Xinyang Tablet inhibits MLK3-mediated pyroptosis to attenuate inflammation and cardiac dysfunction in pressure overload

Xinyang tablet (XYT) has been traditionally used in the treatment of cardiovascular diseases (CVDs). Our previous study indicated that XYT exhibited protective effects in heart failure (HF). The aim of the present study was to determine the protective effects of XYT in pressure overload induced HF and to elucidate its underlying mechanisms of action. We analyzed XYT content using high-performance liquid chromatography (HPLC.). Mice were subjected to transverse aortic constriction (TAC) to generate pressure overload–induced cardiac remodeling and were then orally administered XYT or URMC-099 for 1 week after the operation. HL1 mouse cardiomyoblasts were induced by lipopolysaccharides (LPS) to trigger pyroptosis and were then treated with XYT or URMC-099. We used echocardiography (ECG), hematoxylin and eosin (H&E) staining, Masson's trichrome staining and a terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay to evaluate the effects of XYT. Messenger ribonucleic acid (mRNA) levels of collagen metabolism biomarkers and inflammation-related factors were detected. We determined protein levels of inflammation- and pyroptosis-related signaling pathway members via Western blot (WB). Caspase-1 activity was measured in cell lysate using a Caspase-1 Activity Assay Kit. Subsequently, to define the candidate ingredients in XYT that regulate mixed-lineage kinase-3 (MLK3), we used molecular docking (MD) to predict and evaluate binding affinity with MLK3. Finally, we screened 24 active potential compounds that regulate MLK3 via MD. ECG, H&E staining, Masson's trichrome staining and TUNEL assay results showed that XYT remarkably improved heart function, amelorated myocardial fibrosis and inhibited apoptosis in vivo. Moreover, it reduced expression of proteins or mRNAs related to collagen metabolism, including collagen type 1 (COL1), fibronectin (FN), alpha smooth-muscle actin (α-SMA), and matrix metalloproteinases-2 and -9 (MMP-2, MMP-9). XYT also inhibited inflammation and the induction of pyroptosis at an early stage, as well as attenuated inflammation and pyroptosis levels in vitro. Our data indicated that XYT exerted protective effects against pressure overload induced myocardial fibrosis (MF), which might be associated with the induction of pyroptosis-mediated MLK3 signaling. [Display omitted]