| Objective The deficit of neurological function,disability and even death are mainly caused by cerebral ischemia reperfusion injury in ischemic cerebrovascular disease. By now, there is no effective treatment. We set up the middle cerebral artery in cerebral ischemia reperfusion animal model, to investigate the effects of alpha lipoic acid on the regulation of apoptosis gene Bax, Baxm RNA and JNK3 protein after cerebral ischemia reperfusion injury, and on expression inflammatory cytokine NF- kappa B, TNF-α in protein and in situ hybridization level. To investigate the neuroprotective effects of alpha lipoic acid and the possible underlying mechanisms against cerebral ischemia reperfusion injury. The blood brain barrier permeability of alpha lipoic acid, the level of MMP-2 and MMP-9, and water content of brain tissue were determined after drug administration.Methods The middle cerebral artery cerebral ischemia reperfusion injury animal model was made according to the protocol of Longa, sham operation group insert without filament. Healthy male SD rats were assigned randomly into three groups: the sham operation group(group A),ischemia-reperfusion group(model group, group B), alpha lipoic acid pretreatment group(group C). According to different reperfusion time was divided into 6h, 12 h, 24 h, 48 h 4 groups. Alpha lipoic acid was administered 30 minutes before reperfusion in accordance with the body mass of 20mg/kg by intraperitoneal injection,. Sham operation group and model group were given equal volume of normal saline by intraperitoneal injection. After 2h of cerebral ischemia reperfusion at each time point,we assess the neurological function score using standard Longa method, The volume of cerebral infarction were measured by image analysis software Scion Image. The apoptosis of neurons were detected by TUNEL technique. The level of NF-kappa B, TNF- alpha, MMP-2 and MMP-9 were detected by immunohistochemical method. The level of Bax m RNA, NF- kappa B m RNA, TNF-alpha m RNA were detected by situ hybridization method. The expression level of JNK3 protein were detected by Western Blot method.Results Compared with the sham operation group, in model group and alpha lipoic acid pretreatment group the expression of apoptosis gene Bax and JNK3 protein in m RNA were significantly increased at each time point(P < 0.05). Compared with the model group, neurological function deficit of alpha lipoic acid pretreatment group improved at 24 h, 48 h time point and the volume of cerebral infarction reduced at different time points(P < 0.05); it can also reduce the expression of apoptosis gene Bax m RNA at every time point including the expression of JNK3 protein at 24 h, 48 h timepoints(P< 0.05). Compared with the sham operation group, the expression of NF- kappa B increased at 6h timepoint after reperfusion in model group( P < 0.05), expression of TNF-α increased at 24h(P < 0.05), reached the peak at 3d(P < 0.05). IT was still higher than that in sham operationgroup at 7d(P < 0.05); Compared with the model group, the expression of NF-kappa B and TNF-α decreased at timepoint 24 h, 3d, 7d after cerebral ischemia reperfusion in alpha lipoic acid preintervention group. Comparison of NF- kappa B in alpha-lipoic acid pretreatment group there was no statistical difference( F = 2.245, P > 0.05). Compared with the model group, alpha lipoic acid pretreatment group significantly decreased infarct volume,neurological function deficit score decreased(P<0.05). Evans Blue content markedly increased in model group after cerebral ischemia reperfusion compared with the sham group. Compared with the model group, Evans Blue content decreased significantly in alpha lipoic acid pretrement group at each time point(P < 0.05). MMP-2 positive cells appeared in model group at 24 h after reperfusion, reached peak at 3d~7d. Compared with the model group, the number of MMP-2 positive cells at each time point was significantly lower than that in the alpha lipoic acid pretrement group. MMP-9 positive cells appeared at 12 h in model group,peaked at 2d, The MMP-9 positive cells number in alpha lipoic acid intervention group at each time point was significantly lower than that in the model group.Conclusion Alpha lipoic acid pretreatment can inhibit the expression of apoptosis regulating gene Bax and JNK3 protein, reduce neuron apoptosis after cerebral ischemia reperfusion in rats. Alpha lipoic acid can reduce cerebral infarction volume, improve neurological function. The anti-apoptotic mechanism may be related to inhibit the expression of Bax and MAPK-JNK3 signaling pathway. Inflammatory cytokine NF- kappa B and TNF- alpha highly express after focal cerebral ischemia reperfusion. Alpha lipoic acid pretreatment inhibits inflammatory cytokine expression after brain ischemia reperfusion in rats. Protective effect of the Alpha lipoic acid on cerebral ischemia reperfusion is probably by inhibiting NF-kappa B and TNF- alpha expression,thereby inhibiting the inflammatory reaction in rats with cerebral ischemia reperfusion and improving neurological function. The Evans Blue content in ischemia-reperfusionbrain tissue can reduce significantly in alpha lipoic acid pretreatment group. Alpha lipoic acid reduce the disruption of BBB, the brain water content, cerebral edema. Alpha lipoic acid has a protective effect on ischemic brain tissue. The mechanism is probably through decreasing the expression of MMP-2 and MMP-9, relieving inflammatory reaction. |
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