The Time Course Expression Of 12/15-LOX, P38MAPK And CPLA2 And The Neuroprotective Effect Of Atorvastatin In The Acute Phase Of Cerebral Ischemia In Rats

Objective: Cerebral ischemia is one of the three leading causes of death in the world, with high rate of morbidity, disability and mortality. Brain tissue injuries secondary to ischemia aggravated the illness and inhibited the recovery of patients. Despite advances in the understanding of the pathophysiology of cerebral ischemia in recent years, therapeutic options remain limited. Although thrombolytic therapy with drugs in the acute phase is approved the most definite method for treatment of this disease, its use is still limited by the short therapeutic window, complications derived from the risk of hemorrhage and the potential damage from reperfusion/ischemic injury. Oxidative stress and inflammation are proved to be two important pathophysiologic mechanisms of ischemic stroke. Oxidative stress and inflammation-related genes are dramatically upregulated in neurons after ischemia, providing a new promising treatment strategy. Arachidonic acid(AA)metabolism is one of classical oxidative stress and inflammatory function ways. The signal pathway of mitogen-activated protein kinase(MAPK)mediated by12/15-lipoxygenase(12/15-LOX)is one of the major ways of AA metabolism. LOXs derivatives from AA, such as, hydroxy/hydroperoxyeicosatetraenoic acids(HETE)and leukotrienes(LTs), contribute to the pathophysiologic progress of cerebral ischemia. Atorvastatin is an effective lipid lowering medicine in clinical practice. Increasing evidences have shown that atorvastatin have pleiotropic protective actions that are independent of lipid lowering effect, such as, anti-inflammatory, antioxidant and antitoxic effects. Pre-treatment with atorvastatin can reduce infarct volume and improve neurological deficit in mouse models of cerebral ischemia. Clinical studies have observed that chronic treatment with atorvastatin offers patients a better medium-term clinical evolution. This study is to evaluate the time course expression of 12/15-LOX, p38 mitogen-activated protein kinase(p38 MAPK)and cytosolic phospholipase A2(cPLA2)and the neuroprotective effect of atorvastatin in the acute phase of cerebral ischemia in rats.Methods: Male and healthy Sprague-Dawley rats were subjected to modified permanent middle cerebral artery occlusion(MCAO), as described by Longa previously. Experiment 1 was used to evaluate time course expression of 12/15-LOX, p38MAPK, phosphorylated-p38MAPK(phospho- p38MAPK)and cPLA2 after cerebral ischemia, including normal control group, 3 h, 6 h, 12 h, 24 h, 48 h and 72 h six time points. Experiment 2 was used to detect atorvastatin's neuro-protection in the acute phase of cerebral ischemia. Rats were randomly assigned to four groups: Sham operated group(Sham), MCAO group, Low dose group(MCAO + atorvastatin 10 mg/kg)and High dose group(MCAO + atorvastatin 20 mg/kg). Atorvastatin was administered immediately after MCAO. At 24 h after ischemia neurological deficit was evaluated, brain water content was measured by wet-dry method, infarct size were analyzed with 2, 3, 5- triphenyltetrazolium chloride(TTC)staining. Immunohistochemistry, reverse transcription–polymerase chain reaction(RT-PCR)and western blot were used to analyze the expression of 12/15-LOX, p38MAPK, phospho-p38MAPK and cPLA2. Experiment 3 was used to detect atorvastatin's influence on blood brain barrier(BBB). Rats in this part were assigned to four groups as the same way of experiment 2. Confocal microscope was used to observe the extravascular IgG. Western blot and RT-PCR were used to detect the expression of claudin-5.Results:1 After cerebral ischemia the expression of p38MAPK,p- p38MAPK and cPLA2 was increased at 3 h, reached the peak at 48 h and decreased at 72 h.2 After cerebral ischemia the expression of 12/15-LOX was increased at 12 h, reached the peak at 48 h and decreased at 72 h.3 Rats in MCAO group, high dose group and low dose group performed a right palsy. Neurological deficit score in high dose group was decreased compared with MCAO group(P < 0.05). There was no significant difference in the neurological deficit score between MCAO group and low dose group.4 Both the two doses of atorvastatin decreased the percentage of brain water content in ipsilateral hemispheres after stroke(P < 0.05). Compared with low dose group, high dose group showed more intense decline in the brain water content(P < 0.05).5 Extensive lesion was developed in both striatum and lateral cortex in MCAO group. High dose of atorvastatin significantly reduced the infarct volume after MCAO(P < 0.05). However, there was no significant difference in infarct volume between MACO group and low dose group(P > 0.05).6 High dose of atorvastatin decreased the expression of 12/15-LOX, p38MAPK, p-p38MAPK and cPLA2 after MCAO(P < 0.05). However, this reduction is not in low dose group.7 In sham operated group there was hardly any extravascular IgG in cortex. In MCAO group and low dose group, a great deal of IgG leaked to brain tissue from cerebral circulation. Extravascular IgG were reduecd in high dose group.8 Compared with sham operated group, MCAO induced sharply reduction of claudin-5(P < 0.05). After treatment with high dose of atorvastatin, the expression of claudin-5 in cerebral ischemia was significantly up-regulated(P < 0.05). However, the up-regulation of claudin-5 was not observed in low dose group(P > 0.05).Conclusions: 12/15-LOX, p38MAPK, phospho-p38MAPK and cPLA2 were up-regulated at early stage after cerebral ischemia. Treatment with atorvastatin after stroke could decrease the infarct size and the brain edema. Those effects may be through down-egulation of 12/15-LOX, p38MAPK, phospho-p38MAPK and cPLA2, up-regulation of claudin-5.