Tribulosin protects rat hearts from ischemia/reperfusion injury

Abstract

Aim: To investigate the protective effect of tribulosin, a monomer of the gross saponins from Tribulus terrestris, against cardiac ischemia/reperfusion injury and the underlying mechanism in rats.

Methods: Isolated rat hearts were subjected to 30 min of ischemia followed by 120 min of reperfusion using Langendorff's technique. The hearts were assigned to seven groups: control, ischemia/reperfusion (I/R), treatment with gross saponins from Tribulus terrestris (GSTT) 100 mg/L, treatment with tribulosin (100, 10, and 1 nmol/L) and treatment with a PKC inhibitor (chelerythrine) (1 micromol/L). Infarct size was assessed by triphenyltetrazolium chloride staining. Malondialdehyde (MDA), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) contents as well as superoxide dismutase (SOD) and creatine kinase (CK) activities were determined after the treatment. Histopathological changes in the myocardium were observed using hematoxylin-eosin (H&E) staining. Apoptosis was detected with terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) assay. Bcl-2, Bax, caspase-3, and PKCepsilon protein expression were examined using Western blotting.

Results: Tribulosin treatment significantly reduced MDA, AST, CK and LDH contents, and increased the activity of SOD. The infarct size of I/R group was 40.21% of the total area. GSTT and various concentrations of tribulosin treatment decreased the infarct size to 24.33%, 20.24%, 23.19%, and 30.32% (P<0.01). Tribulosin treatment reduced the myocardial apoptosis rate in a concentration-dependent manner. Bcl-2 and PKCepsilon protein expression was increased after tribulosin preconditioning, whereas Bax and caspase-3 expression was decreased. In the chelerythrine group, Bcl-2 and PKCepsilon expression was decreased, whereas Bax and caspase-3 expression was increased.

Conclusion: Tribulosin protects myocardium against ischemia/reperfusion injury through PKCepsilon activation.

Figure 1

Effect of tribulosin on myocardial infarct of rat hearts after ischemia/reperfusion. Rat hearts were subjected to 30 min ischemia and 120 min reperfusion. Myocardial infarct size was determined by TTC staining, and the percentage of infarct volumes to the left ventricle volumes was calculated. A) Control; B) I/R; C) GSTT 100 mg/L; D) tribulosin 100 nmol/L; E) tribulosin 10 nmol/L; F) tribulosin 1 nmol/L; G) Chelerythrine 1 μmol/L+tribulosin 100 nmol/L. n=6. Mean±SD. ^cP<0.01 vs I/R group.

Figure 2

Effects of tribulosin on pathomorphology of myocardium in ischemia/reperfusion rat hearts. Light micrograph of heart tissue (H&E staining, ×200). A) Control: No focal separation of myocardial fibre; B) I/R: 30-min ischemia and 120-min reperfusion with KH solution. Focal destruction of myocardial fibres with neutrophil infiltration and sarcoplasm agglomeration; C) GSTT 100 mg/L; D) tribulosin 100 nmol/L; E) tribulosin 10 nmol/L; F) tribulosin 1 nmol/L; G) Chelerythrine 1 μmol/L+tribulosin 100 nmol/L.

Figure 3

Effects of tribulosin on apoptosis in ischemia/reperfusion rat hearts by TUNEL staining (×200). A) Control; B) I/R; C) GSTT 100 mg/L; D) tribulosin (100 nmol/L); E) tribulosin (10 nmol/L); F) tribulosin (1 nmol/L); G) Chelerythrine (1 μmol/L)+tribulosin (100 nmol/L). Upper panels display representative histologic images. Lower panels display the percentage of TUNEL-positive cells per high power field. Values are mean±SEM for five individual experiments randomly chosen in each group. ^cP<0.01 vs control group; ^eP<0.05, ^fP<0.01 vs I/R group.

Figure 4

Effects of tribulosin on Bcl-2, Bax, and caspase-3 expression in rat hearts after ischemia/reperfusion. Western blotting analysis of Bcl-2, Bax, and caspase-3 in myocardial abundance are shown (top). β-Tubulin (bottom lanes) was used as control. The lower panels were summary data of Bcl-2, Bax, and caspase-3 expression in myocardial tissue in different groups. (a) Control; (b) I/R; (c) GSTT 100mg/L; (d) tribulosin 100 nmol/L; (e) tribulosin 10 nmol/L; (f) tribulosin 1 nmol/L; (g) Chelerythrine 1 μmol/L+tribulosin 100 nmol/L. Values are expressed as fold changes over the control and are presented as mean±SEM representative of 3 independent experiments. ^bP<0.05, ^cP<0.01 vs control group; ^eP<0.05, ^fP<0.01 vs I/R group.

Figure 5

Effect of tribulosin on PKCɛ of myocardium in ischemia/reperfusion rat hearts (×200). (A) Expression of total-PKCɛ proteins was analyzed by Western blotting. β-Tubulin was immunodetected with a monoclonal antibody as an internal control. The expression levels were measured in the hearts excised from the IR rats at the indicated times. A representative blot of 3 independent experiments is shown. (B) Graphic presentations of PKCɛ expression in myocardial tissue quantified by integrating the volume of image element density. The data presented in each imagine are representative of three separate groups. Values are expressed as fold changes over the control and are presented as mean±SEM. ^fP<0.01 vs I/R group. (C) Expression of phospho-PKCɛ proteins was analyzed by immunohistochemistry. (a) Control; (b) I/R; (c) GSTT 100mg/L; (d) tribulosin 100 nmol/L; (e) tribulosin 10 nmol/L; (f) tribulosin 1 nmol/L; (g) Chelerythrine 1 μmol/L+tribulosin 100 nmol/L. (D) Mean densities from the signals of phospho-PKCɛ were measured. Values are presented as mean±SEM. ^eP<0.05, ^fP<0.01 vsI/R group.

Scheme 1

Chemical structure of tribulosin.