Amelioration of stroke-induced neurological deficiency by lyophilized powder of catapol and puerarin

Abstract

Catalpol and puerarin are active ingredients isolated from Rehmannia glutinosa Libosch and Radix Puerariae, respectively. They are popular in research for their poly-pharmacological effects. This research focused on effect of anti-stroke by lyophilized powder of catalpol and puerarin (C-P) and potential mechanisms. At the beginning of research, C-P was identified and analyzed by HPLC. Neurological function was evaluated by Longa score, neurological complex function score and beam balance score after permanent middle cerebral artery occlusion (PMCAO) in mice. Infarct volume and water content were evaluated after treatment of C-P. Anti-oxidative stress, anti-apoptosis, angiogenesis and neurogenesis were investigated by ELISA, WB and immunohistochemical stain respectively. With treatment of C-P, neurological deficiency of PMCAO mice was ameliorated. Morphologically, infarct volume and water content in ischemic hemisphere were significantly reduced by C-P. In vivo and in vitro, oxidative stress injury was extenuated by C-P. Meanwhile, Caspase-3 was down-regulated and Bxl-2 was up-regulated by C-P in vivo. In addition, C-P enhanced angiogenesis around the infarct of cortex and neurogenesis in the Hippocampal Dentate Gyrus (DG). Hence, C-P ameliorated stroke-induced neurological deficiency through its multiple neuroprotections. What's more, this article provides us a novel formula of active ingredients for stroke.

Keywords: catapol; neurological function.; puerarin; stroke.

Figure 1

High-performance liquid chromatogram of C-P at 210 nm. The first peak is catalpol and the second is puerarin.

Figure 2

Longa score (A), neurological complex function score (B) and beam balance score (C) were tested in PMCAO mice on the 1d, 4d and 7d. (value as mean± SD , n=12, ^##P<0.01 vs sham operation; ^*P<0.05, ^**P<0.01 vs PMCAO).

Figure 3

Morphology observation in stroke mice: (A) Treated with C-P for 5 days, mice were performed TTC stain. And the infarct volume was calculated as percentage of infarct volume to whole brain. (B) Water content was tested on the 3th days of C-P treatment and calculated as follows: water content(%) = (wet weight - dry weight)/ (wet weight). (value as mean ± SD , n=8, ^##P<0.01 vs sham operation; ^*P<0.05, ^**P<0.01 vs PMCAO).

Figure 4

Anti-oxidative stress of C-P in vivo. PMCAO performed for 3 hours, mice were treated with C-P again. The first test was performed at 4h after stroke, and then tested on 1th, 4th and 7th respectively. (value as mean ± SD, n=6, ^##P<0.01 vs sham operation; ^*P<0.05, ^**P<0.01 vs PMCAO).

Figure 5

(A) Cells were co-cultured with CoCl₂ and C-P for 24 hours, and then the cellular viability was tested by MTT. (B) At the same time, the level of NO in culture medium and SOD in cells were tested by ELISA. (value as mean ± SD, n=3, repeated to 3 times ,^##P<0.01 vs sham operation; ^*P<0.05, ^**P<0.01 vs PMCAO).

Figure 6

Cells were co-cultured with CoCl₂ and C-P for 24 hours, and then the expression of Bcl-2, Bax and Caspase-3 were tested by WB. Figures show that C-P has up-regulated Bcl-2 and decreased caspase-3, but has little effect on Bax. (value as mean ± SD, n=3, repeated to 3 times , ^##P<0.01 vs sham operation; ^*P<0.05, ^**P<0.01 vs PMCAO).

Figure 7

Aniogenesis around infarct of cortex. Immunohistochemical stains of BrdU and CD31 were performed on the 1th, 4th, 7th day around infarct of cortex. Cells characterized with brown cytoplasm were counted on the specific time (G-H). (value as mean± SD , n=6, ^##P<0.01 vs sham operation; ^*P<0.05, ^**P<0.01 vs PMCAO).

Figure 8

Neurogenesis in DG of ischemic hemisphere. Immunohistochemical stains of BrdU and Nestin were performed on the 1th, 4th and 7th day after PMCAO and C-P. Cells characterized with brown cytoplasm were counted (G-H). (value as mean± SD , n=6, ^##P<0.01 vs sham operation; ^*P<0.05, ^**P<0.01 vs PMCAO)