Anat Cell Biol.  2019 Dec;52(4):486-497. 10.5115/acb.19.141.

Platycarya strobilacea leaf extract protects mice brain with focal cerebral ischemia by antioxidative property

Affiliations
  • 1Department of Anatomy, Konyang University College of Medicine, Daejeon, Korea. jjzzy@konyang.ac.kr
  • 2Lifetree Co., Ltd., Suwon, Korea.
  • 3Department of Herbal Health and Pharmacy, Joongbu University College of Health and Welfare, Geumsan, Korea. esdoh@joongbu.ac.kr

Abstract

The leaf extract of Platycarya strobilacea (PSL) has long been recognized as possessing various health-promoting activities. However, information on its possible protective effects against ischemic stroke is currently lacking. Here, using a mouse model of focal cerebral ischemia (fCI), we studied the protective potential of an oral supplement of PSL. Mice were randomly divided into four groups: SO, a group subjected to a sham-operation; VEH, pretreated with distilled water and subjected to middle cerebral artery occlusion and reperfusion (MCAO/R); PSL-L and PSL-H, pretreated with low (20 mg/kg) and high (100 mg/kg) doses of PSL, respectively, and subjected to the MCAO/R procedure. PSL was administered via an oral route daily for 8 days prior to surgery. We then measured the infarct volumes and sensorimotor deficits and studied the underlying antioxidant mechanisms by quantifying apoptosis, reactive oxygen species (ROS) generation, oxidative damages, and antioxidant enzymes in the ischemic cortex. The results showed a marked attenuation in infarct volume and sensorimotor deficits in both the PSL-L and PSL-H groups when compared with VEH. The terminal deoxynucleotidyl transferase dUTP nick end labeling and the immunohistochemical detection of the cleaved caspase-3 revealed that PSL could reduce cellular apoptosis in the ischemic lesion in a dose-dependent manner. The dihydroethidium-fluorescence, 4-hydroxynonenal, and 8-hydroxyl-2"²-deoxyguanosine immunoreactivities in the ischemic lesion were markedly attenuated in the PSL-L group compared with the VEH group, indicating that PSL could attenuate ROS generation and the associated oxidative damage in the ischemic cortex. Finally, western blot results indicated that PSL can upregulate levels of heme oxygenase-1 (HO-1), an antioxidant enzyme, in the lesion area. Together, these results suggest that PSL can exert protective effects against fCI, and the mechanism may involve HO-1 upregulation.

Keyword

Platycarya strobilacea leaf; Focal cerebral ischemia; Reactive oxygen species; Antioxidant; Heme oxygenase-1

MeSH Terms

Animals
Apoptosis
Blotting, Western
Brain Ischemia*
Brain*
Caspase 3
DNA Nucleotidylexotransferase
Heme Oxygenase-1
Infarction, Middle Cerebral Artery
Mice*
Reactive Oxygen Species
Reperfusion
Stroke
Up-Regulation
Water
Caspase 3
DNA Nucleotidylexotransferase
Heme Oxygenase-1
Reactive Oxygen Species
Water

Figure

  • Fig. 1 The experimental protocols used in this study. For the VEH, PSL-L, and PSL-H group, vehicle, 20, or 100 mg/kg of PSL were administered daily via an intraoral route 8 times prior to MCAO/R, respectively. For the SO group, a sham-operation was executed instead of the MCAO/R. For in vivo ROS detection, the mice (n=2 per each group) were injected with 10 mg/kg of DHE intravenously just before MCAO/R and sacrificed at 2-hour post-operation for brain sampling. At 24 hours following the MCAO/R or sham-operation, three different motor function tests were conducted (neurological deficit scoring, grip strength test, and inverted screen test; n=10 per each group) and mice were immediately sacrificed for further experimentation. DHE, dihydroethidium; MCAO/R, middle cerebral artery occlusion and reperfusion; PSL, leaf extract of Platycarya strobilacea; PSL-H, pretreated with high doses of PSL; PSL-L, pretreated with low doses of PSL; ROS, reactive oxygen species; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; VEH, pretreated with distilled water and subjected to MCAO/R.

  • Fig. 2 Effects of PSL on infarct volume in a mouse model of focal cerebral ischemia. (A) Representative photographs showing the TTC-stained brain serial sections of the different groups. Upon the TTC staining, infarcted areas appear generally white in color. (B) Quantitation graphs showing the % area of infarction (n=4 per group, ***P<0.001 vs. VEH). Data were represented as mean±SEM. N.S, statistically not significant between indicated groups. PSL, leaf extract of Platycarya strobilacea; TTC, 2,3,5-triphenyltetrazolium chloride; PSL-H, pretreated with high doses of PSL; PSL-L, pretreated with low doses of PSL; SO, a group subjected to a sham-operation; VEH, pretreated with distilled water and subjected to middle cerebral artery occlusion and reperfusion.

  • Fig. 3 Effects of PSL on motor deficits in a mouse model of focal cerebral ischemia. (A) Graph showing the neurological deficit scores of the different groups performed 24 hours after the operation. Dots indicate individual scores. (B) Quantitative graphs showing the results of the grip strength test and (C) the inverted screen test. Data are represented as mean±SEM (n=10 per each group; ***P<0.001 vs. SO; ###P<0.001 vs. VEH; ††P<0.01 and †††P<0.001 vs. PSL-L). PSL, leaf extract of Platycarya strobilacea; PSL-H, pretreated with high doses of PSL; PSL-L, pretreated with low doses of PSL; SO, a group subjected to a sham-operation; VEH, pretreated with distilled water and subjected to middle cerebral artery occlusion and reperfusion.

  • Fig. 4 Effects of PSL on cellular apoptosis in the ischemic cortex of a mouse model of focal cerebral ischemia. Representative images of TUNEL stained ischemic cortices (A) and quantitative graph showing the number of TUNEL-positive (TUNEL+) cells in the cortical lesions of mice of different groups (n=6 per each group; *P<0.05 and **P<0.01 vs. VEH; #P<0.05 vs. PSL-L) (B). Representative images of immunohistochemistry using the cleaved caspase-3 antibody (arrows indicates cleaved caspase-3+ cells) (C) and the quantitative graph showing the number of cleaved caspase-3+ cells in cortical lesions of mice in the different groups (n=6 per each group; ***P<0.001 vs. SO; #P<0.05 and ##P<0.01 vs. VEH; †P<0.05 vs. PSL-L) (D). Scale bar=50 µm. All data are represented as mean±SEM in a randomly chosen HPF. HPF, high-power field; PSL, leaf extract of Platycarya strobilacea; PSL-H, pretreated with high doses of PSL; PSL-L, pretreated with low doses of PSL; SO, a group subjected to a shamoperation; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; VEH, pretreated with distilled water and subjected to middle cerebral artery occlusion and reperfusion.

  • Fig. 5 Effects of 20 mg/kg PSL on intracellular ROS accumulation in the ischemic cortex of a mouse model of focal cerebral ischemia. For this experiment, 10 mg/kg of DHE was injected into the mice via a jugular vein just before the operation. Representative images showing the cortical DHE fluorescence (red) acquired at 2 hours after the operation (scale bar=100 µm) (A) and the quantitative graphs (n=2 per each group; ***P<0.001 vs. SO; ##P<0.01 vs. VEH) (B). Data were represented as MFI±SEMs. Representative images of 4-HNE-immunostained ischemic cortices (scale bar=50 µm) (C) and the quantitative graph showing the numbers of 4-HNE-immunopositive (4-HNE+) cells (n=6 per each group; ***P<0.001 vs. SO; ###P<0.001 vs. VEH) (D). Representative images of 8-OHdG-immunostained ischemic cortices (scale bar=50 µm) (E) and the quantitative graph showing the numbers of 8-OHdG-immunopositive (8-OHdG+) cells (n=6 per each group; ***P<0.001 vs. VEH) (F). All data in (D) and (F) are represented as mean±SEM in a randomly chosen HPF. DHE, dihydroethidium; HPF, high-power field; MFI, mean fluorescence intensity; PSL, leaf extract of Platycarya strobilacea; HPF, high-power field; PSL-L, pretreated with low doses of PSL; ROS, reactive oxygen species; SO, a group subjected to a sham-operation; VEH, pretreated with distilled water and subjected to middle cerebral artery occlusion and reperfusion; 4-HNE, 4-Hydroxynonenal; 8-OHdG, 8-Hydroxyl-2′-deoxyguanosine.

  • Fig. 6 Effects of 20 mg/kg PSL on changes in the ischemic cortical expression of antioxidant enzymes in a mouse model of focal cerebral ischemia. (A) Representative western blot bands for assessing the amount of SOD, GPX, and HO-1 in the ischemic cortical homogenates of the indicated groups and (D) the quantitative graphs showing the band intensities of SOD (B), GPX (C), and HO-1 (D). β-actin was used as a loading control. All data are represented as mean±SEM (n=4 per each group; ***P<0.001 vs. SO; ##P<0.01 vs. VEH). GPX, glutathione peroxidase; HO-1, heme oxygenase-1; N.S, statistically not significant between indicated groups; PSL, leaf extract of Platycarya strobilacea; PSL-L, pretreated with low doses of PSL; SO, a group subjected to a shamoperation; SOD, superoxide dismutase; VEH, pretreated with distilled water and subjected to middle cerebral artery occlusion and reperfusion.


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