YiXin-Shu, a ShengMai-San-based traditional Chinese medicine formula, attenuates myocardial ischemia/reperfusion injury by suppressing mitochondrial mediated apoptosis and upregulating liver-X-receptor α

Abstract

Positive evidence from clinical trials has fueled growing acceptance of traditional Chinese medicine (TCM) for the treatment of cardiac diseases; however, little is known about the underlying mechanisms. Here, we investigated the nature and underlying mechanisms of the effects of YiXin-Shu (YXS), an antioxidant-enriched TCM formula, on myocardial ischemia/reperfusion (MI/R) injury. YXS pretreatment significantly reduced infarct size and improved viable myocardium metabolism and cardiac function in hypercholesterolemic mice. Mechanistically, YXS attenuated myocardial apoptosis by inhibiting the mitochondrial mediated apoptosis pathway (as reflected by inhibition of mitochondrial swelling, cytochrome c release and caspase-9 activity, and normalization of Bcl-2 and Bax levels) without altering the death receptor and endoplasmic reticulum-stress death pathways. Moreover, YXS reduced oxidative/nitrative stress (as reflected by decreased superoxide and nitrotyrosine content and normalized pro- and anti-oxidant enzyme levels). Interestingly, YXS upregulated endogenous nuclear receptors including LXRα, PPARα, PPARβ and ERα, and in-vivo knockdown of cardiac-specific LXRα significantly blunted the cardio-protective effects of YXS. Collectively, these data show that YXS is effective in mitigating MI/R injury by suppressing mitochondrial mediated apoptosis and oxidative stress and by upregulating LXRα, thereby providing a rationale for future clinical trials and clinical applications.

Figure 1. Baseline lipid profile and cardiac function among treatment groups.

(A–C) Plasma levels of TC (A) TG (B) and LDL-C (C) were determined in NC, HF-sham, HF-vehicle, HF-YXS-1, HF-YXS-2 groups by an auto-biochemical analysis system (n = 5 animals per group). (D) Body weight was recorded in indicated groups (n = 10 animals per group). (E) Baseline LVFS was measured by echocardiography before induction of MI/R injury in all groups (n = 6 animals per group). *p < 0.05 or **p < 0.01 versus NC. Abbreviations: TC, total cholesterol; TG, triglyceride; LDL-C, low-density lipoprotein-cholesterol; NC, normal diet fed group; HF-sham, high-cholesterol diet fed group; HF-vehicle, high-cholesterol diet fed group followed by saline treatment; HF-YXS-1, high-cholesterol diet fed group followed by YXS (60 mg·kg⁻¹·d⁻¹) treatment; HF-YXS-2, high-cholesterol diet fed group followed by YXS (120 mg·kg⁻¹·d⁻¹) treatment.

Figure 2. YXS pretreatment reduces myocardial infarct size.

(A) Representative photographs of Evans blue/TTC double stained murine heart slices obtained 24 h after MI/R injury. (B) Distribution of ischemic area, infarcted area and area not at risk in each slice. (C,D) Graphic representation of the LV infarct size (C) and AAR (D) (n = 6–8 animals per group). *p < 0.01 versus sham; ^#p < 0.05 or ^##p < 0.01 versus vehicle. Abbreviation: AAR, area at risk.

Figure 3. YXS pretreatment improves viable myocardium metabolism and cardiac function.

(A) Representative images of Micro-PET, Micro-CT, Micro-PET/CT overlap, and echocardiography. Viable myocardium metabolism was assessed via ¹⁸F-FDG uptake by Micro-PET/CT, and cardiac performance was determined by echocardiography 24 h after reperfusion. (B) Myocardial SUV of ¹⁸F-FDG determined by ¹⁸F-FDG uptake utilizing Micro-PET/CT (n = 5 animals per group). (C) LVFS values were measured by echocardiography (n = 5–6 animals per group). *p < 0.01 versus sham; ^#p < 0.05 or ^##p < 0.01 versus vehicle. Abbreviations: SUV, standardized uptake value; LVFS, left ventricular fractional shortening.

Figure 4. YXS pretreatment suppresses the mitochondrial-mediated apoptosis pathway.

(A) Representative photomicrographs of TUNEL staining (a–l) and transmission electron microscopy images (m–p). MI/R led to significant apoptosis (arrows) and mitochondrial swelling (arrows). Total nuclei were labeled with DAPI (blue), α-actin was labeled in red, and apoptotic nuclei were detected by TUNEL staining (green). (B) Quantitative analysis of apoptotic cells in indicated groups (n = 6 animals per group, n = 4 in sham). (C,D) Activities of caspase-3 (C) and caspase-8/-9/-12 (D) were measured through the specific cleavage of respective substrates in each group (n = 5 animals per group, n = 4 in sham). (E) Protein expression levels of cytosolic Cyto c, Bax, Bcl-2, FAS and CHOP in ischemic/reperfused myocardial tissue were determined by Western blot analysis. The absence of VDAC, a mitochondrial marker, in the cytosolic fraction verified that expression of cytosolic Cyto-C represented specific mitochondrial Cyto-C release. (F–H) Background-subtracted density of the bands for Cyto c, Bax, Bcl-2, FAS and CHOP was normalized against GAPDH and expressed as fold change relative to sham-operated controls (n = 5 animals per group). *p < 0.05 or **p < 0.01versus sham; ^#p < 0.05 or ^##p < 0.01 versus vehicle. Cropped blots were used here and the full-length gels were included in the supplementary information.

Figure 5. YXS pretreatment reduces MI/R-induced oxidative stress.

(A) ROS steady-state levels in ischemic myocardium were measured by in-situ dihydroethidium staining, and images were obtained with confocal microscopy. (B,C) Nitrotyrosine production in ischemic/reperfused myocardium was measured by immunohistochemistry (B) and ELISA (C, n = 5 animals per group). (D) NADPH oxidase activity was measured by lucigenin-enhanced chemiluminescence, and expressed as fold change relative to sham-operated controls (n = 5 animals per group). (E,F) Protein levels of iNOS and gp91^phox were detected by Western blot (E) and subjected to semi-quantitative analysis (F) (n = 5 animals per group). *p < 0.05 or **p < 0.01 versus sham; ^#p < 0.05 or ^##p < 0.01 versus vehicle. Cropped blots were used here and the full-length gels were included in the supplementary information.

Figure 6. YXS pretreatment normalizes expression of antioxidant enzymes.

(A,B) The mRNA levels of GPx1 (A) and SOD1 (B) were determined by real-time quantitative PCR (n = 4 animals per group). (C,D) Representative Western blot bands of GPx1, SOD1 (C) and semi-quantitative analysis (D) (n = 4 animals per group). *p < 0.05 or **p < 0.01 versus sham; ^#p < 0.05 or ^##p < 0.01 versus vehicle. Cropped blots were used here and the full-length gels were included in the supplementary information.

Figure 7. Silencing of cardiac-specific LXRα blunts the beneficial effect of YXS on infarct size.

(A) YXS pretreatment selectively upregulated expression of LXRα, PPARα, PPARβ and ERα in ischemic/reperfused myocardium compared with vehicle treatment (n = 4 animals per group). *p < 0.05 versus sham; ^#P < 0.05 or ^##p < 0.01 versus vehicle. (B) YXS upregulated expression of target genes of LXRα, PPARα, PPARβ and ERα compared with vehicle treatment (n = 4 animals per group). *p < 0.05 or **p < 0.01 versus sham; ^#p < 0.05 or ^##p < 0.01 versus vehicle. C: Representative Western blot bands showing LXRα, PPARα, PPARβ and ERα protein expression at 24 h, 48 h, and 72 h after intramyocardial siRNA delivery (n = 4 animals per group). (D) Real-time q-PCR results showing relative mRNA levels of LXRα, PPARα, PPARβ and ERα after in-vivo siRNA-mediated silencing for the indicated time points; results were expressed as fold change over control siRNA delivery (n = 3 animals per group). *p < 0.01 versus control siRNA. (E) Quantification of AAR (left panel) and infarct size (right panel) in indicated groups (n = 7 animals per group). *p < 0.05 or **p < 0.01 versus vehicle; ^#p < 0.05 versus control siRNA + YXS. Cropped blots were used here and the full-length gels were included in the supplementary information.

Figure 8. Silencing of cardiac-specific LXRα blunts the beneficial effect of YXS on cardiac dysfunction, apoptosis and oxidative stress.

(A) Representative images of echocardiography among different groups (left panel). LVFS values (right panel) were subjected to statistical analysis (n = 6 animals per group). (B) Representative images of TUNEL staining showing that LXRα depletion blunted the effect of YXS on myocardial apoptosis. (C) Quantitative analysis of apoptotic cardiomyocytes among the indicated groups (n = 5 animals per group). (D) Caspase-3 activities in indicated groups (n = 5 animals per group). (E) ROS steady-state levels in ischemic myocardium were measured by in-situ dihydroethidium staining, and images were obtained with confocal microscopy. (F) Nitrotyrosine production in ischemic/reperfused myocardium was measured by ELISA (n = 5 animals per group). (G) NADPH oxidase activity was measured by lucigenin-enhanced chemiluminescence, and expressed as fold change relative to sham-operated controls (n = 5 animals per group). *p < 0.05 or **p < 0.01 versus vehicle; ^#p < 0.05 or ^##p < 0.01 versus control siRNA + YXS.