The Protective Effects And Mechanisims Of Flurbiprofen Axetil On The Injury Of Cerebral Ischemia-Reperfusion In Rats

Background and objective Ischemic cerebrovascular disease is a neurological disorder caused by insufficient blood supply to a particular brain area and has become a common clinically pathological sypmptom. Ischemia and reperfusion (I/R) occurs in a wide range of clinical settings, including trauma, cardiac arrest, cardiac surgery, thrombolysis treatment, organ transplantation and hypovolemic shock with resuscitation. Experimental data obtained from animal models of middle cerebral artery occlusion indicates that inflammation plays a vital role in the pathogenesis of cerebral ischemia and secondary damage. Inflammatory cytokines such as tumor necrosis factorα(TNF-a) and interleukin-1β(IL-1β) as well as adhesion molecules which are produced at the early stage of ischemia promote both the recruitment of neutrophil, their adherence to brain endothelial cells and the resultant activation of inflammatory processes. In various animal models of MCAO, increased neutrophil accumulation has been detected in the microvessels and ischemic cerebral parenchyma,and the hippocampus is one of the most sensitive to ischemia/reperfusion injury in rats. Nuclear factor-KB (NF-κB) is one of the most important transcription factors activated after cerebral ischemia. NF-κB is involved in inflammatory responses that potentiate ischemic injury activating many genes involved in the pathogenesis of cerebral ischemia, such as iNOS, IL-1β, TNF-α, ICAM-1, COX-2, and IL-6. Consequently anti-inflammatory therapies may be of benefit to ischemic cerebrovascular disease outcome.Apoptosis is one of the major mechanisms that lead to cell death after cerebral ischemia and reperfusion. Ischemia followed by restoration of blood flow exacerbates neuronal apoptosis in the cortex and hippocampus. Cerebral ischemia reperfusion injury could induce apoptosis. A large number of TUNEL-positive cells and the alteration of Bax/Bcl-2 protein ratios were observed 24h after reperfusion. It has been suggested that the balance between the protein levels of anti-apoptotic Bcl-2 and pro-apoptotic Bax played an important role in regulating apoptotic cell death. Moreover, Akt activation promotes cell survival by phosphorylation and subsequent inactivation of apoptosis-inducing factors, including the Bcl-2 family member and glycogen synthase kinase GSK-3β.The increased COX-2 activity may contribute to neuro-degeneration by either oxidative stress, or the neurotoxic effect of prostaglandins such as PGA1 and PGE1. Therefore, some studies suggest that NSAIDS might be neural protective in cerebral ischemic conditions. In animal models of stroke, ibuprofen reduces neuronal injury and improves cerebral blood flow and neurological outcome in global ischemia and decreases infarct size in focal ischemia. The COX inhibitor flurbiprofen, which utilizes a lipid microsphere drug delivery system, may facilitate effective target therapy. In addition, the lipid microsphere easily permeates cell membrane and greatly promotes the absorption of the drug and results in shortened onset time. Their primary mechanism of action is through the inhibition of COX activity in the arachidonic acid cascade which in turn inhibits the biosynthesis of prostaglandin (PG), which is involved in causing inflammation of cerebral ischemia/reperfusion injury. The aim of this study was to investigate the protective effects and mechanisims of flurbiprofen axetil on the injury of cerebral ischemia-reperfusion in rats from apoptosis and inflammation.Methods Healthy clean male Wistar rats weighing 260-320 g were randomLy divided into five groups:sham group (group S), ischemia/reperfusion (I/R) group (group I/R),5 mg/kg,10 mg/kg dose of flurbiprofen axetil groups (group FA-L and group FA-H) and 1 mL/kg lipo-microballoons group (group V). All drags were administered via tail-vein injection at the onset of reperfusion. Focal cerebral ischemia/reperfusion model was conducted by occluding left middle cerebral artery for 2 hours followed by 24 hours of reperfusion. There are four parts in this study:1. The suture method was used to block the middle cerebral artery for 120 mins, and then a reperfusion for 24 hours was performed in rats. We evaluated the influence of flurbiprofen on cerebral infarction volume, neurological outcome and pathological issue change following focal cerebral ischemia-reperfusion (I/R) injury in rats. The hematoxylin-eosin (HE) staining was used to observe pathological changes of ischemic hippocampus. Zea-Longa method was used to assessment the neurological disfunction scores. Triphenyltetrazolium chloride (TTC) staining was used to measure cerebral infarction size.2. To investigate the effects of flurbiprofen on myeloperoxidase (MPO) activity and cytokines (IL-1β, TNF-α, IL-6) gene expression in ischemic hippocampus following focal cerebral ischemia-reperfusion (I/R) injury in rats.10% hippocampus homogenate was prepared to analyze MPO activity by spectrophotometry. Pro inflammation cytokine TNF-α, IL-1βand IL-6 were analyzed by RT-PCR.3. To investigate the effects of flurbiprofen on NF-κB activity and the level of phospho-P38 in ischemic hippocampus following focal cerebral ischemia-reperfusion (I/R) injury in rats. The expression of NF-κB (p65) in ischemic hippocampus was detected by immunohistochemistry assay and western blot assay. Western blot was used to determine the phospho-P38.4. The present study aimed to show that administration of flurbiprofen may provide neuroprotection in focal cerebral ischemia-reperfusion injury and this neuroprotection is associated with inhibition of apoptosis mediated by activation of the Akt/GSK-3b pathway. The number of apoptotic neurons in the ischemic penumbra region was counted using TUNEL. Western blot was used to test the level of Bcl-2, Bax, phospho-Akt and phospho-GSK3βin the ischemic penumbra.Results1. The neurologic deficit scores were significantly higher in both group I/R and group V than in group S and group FA 24 hours after reperfusion, and significant differences were found in neurological deficits between the 5 mg·kg-1 and 10 mg·kg-1 groups (P<0.05). No great differences were found in neurological deficits between group I/R and group V (P>0.05). The infarct volumes in group FA were significantly smaller than those of group I/R and group V (P<0.05). Furthermore, there was a significant difference in infarct volume between 5 mg·kg-1 and 10 mg·kg-1 groups (P<0.05).The results of HE staining showed that the ischemic damage of the group I/R and group V was great and the group S and group FA had a light pathological damage.2. The mRNA levels of the inflammatory cytokines (TNF-α, IL-6,IL-1β) and MPO activity were greatly increased in group I/R and group V compared with the group S (P<0.05). However, treatment with flurbiprofen in doses of 5 mg·kg-1 and 10 mg·kg-1 significantly relieved the increase of these variables induced by MCAO. Furthermore, significant differences were found in these variables between group FA-L and group FA-H (P< 0.05).3. The expression of NF-κB (p65) in ischemic hippocampus was detected by immunohistochemistry assay and western blot assay. NF-κB (p65) was predominantly located in the nucleus in group I/R and group V and flurbiprofen can significantly inhibit its translocation from cytoplasm into the nucleus (P<0.05). Furthermore, through western blot assay of nuclear extract, the level of p-NF-KB (p65) in group I/R and group V was markedly higher than that in the sham and flurbiprofen groups (P<0.05).4. TUNEL positive cells and expression of Bax were significantly higher and expression of Bcl-2 were significantly lower in the ischemic penumbra in group I/R and group V than in group S at 24 h after reperfusion (P<0.05).However, TUNEL positive cells and expression of Bax were significantly lower and expression of Bcl-2 in the ischemic penumbra were significantly higher in group FA than in group I/R and group V (P<0.05). The present study showed that phospho-Akt level in ischemic penumbra was increased significantly after administration of flurbiprofen, whereas the level of phospho-GSK3βsignificantly decreased compared with compared with group I/R and group V(P<0.05). No significant differences in the levels of phospho-Akt and phospho-GSK3P were found between flurbiprofen 5 and 10 mg/kg groups (P>0.05). Conclosion1. The modified suture is reliable in making MCAO model in rats. Flurbiprofen could not only reduce infarct volume and pathological damage but also improve the neurological outcome in the rat model after cerebral ischemia.2. Flurbiprofen can inhibit the inflammatory response in the hippocampus of rats exposed to cerebral ischemia/reperfusion by limiting the increase of MPO activity and cytokines gene expression.3. Flurbiprofen can significantly inhibit NF-κB (p65) translocation from cytoplasm into the nucleus and reduce the levelof p-NF-KB (p65) and p-P38 protein in the hippocampus of rats.4. Flurbiprofen protected against cerebral ischemia/reperfusion injury by reducing apoptosis via up-regulation of Bcl-2 expression and down-regulation of Bax expression in rats. These effects may be partly due to the activation of Akt/GSK-3βsignaling pathway.