Acorus tatarinowii Schott extract reduces cerebral edema caused by ischemia-reperfusion injury in rats: involvement in regulation of astrocytic NKCC1/AQP4 and JNK/iNOS-mediated signaling

Abstract

Background: This study aimed to evaluate the effects of the Acorus tatarinowii Schott [Shi Chang Pu (SCP)] extract administered at the start of 2 h of middle cerebral artery occlusion (MCAo), followed by 3 d of reperfusion, and to determine mechanisms involved in anti-edema effects in the penumbra of the cerebral cortex.

Method: Rats were intraperitoneally administered the SCP extract at a dose of 0.25 g/kg (SCP-0.25 g), 0.5 g/kg (SCP-0.5 g), or 1 g/kg (SCP-1 g) at the start of MCAo.

Result: SCP-0.5 g and SCP-1 g treatments effectively reduced the cerebral infarct size, ameliorated cerebral edema, reduced blood-brain barrier permeability, and restored neurological function. SCP-0.5 g and SCP-1 g treatments markedly downregulated the levels of glial fibrillary acidic protein, Na⁺-K⁺-2Cl^- cotransporter type 1 (NKCC1), aquaporin 4 (AQP4), phospho-c-Jun N-terminal kinase (p-JNK)/JNK, inducible nitric oxide synthase (iNOS), 3-nitrotyrosine, intercellular adhesion molecule-1 (ICAM-1), matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor-A (VEGF-A), and zonula occluden-1 (ZO-1) and upregulated ZO-3 expression in the penumbra of the cerebral cortex 3 d after reperfusion.

Conclusions: SCP-0.5 g and SCP-1 g treatments exert neuroprotective effects against cerebral infarction and cerebral edema partially by mitigating astrocytic swelling and blood-brain barrier disruption. Moreover, the anti-cerebral edema effects of SCP extract treatments are possibly associated with the downregulation of astrocytic NKCC1/AQP4 and JNK/iNOS-mediated ICAM-1/MMP-9 signaling in the penumbra of the cerebral cortex 3 d after reperfusion.

Keywords: Acorus tatarinowii Schott; Aquaporin 4; C-Jun N-terminal kinase; Cerebral edema; Intercellular adhesion molecule-1; Zonula occluden-1.

Fig. 1

HPLC profiles of the a standard solution and b SCP extract solution. AU, Absorbance unit

Fig. 2

Representative coronal brain sections (S1–S6) among the experimental groups 3 d after reperfusion. In TTC stained brain sections, white color indicated the infarct area and dark-red color indicated the normal brain tissue. Scale bar = 1 cm

Fig. 3

Effects of SCP-0.5 g and SCP-1 g treatments on cerebral infarction, neurological deficits, and cerebral edema 3 d after reperfusion. a The percentage of cerebral infarct areas in the Sham, Model, SCP-0.25 g, SCP-0.5 g, and SCP-1 g groups was evaluated 3 d after reperfusion. b Neurological deficit scores of the Sham, Model, SCP-0.25 g, SCP-0.5 g, and SCP-1 g groups were examined 1 and 3 d after reperfusion. c Water content in right cerebral hemispheres in the Sham, Model, SCP-0.25 g, SCP-0.5 g, and SCP-1 g groups was evaluated 3 d after reperfusion. Data are expressed as mean ± standard deviation. *P < 0.05 compared with the Sham group; #P < 0.05 compared with the Model group

Fig. 4

Effects of SCP-0.5 g and SCP-1 g treatments on BBB permeability in right cortical regions. Representative images (a) and (b) show EBD extravasation in right cortical regions in the Sham, Model, SCP-0.25 g, SCP-0.5 g, and SCP-1 g groups 3 d after reperfusion. c EBD extravasation was examined in the selected right cortical regions among the experimental groups. Scale bar = 5 mm. EBD, Evans blue dye. *P < 0.05 compared with the Sham group; #P < 0.05 compared with the Model group

Fig. 5

Effects of SCP-0.5 g and SCP-1 g treatments on cytosolic GFAP, NKCC1, p-JNK, and JNK expression in the penumbra of the cerebral cortex. a Representative images show cytosolic GFAP, NKCC1, p-JNK, and JNK expression in the penumbra of the cerebral cortex in the Sham, Model, SCP-0.25 g, SCP-0.5 g, and SCP-1 g groups 3 d after reperfusion. The ratios of b GFAP/actin, c NKCC1/actin, and d p-JNK/JNK expression were examined in the penumbra of the cerebral cortex among the experimental groups. Actin levels were used as internal controls. Cyto, cytosolic fraction. *P < 0.05 compared with the Sham group; #P < 0.05 compared with the Model group

Fig. 6

Effects of SCP-0.5 g and SCP-1 g treatments on cytosolic iNOS, VEGF-A, ZO-1, ZO-2, and ZO-3 expression in the penumbra of the cerebral cortex. a Representative images show cytosolic iNOS, VEGF-A, ZO-1, ZO-2, and ZO-3 expression in the penumbra of the cerebral cortex in the Sham, Model, SCP-0.25 g, SCP-0.5 g, and SCP-1 g groups 3 d after reperfusion. The ratios of b iNOS/actin, c VEGF-A/actin, d ZO-1/actin, e ZO-2/actin, and f ZO-3/actin expression were examined in the penumbra of the cerebral cortex among the experimental groups. *P < 0.05 compared with the Sham group; #P < 0.05 compared with the Model group

Fig. 7

Effects of SCP-0.5 g and SCP-1 g treatments on NKCC1 and AQP4 expression in the penumbra of the cerebral cortex. Representative images show a NKCC1 and b AQP4 expression in the penumbra of the cerebral cortex in the Sham, Model, SCP-0.25 g, SCP-0.5 g, and SCP-1 g groups 3 d after reperfusion. c The dotted line square in a TTC-stained coronal brain section reveals the region in which the immunopositive cells are counted. CP, cortical penumbra. Dotted line square = 1 mm². The bar graphs display the numbers of d NKCC1- and e AQP4-positive cells in the penumbra of the cerebral cortex among the experimental groups. *P < 0.05 compared with the Sham group; #P < 0.05 compared with the Model group. Arrows in (a) and (b) point to NKCC1- and AQP4-positive cells, respectively. Scale bars = 10 μm and 2 mm for (b) and (c), respectively

Fig. 8

Effects of SCP-0.5 g and SCP-1 g treatments on 3-NT and ICAM-1 expression in the penumbra of the cerebral cortex. Representative images show a 3-NT and b ICAM-1 expression in the penumbra of the cerebral cortex in the Sham, Model, SCP-0.25 g, SCP-0.5 g, and SCP-1 g groups 3 d after reperfusion. The bar graphs display the numbers of c 3-NT- and d ICAM-1-positive cells in the penumbra of the cerebral cortex among the experimental groups. *P < 0.05 compared with the Sham group; #P < 0.05 compared with the Model group. Arrows in (a) and (b) point to 3-NT- and ICAM-1-positive cells, respectively. Scale bar = 10 μm

Fig. 9

Effects of SCP-0.5 g and SCP-1 g treatments on MMP-9 and ZO-1 expression in the penumbra of the cerebral cortex. Representative images show a MMP-9 and b ZO-1 expression in the penumbra of the cerebral cortex in the Sham, Model, SCP-0.25 g, SCP-0.5 g, and SCP-1 g groups 3 d after reperfusion. The bar graphs display the numbers of c MMP-9- and d ZO-1-positive cells in the penumbra of the cerebral cortex among the experimental groups. *P < 0.05 compared with the Sham group; #P < 0.05 compared with the Model group. Arrows in (a) and (b) point to MMP-9- and ZO-1-positive cells, respectively. Scale bar = 10 μm

Fig. 10

Expression of GFAP-, NKCC1-, AQP4-, ICAM-1-, and ZO-1-positive cells in the penumbra of the cerebral cortex. Arrows in (a), (b), (d), (e), (g), and (h) point to GFAP-, NKCC1-, AQP4-, NKCC1-, ICAM-1-, and ZO-1-positive cells, respectively, in the penumbra of the cerebral cortex. The arrow and arrowhead in (c) point to GFAP- and NKCC1-positive cells, respectively. Arrows in (f) and (i) point to AQP4/NKCC1 and ICAM-1/ZO-1 double-labeled cells, respectively. Scale bar = 50 μm