The cardioprotective effects of citric Acid and L-malic Acid on myocardial ischemia/reperfusion injury

Abstract

Organic acids in Chinese herbs, the long-neglected components, have been reported to possess antioxidant, anti-inflammatory, and antiplatelet aggregation activities; thus they may have potentially protective effect on ischemic heart disease. Therefore, this study aims to investigate the protective effects of two organic acids, that is, citric acid and L-malic acid, which are the main components of Fructus Choerospondiatis, on myocardial ischemia/reperfusion injury and the underlying mechanisms. In in vivo rat model of myocardial ischemia/reperfusion injury, we found that treatments with citric acid and L-malic acid significantly reduced myocardial infarct size, serum levels of TNF-α, and platelet aggregation. In vitro experiments revealed that both citric acid and L-malic acid significantly reduced LDH release, decreased apoptotic rate, downregulated the expression of cleaved caspase-3, and upregulated the expression of phosphorylated Akt in primary neonatal rat cardiomyocytes subjected to hypoxia/reoxygenation injury. These results suggest that both citric acid and L-malic acid have protective effects on myocardial ischemia/reperfusion injury; the underlying mechanism may be related to their anti-inflammatory, antiplatelet aggregation and direct cardiomyocyte protective effects. These results also demonstrate that organic acids, besides flavonoids, may also be the major active ingredient of Fructus Choerospondiatis responsible for its cardioprotective effects and should be attached great importance in the therapy of ischemic heart disease.

Figure 1

A representative N-BT staining of infarct size. The normal myocardium was stained dark, and the ischemic area was stained red. (a) Citric acid; (b) L-malic acid.

Figure 2

Effects of citric acid (a) and L-malic acid (b) on serum levels of TNF-α following myocardial ischemia/reperfusion. Data are shown as mean ± SD. ^## P < 0.01 versus sham, **P < 0.01, *P < 0.05 versus model control (n = 10).

Figure 3

Effects of citric acid (a) and L-malic acid (b) on platelet aggregation induced by ADP following myocardial ischemi-reperfusion. Data are shown as mean ± SD. ^# P < 0.05 versus sham, **P < 0.01 versus model control (n = 10).

Figure 4

Effects of citric acid (a) and L-malic acid (b) on LDH release. Cardiomyocytes were subjected to 3 h hypoxia followed by 2 h reoxygenation with or without treatment. LDH activities in the hypoxia and the reoxygenation media and in the cell lysates were measured. Data are shown as mean ± SD. ^## P < 0.01 versus normal control, **P < 0.01,*P < 0.05 versus model control (n = 3).

Figure 5

Effects of citric acid (a) and L-malic acid (b) on H/R-induced cardiomyocyte apoptosis. Cardiomyocytes were subjected to 3 h hypoxia and 6 h reoxygenation in the presence or absence of citric acid or L-malic acid. Data are shown as mean ± SD. ^## P < 0.01 versus normal control, **P < 0.01, *P < 0.05 versus model control (n = 3).

Figure 6

Effects of citric acid and L-malic acid on expression levels of cleaved caspase-3 (fold increase relative to normal control levels) after cardiomyocytes subjected to 3 h hypoxia followed by 6 h reoxygenation. Data are shown as mean ± SD. ^## P < 0.01 versus normal control, **P < 0.01, *P < 0.05 versus model control. Results are representative of three independent experiments.

Figure 7

Citric acid and L-malic acid activate PI/3K and phosphorylation of Akt. A representative western blot analysis of total Akt and phosphorylation of Akt at Ser473 after cardiomyocytes were subjected to 3 h hypoxia followed by 6 h reoxygenation. Data are shown as mean ± SD. *P < 0.05 versus model control. Results are representative of three independent experiments.