Fig 1.
Effects of HHC on brain infarction and neurological outcomes after cerebral I/R in rats.
HHC was administered upon initiation of the reperfusion. (A) The regional cerebral blood flow (rCBF) observed during pre-occlusion, occlusion, and reperfusion. (B) The neurological deficit scores after cerebral I/R. (C) The TTC staining of series coronal brain section (2 mm thick) after 24-h reperfusion. The infarct region is in white color, whereas the non-ischemic region appears in red color. (D) The TTC staining of rat treatment with 40 mg/kg HHC and vehicle group at 2 h, 4 h, 6 h, 12 h and 48 h after reperfusion. (E) The percentage of infarct volume after I/R in different groups. (F) The percentage of infarct volume after I/R in various reperfusion time. All the data are presented as mean±SD. *** P < 0.001 versus sham group. ### P < 0.001 versus vehicle group.
Fig 2.
The representative histopathological changes of the brain at 24 h after focal cerebral I/R in stroke rats.
The brain tissues were stained with H&E and visualized with a light microscope (20× and 40×). In the sham group, the structure of most of the neurons in the cortex and the striatum were clear (thin arrow). The neurons of the I/R group were found to present shrinkage, chromosome condensation, and nuclear pyknosis, and showed increased intercellular space (thick arrow). The number of intact neurons in the rats that were treated with HHC was found to have increased.
Fig 3.
HHC reduces markers of lipid peroxidation in the ischemic tissue.
The effects of HHC on the MDA level in different groups. The data are presented as mean±SD (n = 6). *** P < 0.001versus sham group. # P < 0.05 versus vehicle group, and ### P < 0.001 versus vehicle group.
Fig 4.
The effect of HHC on inflammation in cerebral I/R rats.
(A) Representative of the NF-κB (p65) and the COX-2 protein expressions, as examined by western blot analysis. (B) The quantitative results of the expressions of the NF-κB (p65) proteins in each of the groups. (C) The quantitative results of the expressions of the COX-2 proteins in each of the groups. (D) The effect of HHC on the NO level in stroke rats. The data are expressed as mean±SD. ** P < 0.01 versus sham group, and *** P < 0.001 versus sham group. # P < 0.05 versus vehicle group, and ### P < 0.001 versus vehicle group.
Fig 5.
The effect of HHC on the Nrf2 signaling pathway in cerebral I/R injury rats.
(A) Representative of the Nrf2 (nucleus) and the HO-1 protein expressions, as examined by western blotting. (B) The quantitative results of the expressions of the Nrf2 proteins in each of the groups. (C) The quantitative results of the expressions of the HO-1 proteins in each of the groups. (D) The effect of HHC on SOD activity in cerebral I/R injury rats. (E) The effect of HHC on GSH in cerebral I/R injury rats. (F) The effect of HHC on GSH-Px in cerebral I/R injury rats. The data are presented as mean±SD. ** P < 0.01 versus sham group, and *** P < 0.001 versus sham group. # P < 0.05 versus vehicle group, ## P < 0.01 versus vehicle group, and ### P < 0.001 versus vehicle group.
Fig 6.
The effect of HHC on the apoptosis pathway in stroke rats.
(A) Representative of the Bax, the Bcl-XL, and the cleaved caspase-3 protein expressions in the penumbra cortex, as determined by western blot analysis. (B) The quantitative results of the expressions of the Bcl-XL proteins in each of the groups. (C) The quantitative results of the expressions of the Bax protein in each of the groups. (D) The quantitative results of the expressions of the cleaved caspase-3 protein in each of the groups. The data are presented as mean±SD. ** P < 0.01 versus sham group, and *** P < 0.001 versus sham group. # P < 0.05 versus vehicle group and ## P < 0.01 versus vehicle group.
Fig 7.
The effect of HHC on I/R-induced neuronal apoptosis.
(A) The apoptotic cells examined by TUNEL staining and which were visualized with a light microscope (20×). The arrows show TUNEL-positive cells. (B) The quantitative analysis of TUNEL-positive cells in each of the groups. The data are presented as mean±SD. ***P < 0.001 versus sham group. ### P < 0.001 versus vehicle group.