Lingbao Huxin Pill Alleviates Apoptosis and Inflammation at Infarct Border Zone through SIRT1-Mediated FOXO1 and NF- κ B Pathways in Rat Model of Acute Myocardial Infarction

OBJECTIVE To investigate whether Lingbao Huxin Pill (LBHX) protects against acute myocardial infarction (AMI) at the infarct border zone (IBZ) of myocardial tissue by regulating apoptosis and inflammation through the sirtuin 1 (SIRT1)-mediated forkhead box protein O1 (FOXO1) and nuclear factor-κ B (NF-κ B) signaling pathways. METHODS Six-week-old Wistar rats with normal diet were randomized into the sham, the model, Betaloc (0.9 mg/kg daily), LBHX-L (0.45 mg/kg daily), LBHX-M (0.9 mg/kg daily), LBHX-H (1.8 mg/kg daily), and LBHX+EX527 (0.9 mg/kg daily) groups according to the method of random number table, 13 in each group. In this study, left anterior descending coronary artery (LADCA) ligation was performed to induce an AMI model in rats. The myocardial infarction area was examined using a 2,3,5-triphenyltetrazolium chloride solution staining assay. A TdT-mediated dUTP nick-end labeling (TUNEL) assay was conducted to assess cardiomyocyte apoptosis in the IBZ. The histopathology of myocardial tissue at the IBZ was assessed with Heidenhain, Masson and hematoxylineosin (HE) staining assays. The expression levels of tumor necrosis factor α (TNF-α), interleukin (IL)-6, IL-1 β, and intercellular adhesion molecule-1 were measured using enzyme-linked immunosorbent assays (ELISAs). The mRNA expressions of SIRT1 and FOXO1 were detected by real-time qPCR (RT-qPCR). The protein expressions of SIRT1, FOXO1, SOD2, BAX and NF- κ B p65 were detected by Western blot analysis. RESULTS The ligation of the LADCA successfully induced an AMI model. The LBHX pretreatment reduced the infarct size in the AMI rats (P<0.01). The TUNEL assay revealed that LBHX inhibited cardiomyocyte apoptosis at the IBZ. Further, the histological examination showed that the LBHX pretreatment decreased the ischemic area of myocardial tissue (P<0.05), myocardial interstitial collagen deposition (P<0.05) and inflammation at the IBZ. The ELISA results indicated that LBHX decreased the serum levels of inflammatory cytokines in the AMI rats (P<0.05 or P<0.01). Furthermore, Western blot analysis revealed that the LBHX pretreatment upregulated the protein levels of SIRT1, FOXO1 and SOD2 (P<0.05) and downregulated NF- κ B p65 and BAX expressions (P<0.05). The RT-qPCR results showed that LBHX increased the SIRT1 mRNA and FOXO1 mRNA levels (P<0.05). These protective effects, including inhibiting apoptosis and alleviating inflammation in the IBZ, were partially abolished by EX527, an inhibitor of SIRT1. CONCLUSION LBHX could protect against AMI by suppressing apoptosis and inflammation in AMI rats and the SIRT1-mediated FOXO1 and NF- κ B signaling pathways were involved in the cardioprotection effect of LBHX.