Carthamus tinctorius L. Extract ameliorates cerebral ischemia-reperfusion injury in rats by regulating matrix metalloproteinases and apoptosis

Abstract

We investigate the protective effect of Carthamus tinctorius L. (CTL, also known as Honghua in China or Safflower) on cerebral ischemia-reperfusion and explored the possible mechanisms on regulating apoptosis and matrix metalloproteinases (MMPs). High-performance liquid chromatography method with diode array detection analysis was established to analyze the components of CTL. Middle cerebral artery occlusion rats model was established to evaluate Neurological Function Score and hematoxylin-eosin staining, as well as triphenyltetrazolium was used to examine the infarction area ratio. Transferase-mediated dUTP nick-end labeling was performed for the apoptosis. Apoptosis-related factors, including B-cell lymphoma-2 (Bcl-2), Bax and Caspase3, and MMPs-related MMP2, MMP9, tissue inhibitor of metalloproteinases 1 (TIMP1) in ischemic brain, were assayed by Western blot, reverse transcription polymerase chain reaction, and immunohistochemistry. The data showed that CTL (2, 4 g crude drug/kg/d) treatment could significantly reduce the ischemic damage in brain tissue and improve a significant neurological function score. In addition, CTL could also attenuate apoptosis degree of brain tissues and regulate Bcl-2, Bax, and Caspase 3 and also have a significant decrease on MMP-9 expression, followed by a significant increase of TIMP1 protein expression. These findings indicated that regulation of CTL on apoptosis and MMPs contributed to its protective effect on ischemia/reperfusion injury.

Keywords: Apoptosis; Carthamus tinctorius L.; cerebral ischemia-reperfusion; matrix metalloprotein.

Figure 1

Pictorial diagram of middle cerebral artery occlusion rat model

Figure 2

High-performance liquid chromatography for Carthamus tinctorius L. (a) and reference compounds (b) hydroxysafflor yellow A (1) and safflower yellow B (2)

Figure 3

Neurological function of Carthamus tinctorius L. on cerebral ischemia in rats. (A) Infarction behavior of rats; (B) Hematoxylin and eosin staining for brain tissues of cerebral ischemic rats, a. Normal, b. Sham, c. Model, d. positive drug Nimodipine group (7.53 mg/kg/d), e. high dose Carthamus tinctorius L. group (4 g crude drug/kg/d), f. low dose Carthamus tinctorius L. group (2 g crude drug/kg/d); (C) Neurological deficit scores of cerebral ischemic rats on the 1^st day, 7^th day, and 14^th day; Magnification × 200. Data are presented as means ± standard deviation (n = 8). *P < 0.05, **P < 0.01, 7^th day or 14^th day versus 1^st day

Figure 4

Effect of Carthamus tinctorius L. on infarction aera ratio of rats after cerebral ischemia. (A) Triphenyltetrazolium for brain tissues; (B) infarction aera ratio. Data are presented as means ± standard deviation (n = 6). ^##P < 0.01, vs. Sham; *P < 0.05, **P < 0.01, versus model

Figure 5

Regulation of Carthamus tinctorius L. on apoptosis of brain tissues of rats after cerebral ischemia. (A) Transferase-mediated dUTP nick-end labeling assay for apoptosis; (B) Western blotting for apoptosis-related factor levels including B-cell lymphoma-2, Bax, and Caspase3; (C) Reverse transcription polymerase chain reaction for messenger RNA levels of B-cell lymphoma-2, Bax and Caspase3. Nimodipine of 7.53 mg/kg/d was used as positive drug. Data are presented as means ± standard deviation (n = 6). ^#P < 0.05, ^##P < 0.01, versus Sham; *P < 0.05, **P < 0.01, versus model

Figure 6

Effect of Carthamus tinctorius L. on matrix metalloproteinases in brain tissue of cerebral ischemic rats. (A) Immunohistochmeistry for matrix metalloproteinase-9 level; (B) Western blotting for matrix metalloproteinase-2, matrix metalloproteinase-9, and tissue inhibitor of metalloproteinases 1 protein expressions. Data are presented as means ± standard deviation (n = 6). ^#P < 0.05, ^##P < 0.01, versus Sham; *P < 0.05, **P < 0.01, versus model