Protective effect of troxerutin and cerebroprotein hydrolysate injection on cerebral ischemia through inhibition of oxidative stress and promotion of angiogenesis in rats

Abstract

Brain ischemia, including cerebral ischemia and cerebrovascular ischemia, leads to poor oxygen supply or cerebral hypoxia, and causes brain tissue death or cerebral infarction/ischemic stroke. The troxerutin and cerebroprotein hydrolysate injection (TCHI), is widely applied in China to improve blood supply in ischemic brain tissues and to enhance neuroprotective effects in clinical practice. However, the benefits and detailed underlying mechanism elaborating the effectiveness of TCHI in cerebrovascular diseases require further investigation. Therefore, in the present study, experimental in vivo and in vitro models were employed to investigate the potential mechanisms of TCHI on cerebral ischemic injury. The results demonstrated that TCHI increased the lactate dehydrogenase levels in the brain homogenate and conversely decreased lactic acid levels. TCHI was further observed to significantly increase superoxide dismutase activity and decrease malondialdehyde levels in ischemic brain tissues. In addition, TCHI significantly induced vascular maturation processes, including proliferation, adhesion, migration and tube formation in cultured human umbilical vein endothelial cells. Additionally, TCHI significantly stimulated microvessel formation in the rat aortic ring and chick chorioallantoic membrane assays. Taken together, these results provided strong evidence that TCHI stimulated angiogenesis at multiple steps, and indicated that TCHI attenuated cerebral ischemic damage through the amelioration of oxidative stress and promotion of angiogenesis.

Figure 1.

Effect of TCHI on neurological scores and infarct volume in MCAO rats. Neurological scores at (A) 6 h and (B) 24 h following MCAO are displayed. (C) Coronal sections from ischemic rat brains stained with triphenyltetrazolium chloride. (D) Cerebral infarct area was decreased following TCHI treatment in rats with an MCAO. Data are expressed as the mean ± standard error of the mean (n=10-11/group). **P<0.01 vs. sham group; ^#P<0.05 and ^##P<0.01, vs. MCAO group; ^▲P<0.05 and ^▲▲P<0.01 vs. edaravone group. TCHI, troxerutin and cerebroprotein hydrolysate injection; MCAO, middle cerebral artery occlusion.

Figure 2.

Effect of TCHI on SOD, MDA, LD and LDH levels following MCAO. Representative levels of (A) SOD, (B) MDA, (C) LD and (D) LDH at 24 h following MCAO. Data are expressed as the mean ± standard error of the mean (n=10-11/group). **P<0.01 vs. sham group; ^##P<0.01 vs. MCAO group; ^▲P<0.05 and ^▲▲P<0.01 vs. edaravone group. TCHI, troxerutin and cerebroprotein hydrolysate injection; SOD, superoxide dismutase; MDA, malondialdehyde; LD, lactic acid; LDH, lactate dehydrogenase; MCAO, middle cerebral artery occlusion.

Figure 3.

Effects of TCHI on human umbilical vein endothelial cell proliferation and tube formation. (A) Cell proliferation following exposure to TCHI at 2, 10, 50 or 250 µg/ml for 24 h was assessed by an MTT assay. (B) Quantification and (C) cell images (magnification, ×100) of capillary-type tube formation in cells cultured on a layer of Matrigel and incubated with medium containing 2, 10, 50 or 250 µg/ml TCHI at 37°C for 24 h. Data are expressed as the mean ± standard error of the mean. *P<0.05, **P<0.01 and ***P<0.001, vs. control group. TCHI, troxerutin and cerebroprotein hydrolysate injection; MTT, methylthiazolyldiphenyl-tetrazolium bromide; OD, optical density.

Figure 4.

Effects of TCHI on HUVEC adhesion and migration. (A) HUVECs were exposed to TCHI at a concentration of 2, 10, 50 or 250 µg/ml for 24 h, seeded on a Matrigel-coated 24-well plate, incubated for 1 h and observed under a microscope. (B) Adhesion and (C) migration of HUVECs were significantly enhanced by TCHI treatment. (D) Cell migration was examined by a wound healing assay, during which the confluent HUVEC monolayer was wounded with a 200-µl pipette tip and treated with TCHI (2, 10, 50 or 250 µg/ml). At 0, 12 and 24 h, wound healing was visualized with a digital camera. *P<0.05 and **P<0.01 vs. Control. TCHI, troxerutin and cerebroprotein hydrolysate injection; HUVEC, human umbilical vein endothelial cells.

Figure 5.

(A) TCHI promoted angiogenesis in the CAM model. (B) Blood vessel density was significantly increased following exposure to 5 ng/ml bFGF or 10 ng/ml TCHI. **P<0.01 vs. control group. TCHI, troxerutin and cerebroprotein hydrolysate injection; CAM, chick chorioallantoic membrane; bFGF, recombinant bovine basic fibroblast growth factor.

Figure 6.

(A) TCHI promoted aortic ring sprouting after 6 and 9 days of treatment. (B) Increased number of microvessels was detected in the rat aortic ring model. *P<0.05 and **P<0.01 vs. control group. TCHI, troxerutin and cerebroprotein hydrolysate injection.

Figure 7.

TCHI promoted the expression of integrin β3 mRNA on human umbilical vein endothelial cells. (A) Reverse transcription-quantitative polymerase chain reaction assay results and (B) quantified mRNA expression of integrin β3 are shown in cells were treated with TCHI for 24 h. TCHI induced a significant increase in integrin β3 mRNA expression. Data are expressed as the mean ± standard error of the mean. **P<0.01 vs. control group. TCHI, troxerutin and cerebroprotein hydrolysate injection.

Figure 8.

Mechanisms of action illustrating the protective effect of TCHI on neurological function following stroke. TCHI, troxerutin and cerebroprotein hydrolysate injection; ROS, reactive oxygen species; SOD, superoxide dismutase; MDA, malondialdehyde; LD, lactic acid; LDH, lactate dehydrogenase.