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. 2017 Sep 5;7(1):10556.
doi: 10.1038/s41598-017-11181-5.

Dihydrocapsaicin Attenuates Blood Brain Barrier and Cerebral Damage in Focal Cerebral Ischemia/Reperfusion via Oxidative Stress and Inflammatory

Adchara Janyou  1 , Piyawadee Wicha  1 , Jinatta Jittiwat  2 , Apichart Suksamrarn  3 , Chainarong Tocharus  1 , Jiraporn Tocharus  4
Affiliations

Dihydrocapsaicin Attenuates Blood Brain Barrier and Cerebral Damage in Focal Cerebral Ischemia/Reperfusion via Oxidative Stress and Inflammatory

Adchara Janyou et al. Sci Rep. .

Abstract

This study investigated the effect of dihydrocapsaicin (DHC) on cerebral and blood brain barrier (BBB) damage in cerebral ischemia and reperfusion (I/R) models. The models were induced by middle cerebral artery occlusion (MCAO) for 2 h followed by reperfusion. The rats were divided into five groups: sham, or control group; vehicle group; and 2.5 mg/kg, 5 mg/kg, and 10 mg/kg BW DHC-treated I/R groups. After 24 h of reperfusion, we found that DHC significantly reduced the area of infarction, morphology changes in the neuronal cells including apoptotic cell death, and also decreased the BBB damage via reducing Evan Blue leakage, water content, and ultrastructure changes, in addition to increasing the tight junction (TJ) protein expression. DHC also activated nuclear-related factor-2 (Nrf2) which involves antioxidant enzymes like superoxide dismutase (SOD) and glutathione peroxidase (GPx), and significantly decreased oxidative stress and inflammation via down-regulated reactive oxygen species (ROS), NADPH oxidase (NOX2, NOX4), nuclear factor kappa-beta (NF-ĸB), and nitric oxide (NO), including matrix metalloproteinases-9 (MMP-9) levels. DHC protected the cerebral and the BBB from I/R injury via attenuation of oxidative stress and inflammation. Therefore, this study offers to aid future development for protection against cerebral I/R injury in humans.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Decrease in neurological deficit scores and area of infarction in DHC-treated I/R. (A and B) Representative of core temperature and heart rate. (C) Representative of neurological deficit scores after 24 h reperfusion. (D and F) Percentage of infarct volume. (E) Regional cerebral blood flow was monitored before ischemia/reperfusion, during ischemia, and after reperfusion. Representative of TTC-stained coronal section after 24 h of reperfusion. The data are the mean ± S.E.M from three independent experiments (*** p < 0.001 compared with the sham group; and # p < 0.05, ## p < 0.01, and ### p < 0.001 compared with the vehicle group).
Figure 2
Figure 2
Effect of DHC on histopathology. (A) H&E-stained cerebral cortex of I/R brain after 24 h of reperfusion (20×). (B) Striatum (20×). The images were visualized with a light microscope (scale bar = 50 µm). The black arrow represents the pyknotic nucleus.
Figure 3
Figure 3
Effect of DHC on TUNEL staining in cerebral I/R rats. (A and B) Representative of TUNEL-stained apoptotic cell of I/R brain after 24 h of reperfusion, shown as positive brown particles (4× and 20×). (C) Apoptotic index (AI). The images were visualized with a light microscope (scale bar = 50 µm). The data are the mean ± S.E.M from three independent experiments (*** p < 0.001 compared with the sham group and ### p < 0.001 compared with the vehicle group).
Figure 4
Figure 4
Administration of DHC protected BBB permeability and increased the level of TJ proteins. (A) The images represent the Evan Blue injected brain after 24 h of reperfusion, and the absorbance of the Evan Blue leakage is shown as OD620nm/g. (B) Percentage of brain water content. (C) Representative of western blot analysis of occludin and claudin in cerebral I/R rats at 24 h after reperfusion. (D) Quantitative analysis of protein expressions of occludin and claudin normalized using β-actin. The data are the mean ± S.E.M from three independent experiments (* p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the sham group; and # p < 0.05, ## p < 0.01, and ### p < 0.001 compared with the vehicle group).
Figure 5
Figure 5
DHC-treated I/R reduced the ultrastructure changes in the BBB. Transmission electron micrographs showing the ultrastructure of the BBB in cerebral I/R rats at 24 h after reperfusion. Swelling of astrocytic foot plate (AS); black arrow shows microvillous formation. The images were visualized with TEM at a magnification of 6000× (scale bar = 1 µm).
Figure 6
Figure 6
Administration of DHC attenuated oxidative stress via down-regulating the expressions of NOX2, NOX4, ROS, and lipid peroxidation. (A) Representative of ROS production. (B) Representative of MDA product of I/R brain after 24 h of reperfusion. (C) Representative of western blot analysis of NOX2 and NOX4 in cerebral I/R rats at 24 h after reperfusion. (D) Quantitative analysis of protein expression of NOX2 and NOX4 normalized using β-actin. The data are the mean ± S.E.M from three independent experiments (** p < 0.01 and *** p < 0.001 compared with the sham group; and # p < 0.05, ## p < 0.01, and ### p < 0.001 compared with the vehicle group).
Figure 7
Figure 7
Effect of DHC on the inflammatory pathway through down-regulating NO production, NF-κB (p65 subunits), and MMP-9 level. (A) NO production is shown as % of control after 24 h of reperfusion. (B) Representative of western blot analysis of MMP-9 and p65 in cerebral I/R rats at 24 h after reperfusion. Quantitative analysis of protein expressions of MMP-9 and p65 normalized using β-actin. (C) Representative of western blot analysis of TRPV1 in cerebral I/R rats at 24 h after reperfusion. Quantitative analysis of protein expressions of TRPV1 normalized using β-actin. The data are the mean ± S.E.M from three independent experiments (** p < 0.01 and *** p < 0.001 compared with the sham group; and # p < 0.05 and ## p < 0.01 compared with the vehicle group).
Figure 8
Figure 8
Effect of DHC on the Nrf2 signaling pathway via upregulating Nrf2 and NQO1 levels and promoting SOD and GPx activities. (A and B) SOD and GPx activities in cerebral I/R rats after 24 h of reperfusion. (C) Representative of western blot analysis of Nrf2 and NQO1 in cerebral I/R rats at 24 h after reperfusion. (D) Quantitative analysis of protein expressions of Nrf2 and NQO1 normalized using β-actin. The data are the mean ± S.E.M from three independent experiments (* p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the sham group; and # p < 0.05, ## p < 0.01, and ### p < 0.001 compared with the vehicle group).
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