Protective Effects Of 1β-hydroxyalantolactone On Cerebral Ischemia And Reperfusion Injury

Objective: Stroke has become one of the major causes of death and disability worldwide. With the complex factors of society, the incidence of stroke is rising. To restore blood flow is the key way to treat stroke clinically, but it will also lead to reperfusion injury at the same time. Ischemia induces many inflammatory responses during cerebral ischemia-reperfusion and it results in microvascular function disorders to damage the brain tissue further. Since the pathogenesis of cerebral ischemia is complex, the specific mechanism remains unclear and there is short of useful drug to control ischemia clinically. To find a series of new drugs to cure stroke and to clarify the mechanism of the action on cardiovascular and cerebrovascular disease has become a hotspot worldwide. Inula lineariifolia, a traditional Chinese medicine used clinical.1β-hydroxyalantolactone is a new compound and extracted from Inula lineariifolia. The protective role and its mechanism of 1β-hydroxyalantolactone on cerebral ischemiareperfusion are studied in the paper.Methods: 1. Animal model: Middle cerebral artery occlusion(MCAO) model was made during the experiments. The model was brain ischemia for 2h and reperfusion for24 h.2. Rats or mice were divided into four groups: sham group, model group, low dose group of 1β-hydroxyalantolactone(25mg/kg) and high dose group of 1β-hydroxyalantolactone(50mg/kg).3. Administration method: Rats or mice were intragastric administrated with 1β-hydroxyalantolactone when MCAO model was made, and dosed twice after reperfusion for 8h.4. Detection index:4.1 Detection index on protective effects of 1β-hydroxyalantolactone in cerebral ischemia and reperfusion injury.When the rats or mice survived after brain ischemia for 2h and perfusion 24 h, the changes of ethology scores, content of water in brain tissue and cerebral infarction volumes were measured, HE staining and transmission electron microscope were used to detect the pathological and ultrastructure changes of ischemia tissues and TUNEL staining was used to detect apoptotic cells in rat brain tissues.4.2 Detection index on protective mechanism of 1β-hydroxyalantolactone in cerebral ischemia and reperfusion injury.(1)The expression of Peroxiredoxin1 and Peroxiredoxin5 were measured by Real-time PCR, Western Blot and TUNEL staining in each group.(2) The expression of IL-1β 、 TNF-α 、 IFN-γ 、 IL-6 、 ICAM-1 and MMP-9 were measured by Real-time PCR in each group.Results: 1. Results about protective effects of 1β-hydroxyalantolactone in cerebral ischemia and reperfusion injury.1.1 In the low dose group and high dose of 1β-hydroxyalantolactone, the ethology scores of MCAO rats were reduced from 2.5 to 1.8(p<0.05) and 1.4(p<0.01)respectively; water content of brain of MCAO rats were reduced from 83.52% to82.15%(p<0.05) and 80.89%(p<0.01) respectively and cerebral infarction areas of MCAO rats were reduced from 31.11% to 23.43%(p<0.05) and 19.79%(p<0.01)respectively. HE staining showed that 1β-hydroxyalantolactone could reduce the pyknosis and dissolution of nerve cells, decrease the edema of intercellular substance,relieve the softening lesion, lower the infiltrate of neutrophile granulocyte, shrink the necrotic tissues in a dose-dependent manner. Results of transmission electron microscope showed that 1β-hydroxyalantolactone reduced the brain injury when compared with model group, reduced the endoplasmic reticulum expansion, prevented the glial cytoplasmic from falling off and decreased the aggregation of nuclear chromatin. It is suggested that 1β-hydroxyalantolactone had protective effects on cerebral ischemia and reperfusion injury.1.2 In the low dose group and high dose of 1β-hydroxyalantolactone, the ethology scores of MCAO mice wrer reduced, from 2.71 to 2.14(p<0.05) and 1.57(p<0.01)respectively; water content of brain of MCAO mice were reduced from 79.61% to78.52%(p<0.05) and 77.92%(p<0.01) and cerebral infarction areas of MCAO mice were reduced from 30.70% to 23.93 and 21.90%(p<0.05). HE staining showed that 1β-hydroxyalantolactone reduced the pyknosis and dissolution of cortical pyramidal cell and parenchymal nerve cell, decreased the edema of intercellular substance, shrinked the necrotic tissues. Results of transmission electron microscope showed that the synaptic cleft become visible, endoplasmic reticulum get rich,cells become tight and junction in 1β-hydroxyalantolactone group. It is suggested that 1β-hydroxyalantolactone had protective effects on cerebral ischemia and reperfusion injury.1.3 TUNEL staining showed that apoptosis increased in model group, while apoptosis was significantly reduced in a dose-dependent manner after administrated with 1β-hydroxyalantolactone. It showed that 1β-hydroxyalantolactone reduced cerebral ischemia-reperfusion injury.2. Results about protective mechanisms of 1β-hydroxyalantolactone in cerebral ischemia and reperfusion injury.2.1 Immunofluorescence staining showed that the expression of Prx1, Prx5 and apoptotic cells increased in MCAO group. The results of immunofluorescent localization between Prx and apopotosis cells showed that damaged cells released Prx1 and Prx5 into intercellular substance Prx1 and Prx5 were reduced in 1β-hydroxyalantolactone group when compared with the model group(p < 0.05).Compared with the sham group, the expression of Prx1 and Prx5 was higher in the model group(p<0.05) in m RNA and protein level, while they were decreased after administrated with 1β-hydroxyalantolactone(p < 0.05). It is suggested that 1β-hydroxyalantolactone reduced the expression of Prx1 and Prx5 in intercellular space to decrease the damage of brain.2.2 Compared with the sham group, the expression of IL-1β、TNF-α、IFN-γ、IL-6、ICAM-1 and MMP-9 were higher in the model group(p < 0.01), while they were reduced when administrated with 1β-hydroxyalantolactone(p < 0.05). It showed that1β-hydroxyalantolactone exerted protective effects via decreasing the expression of inflammation cytokines(IL-1β、TNF-α、IFN-γ and IL-6), adhesion molecule(ICAM-1)and matrix metalloproteinases(MMP-9).Conclusions: 1. 1β-hydroxyalantolactone has protective effect in cerebral ischemiareperfusion injury.2. 1β-hydroxyalantolactone exerts protective effects iby decreasing the level of Prx1 and Prx5, reducing the expression of inflammatory cytokines( IL-1β、TNF-α、IFN-γand IL-6)、adhension molecules(ICAM-1) and matrix metalloproteinases(MMP-9).