Neuroprotective mechanisms of puerarin in middle cerebral artery occlusion-induced brain infarction in rats

Abstract

Puerarin, a major isoflavonoid derived from the Chinese medical herb Radix puerariae (kudzu root), has been reported to be useful in the treatment of various cardiovascular diseases. In the present study, we examined the detailed mechanisms underlying the inhibitory effects of puerarin on inflammatory and apoptotic responses induced by middle cerebral artery occlusion (MCAO) in rats. Treatment of puerarin (25 and 50 mg/kg; intraperitoneally) 10 min before MCAO dose-dependently attenuated focal cerebral ischemia in rats. Administration of puerarin at 50 mg/kg, showed marked reduction in infarct size compared with that of control rats. MCAO-induced focal cerebral ischemia was associated with increases in hypoxia-inducible factor-1alpha (HIF-1alpha), inducible nitric oxide synthase (iNOS), and active caspase-3 protein expressions as well as the mRNA expression of tumor necrosis factor-alpha (TNF-alpha) in ischemic regions. These expressions were markedly inhibited by the treatment of puerarin (50 mg/kg). In addition, puerarin (10-50 microM) concentration-dependently inhibited respiratory bursts in human neutrophils stimulated by formyl-Met-Leu-Phe. On the other hand, puerarin (20-500 microM) did not significantly inhibit the thiobarbituric acid-reactive substance reaction in rat brain homogenates. An electron spin resonance (ESR) method was conducted on the scavenging activity of puerarin on the free radicals formed. Puerarin (200 and 500 microM) did not reduce the ESR signal intensity of hydroxyl radical formation. In conclusion, we demonstrate that puerarin is a potent neuroprotective agent on MCAO-induced focal cerebral ischemia in vivo. This effect may be mediated, at least in part, by the inhibition of both HIF-1alpha and TNF-alpha activation, followed by the inhibition of inflammatory responses (i.e., iNOS expression), apoptosis formation (active caspase-3), and neutrophil activation, resulting in a reduction in the infarct volume in ischemia-reperfusion brain injury. Thus, puerarin treatment may represent a novel approach to lowering the risk of or improving function in ischemia-reperfusion brain injury-related disorders.

Figure 1

Effect of puerarin in middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia in rats. (A) Coronal sections of TTC-stained brains, (B) dose-response effect of puerarin, and (C) infarct area at various distances from the frontal pole in 24 h after MACO-reperfusion rats. Cerebral infarction in sham-operated (sham) or MACO-reperfusion rats is from a representative animal that received solvent (solvent; cremophor: ethanol: normal saline at 1: 1: 4) or puerarin (25 and 50 mg/kg) intraperitoneally. (B) Infarct volumes were calculated as described in "Methods", and data are presented as a superimposed scatterplot showing the infarct volume for each animal in the group as well as the means ± S.E.M. (n = 5). ***P < 0.001 compared to the sham-operated group; ^###P < 0.001 compared to the solvent-treated group. (C) Forebrain profiles of the infarct area at various distances from the frontal pole as described in "Methods". Each point (○, solvent-treated group; △, puerarin 50 mg/kg-treated group) and vertical bar represent the means ± S.E.M. (n = 5). *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the solvent-treated group.

Figure 2

Effect of puerarin in middle cerebral artery occlusion (MCAO)-induced regional cerebral blood flow (rCBF) and neurobehavioral deficits in rats. (A) rCBF of solvent- and puerarin (50 mg/kg)-treated rats were measured by laser Doppler flowmeter in the MCA-supplied cortex. The value of rCBF were recorded every 10 min after MCAO and reperfusion periods. Each point (○, solvent-treated group; △, puerarin 50 mg/kg-treated group) and vertical bar represent the means ± S.E.M. (n = 6). (B) Neurobehavioral scores of solvent- and puerarin (50 mg/kg)-treated rats were recorded at 1 and 24 h after MCAO. Each point (○, solvent-treated group; △, puerarin 50 mg/kg-treated group) and vertical bar represent the means ± S.E.M. (n = 5).

Figure 3

Effects of puerarin on the expressions of (A) hypoxia-inducible factor-1α (HIF-1α) and (B) inducible nitric oxide synthase (iNOS) in cerebral homogenates 24 h after middle cerebral artery occlusion (MCAO)-reperfusion injury in rats. Fresh brains from sham-operated (lane 1), solvent-treated (lane 2), and puerarin (50 mg/kg)-treated (lane 3) rats were removed and sectioned coronally into four sequential parts from the frontal lobe to the occipital lobe. The third part of the four sequential parts of the ischemic-injured hemisphere was separately collected, homogenized, and centrifuged. The supernatant (50 μg protein) was then subjected to SDS-PAGE, and transferred onto membranes for analysis of HIF-1α and iNOS expressions. The results are representative examples of three similar experiments. Data are presented as the means ± S.E.M. **P < 0.01 compared to the sham-operated group (lane 1); ^#P < 0.05 and ^##P < 0.01 compared to the solvent-treated group (lane 2). Equal loading in each lane is demonstrated by similar intensities of α-tubulin.

Figure 4

Effects of puerarin on the expressions of (A) active caspase-3 protein and (B) tumor necrosis factor (TNF)-α mRNA in cerebral homogenates 24 h after middle cerebral artery occlusion (MCAO)-reperfusion injury in rats. (A) Fresh brains from sham-operated, solvent-treated, and puerarin (50 mg/kg)-treated rats were removed, and sectioned coronally into four sequential parts from the frontal lobe to the occipital lobe. The third part of the four sequential parts of the ischemic-injured hemisphere was separately collected, homogenized, and centrifuged. The supernatant (50 μg protein) was then subjected to SDS-PAGE, and transferred onto membranes for analysis of active caspase-3 expression. The results are representative examples of three similar experiments. Data are presented as the means ± S.E.M. *P < 0.05 compared to the sham-operated group (lane 1); ^#P < 0.05 compared to the solvent-treated groups (lane 2). Equal loading in each lane is demonstrated by similar intensities of α-tubulin. (B) Fresh brains from sham-operated, solvent-treated, and puerarin (50 mg/kg)-treated rats were removed, homogenized, and centrifuged from the ipsilateral cortex, followed by analysis of TNF-α mRNA expression by RT-PCR as described in "Methods". The GAPDH level was used to normalize the amount of the cDNA template used in each PCR reaction. The results are representative examples of three similar experiments.

Figure 5

Effects of puerarin on (A) respiratory bursts in human neutrophils, and (B) the antioxidation in thiobarbituric acid-reactive substance (TBARS) in rat brain homogenate. (A) Washed neutrophil suspensions (2 × 10⁶ cells/ml) were preincubated with the solvent control (0.5% DMSO) or various concentrations of puerarin (10, 20, and 50 μM) in the presence of lucigenin (100 μM), followed by the addition of fMLP (800 nM) to trigger neutrophil respiratory bursts. Data are presented as a percent inhibition of the solvent control (means ± S.E.M., n = 4). (B) Brain homogenates were preincubated with solvent (0.5% DMSO) or various concentrations of puerarin (20~500 μM) for 10 min followed by the addition of Fe²⁺ (200 μM). Results are presented as the absorbance at 532 nm/mg protein in brain homogenates. Data are presented as the means ± S.E.M. (n = 4). **P < 0.01 compared with the solvent group (DMSO only).

Figure 6

Effect of puerarin on the free radical-scavenging activity in the H₂O₂/NaOH/DMSO system. The signal of hydroxyl radical peaks was observed in electron spin resonance (ESR) experiments. (A) Typical ESR spectra in the presence of solvent control (0.5% DMSO) or puerarin (B, 200 μM), (C, 500 μM) in the H₂O₂/NaOH/DMSO system, the spectrum is a representative example of four similar experiments.