofluorescence and qRT-PCR assay showed that RAF inhibited p38 MAPK, Bax, and Bcl-2 proteins in mice myocardium. Western blot results validated that the expressions of key proteins were inhibited by RAF. Also, the apoptotic cells and apoptosis-related proteins were dramatically reduced by RAF in vivo and in vitro. Besides, RAF and PAF were analyzed by LCMS-IT-TOF to identify the main compounds and to demonstrate the difference between them. The results showed that a total of 14 alkaloids were identified, which indicated that the isoquinoline alkaloids were the main ingredients in RAF may contributing to the cardioprotective effect in mice.

Conclusions
RAF improves cardiac function by inhibiting apoptosis via p38 MAPK signaling pathway, and RAF contributes to the effect against myocardial ischemic injury of TE in mice, which provides a substantial reference for the clinical application against ischemia heart disease and quality control of CH.

Graphical Abstract