Ameliorative effects of Gualou Guizhi decoction on inflammation in focal cerebral ischemic-reperfusion injury

Abstract

Gualou Guizhi decoction (GLGZD) is a well-established Traditional Chinese Medicinal formulation which has long been used to treat stroke in a clinical setting in China. The present study investigated the ameliorative effects of GLGZD on inflammation in focal cerebral ischemic-reperfusion injury. A rat model of middle cerebral artery occlusion (MCAO) was employed. Rats were administrated GLGZD (7.2 and 14.4 g/kg per day) or saline as control 2 h after reperfusion and daily over the following seven days. Neurological deficit score and screen test were evaluated at 1, 3, 5 and 7 days after MCAO. Brain infarct size and brain histological changes were observed via 2,3,5-triphenyltetrazolium chloride staining and regular hematoxylin & eosin staining. Furthermore, inflammation mediators and nuclear factor-κB (NF-κB) were investigated using ELISA and immunohistochemistry. GLGZD treatment significantly improved neurological function, ameliorated histological changes to the brain and decreased infarct size in focal cerebral ischemic-reperfusion injury. GLGZD was found to significantly reduce interleukin (IL)-1, tumor necrosis factor-α and NF-κB levels, while increasing levels of IL-10. In conclusion, the present study suggested that GLGZD has a neuroprotective effect on focal cerebral ischemic-reperfusion injury and this effect is likely to be associated with the anti-inflammatory function of GLGZD.

Figure 1

Liquid chromatograms of standard and sample. (A) Liquid chro-matogram of the standard, where 1 is peoniflorin, 2 is liquiritigenin, 3 is liquiritin, 4 is cinnamic acid, 5 is cinnamaldehyde and 6 is glycyrrhizic acid. (B) Liquid chromatogram of Gualou Guizhi decoction.

Figure 2

Effect of GLGZD on the development of behavioral abnormalities following MCAO. Neurological score was taken as index. Values are expressed as the mean ± standard deviation in each group. 0 indicates no neurological symptoms, 1 indicates inability fully flex the left forepaw, 2 indicates rotating while crawling and falling to the contralateral side, 3 indicates inability to walk unaided and 4 indicates unconsciousness. ^**P<0.01 vs. sham-surgery group, ^#P<0.05, ^##P<0.01, vs. MCAO model group. MCAO, middle cerebral artery occlusion; GLGZD, Gualou Guizhi decoction; GLGZD-1, rats that were administered 7.2 g/kg GLGZD; GLGZD-2, rats that were administered 14.4 g/kg GLGZD.

Figure 3

Effect of GLGZD on cerebral infarction in MCAO model rats. (A) Representative 2,3,5-triphenyltetrazolium chloride-stained brain coronal sections of the four groups. The normal tissue was stained to red-blue and the infarct area was stained to pale pink. (B) Infarction rate was quantified using the Motic Med 6.0 system. Values are expressed as the mean ± standard deviation from five individual rats in each group. ^*P<0.05, vs. sham-surgery group, ^#P<0.05, vs. model group. MCAO, middle cerebral artery occlusion; GLGZD, Gualou Guizhi decoction; GLGZD-1, rats that were administered 7.2 g/kg GLGZD; GLGZD-2, rats that were administered 14.4 g/kg GLGZD.

Figure 4

Effect of GLGZD on the histopathological changes in brain tissue following MCAO or treated with GLGZD. Representative images are shown (hematox-ylin and eosin stained; magnification, ×200). MCAO, middle cerebral artery occlusion; GLGZD, Gualou Guizhi decoction; GLGZD-1, rats that were administered 7.2 g/kg GLGZD; GLGZD-2, rats that were administered 14.4 g/kg GLGZD.

Figure 5

Immunohistochemistry for NF-κB and TNF-α in the brain of rats following MCAO or treated with GLGZD. Representative images are shown (magnification, ×100). The positive cells were stained dark. MCAO, middle cerebral artery occlusion; GLGZD, Gualou Guizhi decoction; NF, nuclear factor; TNF, tumor necrosis factor; GLGZD-1, rats that were administered 7.2 g/kg GLGZD; GLGZD-2, rats that were administered 14.4 g/kg GLGZD.