Protective mechanism of shenmai on myocardial ischemia-reperfusion through the energy metabolism pathway

Abstract

Shenmai (SM) injection has been reported to attenuate ischemia-reperfusion (I/R) injury, but its effect on energy metabolism during I/R and the underlying mechanism remain unknown. To explore the protective mechanism of SM on ischemic cardiomyopathy, primary cardiomyocytes from SD rats were treated with SM, total saponins of Panax ginseng (TSPG), L-carnitine (LC) and trimetazidine (TMZ). Changes in glucose, free fatty acids (FFAs), pyruvic acid (PA), lactic acid (LA) and intracellular ATP capacity were observed with the appropriate assays. For each treatment group, the key enzymes and transporters of myocardial energy metabolism were detected and compared via Western blot. Furthermore, impairments after I/R were assessed by examining cardiomyocyte apoptosis and LDH and PK activity in the culture medium. Our results indicated that SM and TSPG markedly alleviated the decrease in key enzymes and transporters and the utilization of metabolic substrates following I/R, while SM prevented aberrant apoptosis and restored the depleted ATP resulting from I/R. Notably, the effects of SM were superior to those of its main components TSPG, LC and TMZ. Thus, the protective effect of SM in ischemic cardiomyopathy may be mediated by the upregulation of key enzymes and restoration of the depleted ATP content in the energy metabolism process.

Keywords: Shenmai; cardiomyocytes; energy metabolism; ischemia-reperfusion; total saponins of Panax ginseng.

Figure 1

Observation of cardiomyocytes under confocal microscopy. Anti-labeled actin conjugated by second antibody is shown in yellowish green (B); nuclei are labeled by DAPI in blue (A). Combined fluorescence of the cytoplasm and nuclei of cardiac muscle cells (C). Cardiomyocyte purity was calculated by observing 5 random fields of view and counting 200 cells in each field (97.1±1.9)%.

Figure 2

HPLC fingerprint of Shenmai injection and ginsenosides Re, Rg1 and Rb1 as reference standards. A. Chromatogram of reference standards: 1 represents ginsenoside Re; 2 represents ginsenoside Rg1; 3 represents ginsenoside Rb1. B. Representative chromatogram of Shenmai injection samples: No. 1-3 represent the common peaks with reference standards Re, Rg1 and Rb1, respectively.

Figure 3

Injury of cardiomyocytes in different treatment groups was detected by LDH measurements and PK release levels. A. Release level of LDH in medium of different treatment groups. B. Release level of PK in medium of different treatment groups. The data are expressed as the mean ± SD. The experiments were repeated 10 times (n=10). A. **P<0.01 compared with the normal group; #P<0.01, ##P<0.01, &P<0.01, &&P<0.01 compared with the I/R group; #P<0.01 compared with the I/R+TSPG group. B. **P<0.01 compared with the normal group; #P<0.01, ##P<0.01, &P<0.01, &&P<0.05 compared with the I/R group; #P<0.01 compared with the I/R+TSPG group. N, normoxia.

Figure 4

Production and utilization of energy in cardiomyocytes presented by intracellular ATP levels and ADP/ATP ratios in normoxic and I/R conditions following SM, LC, TSPG and TMZ treatment. A. The intracellular ATP levels of the different treatment groups. B. The ADP/ATP ratios of the different treatment groups. The data are expressed as the mean ± SD. The experiments were repeated 10 times (n=10). A. **P<0.01 compared with the normal group; #P<0.01, &P<0.05, &&P<0.01 compared with the I/R group; #P<0.01 compared with the I/R+TSPG group. B. **P<0.01 compared with the normal group; #P<0.01, ##P<0.01, &P<0.01, &&P<0.01 compared with the I/R group; #P<0.01 compared with the I/R+TSPG group. N, normoxia.

Figure 5

Effects of SM, LC, TSPG and TMZ on CD36, CPT1B, ACADL, ACADM, UCP3, OGDH, PFKM, GLUT4, citrate synthetase and isocitrate dehydrogenase mRNA expression following I/R in cardiomyocytes. (A) Evaluation of FFA metabolism-related gene expression after I/R injury by RT-PCR analysis. (B) Evaluation of glucose metabolism-related gene expression after I/R injury by RT-PCR analysis. Beta-actin was used as the loading control. Data are expressed as the mean ± standard deviation (n=6). (A) mRNA levels of CD36, CPT1B, ACADL, ACADM and UCP3, **P<0.01 vs. N; &&P<0.01 vs. I/R. (A. CD36, CPT1B, ACADL, UCP3) ##P<0.05 vs. I/R; #P<0.05 vs. I/R. (A. CD36, ACADL) &P<0.05 vs. I/R. (A. ACADM) #P>0.05, ##P>0.05 and &P>0.05 vs. I/R. (B) mRNA levels of OGDH, PFKM, GLUT4, citrate synthetase and isocitrate dehydrogenase, **P<0.01 vs. N; &&P<0.01 vs. I/R. (B. OGDH, GLUT4) #P<0.05, ##P<0.05 and &P<0.05 vs. I/R. (B. OGDH, PFKM, citrate synthetase and isocitrate dehydrogenase) &&P<0.05 vs. I/R+TSPG; (B. PFKM, citrate synthetase and isocitrate dehydrogenase) ##P>0.05 vs. I/R. N, normoxia.

Figure 6

Expression of key enzymes and transporters in the fatty acid metabolism pathway of cardiomyocytes in the different treatment groups. A. The protein expression levels of CD36, CPT1B, ACADL, ACADM and UCP3 were evaluated by Western blot analysis; beta-actin served as the control. Quantification of protein bands with image analysis. B-F. The protein levels of CD36, CPT1B, ACADL, ACADM and UCP3 are shown as ratios relative to the loading control (beta-actin) and are presented as the mean ± SD. (n=6). The corresponding one-way ANOVA and LSD test results for each protein are shown. B-D, F. **P<0.01 compared the normal group; &&P<0.01 and ##P<0.05 compared with the I/R group. E. Protein levels of ACADM, **P<0.01 compared with the normal group, &&P<0.01 and ##P>0.05 compared with the I/R group. N, normoxia.

Figure 7

Effects of SM, LC, TSPG and TMZ on the OGDH, PFKM, GLUT4, citrate synthetase and isocitrate dehydrogenase levels following I/R in cardiomyocytes; beta-actin was used as the loading control. (A) Western blot analysis of the OGDH, PFKM, GLUT4, citrate synthetase, isocitrate dehydrogenase and beta-actin levels. (B-F) Quantification of the (B) OGDH, (C) PFKM, (D) GLUT4, (E) citrate synthetase and (F) isocitrate dehydrogenase levels. Data are expressed as the mean ± standard deviation (n=6). (B-F) **P<0.01 vs. N; &&P<0.01 vs. I/R; (B, D) #P<0.05, ##P<0.05 and &P<0.05 vs. I/R; (B, C, E, F) &&P<0.05 vs. I/R+TSPG; (C, E, F) **P>0.05 vs. I/R+TSPG. N, normoxia.

Figure 8

Effects of SM, LC, TSPG and TMZ on I/R-induced cardiomyocyte apoptosis. A-F. Cardiomyocytes after I/R treatment with SM, LC, TSPG and TMZ were stained with Annexin V-FITC/PI and analyzed for apoptosis using a flow cytometer. Cardiomyocytes in regions Q4 and Q2 represent early- and late-apoptotic cardiomyocytes, respectively. G. The percentages of apoptotic cells were calculated and compared. Data are expressed as the mean ± standard deviation (n=3). **P<0.01 vs. N; #P<0.01 vs. I/R; &P<0.01 vs. I/R+SM; &P<0.05 vs. I/R. N, normoxia.

Figure 9

Effects of SM, LC, TSPG and TMZ on energy substrates in cardiomyocytes following I/R. A-D. Detection of the FFA, glucose, LA and PA levels in culture medium under normoxic or I/R conditions following SM, LC, TSPG and TMZ treatment. Data are expressed as the mean ± standard deviation (n=10). A. **P<0.01 vs. N; #P<0.01, ##P<0.01, &P<0.01, &&P<0.05 vs. I/R; **P<0.05 vs. I/R+TSPG. B. **P<0.01 vs. N; #P<0.01 vs. I/R; C. **P<0.01 vs. N; #P<0.01, ##P<0.01, &P<0.01, &&P<0.01 vs. I/R; **P<0.01 vs. I/R+TSPG. D. *P>0.05, #P>0.05, ##P>0.05, &P>0.05, &&P>0.05 vs. I/R; N, normoxia.