Protective Effects And Mechanisms Of Isoliquiritigenin On Cerebral Ischemia-reperfusion In Rats And Mice

Isoliquiritigenin (ISL), one of the components in the root of Glycyrrhiza glabra, is a member of the flavonoids, which has shown a wide range of therapeutic activities, such as vasorelaxant effect, antioxidant, anti-platelet, anti-tumor, anti-allergic, antiviral activities and estrogenic properties. In this thesis, the protective effects of ISL against cerebral ischemia-reperfusion injury and its mechanisms in rats and mice were investigated. This study included three parts as follows:1. An RP-HPLC (reversed-phase high performance liquid chromatography) method was established to determine the contents of adenine nucleotides in brain tissue samples. The levels of ATP, energy charge (EC) and total adenine nucleotides (TAN) of brain energy state were analyzed by RP-HPLC. The Hypersil C₁8 (4.6 mm × 250 mm, 5 μm) column was used, the mobile phase was 100 mmol · L^-1 KH₂PO₄ K₂HPO₄ buffer solution (pH 6.0), the flow rate was 0.7 ml · min^-1, the column temperature was 25℃, the detection wavelength was 254 nm. The linear range was 1.656 - 212.0 ug · ml^-1 (r = 0.9999) for ATP, 1.625 208.0 ug · ml^-1 (r = 0.9999) for ADP and 1.281 164.0 ug · ml^-1 (r = 0.9999) for AMP. The average recoveries (n = 6) were 101.3% (RSD = 1.0%) for ATP, 99.0% (RSD =1.4%) for ADP and 97.6% (RSD = 1.6%) for AMP. The results indicated that this analytical method is simple, sensitive, accurate and reliable.2. The protective effects of ISL against cerebral ischemia-reperfusion injury in mice were studied. The cerebral ischimia-reperfusion models in mice were made by repeated occlusion of bilateral common carotid arteries and reperfusion. To observe the hypoxia tolerance ability by means of recording the respiratory duration after cutting heads in repeated cerebral ischemia-reperfusion mice. The whole blood viscosity, hematocrit and blood caogulation time were measured. Step-down test and Y-maze task were used to examine the learning and memory abilities in mice. The brain energy state was analyzed by RP-HPLC. Pathological changes of brain tissues were studied. ISL 20, 40 mg · kg^-1 ig significantly prolonged the respiratory duration after head-cutting (P<0.01). ISL 20, 40 mg · kg^-1 ig significantly decreased the whole blood viscosity and hematocrit (P<0.01),prolonged the blood caogulation time (P<0.01). ISL (10⁴0 mg ? kg"1 ig) reduced the number of errors and extended the latency of memory in step-down test, decreased the number of cumulated stimulation and increased the number of right choices in Y-maze task. ISL (10 40 mg -kg"1 ig) improved the levels of ATP, EC and TAN of brain energy state in a dose-dependent manner. ISL mitigated pathological damage of neuron after cerebral ischemia-reperfusion in mice. In conclusion, ISL significantly improved cognition dysfunction, mitigated the abnormal hemorheological changes and improved brain energy metabolism in repeated cerebral ischemia-reperfusion mice. These results suggest that ISL has protective effects against cerebral ischemia-reperfusion injury in mice.3. The protective effects of ISL were investigated in middle cerebral artery occlusion (MCAO) induced focal cerebral ischemia-reperfusion injury in rats. Male Sprague-Dawley rats were subjected to 2 h right MCAO via the intraluminal filament technique and 22 h reperfusion. In this study, the neurological deficit score, infarct size, cerebral water content, brain Na+K+ATPase activity, malondialdehyde (MDA) content and antioxidant enzymes activities were measured. ISL (5-²0 mg ? kg'1 ig)significantly reduced the infarct size and water content, and produced significant reduction in neurological deficits. ISL 10, 20 mg ? kg'1 ig significantly protected brain Na+K+ATPase activity (PO.01). ISL 10, 20 mg ? kg"1 ig significantly inhibited the increases of brain MDA content and prevented the activities of brain superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) from declines caused by cerebral ischemia-reperfusion (PO.01). All these findings suggest that ISL has the protective potential against focal ischemia-reperfusion injury in rats. Its protective effects may be related to inhibition of lipid peroxidation, protection of Na+K+ATPase and modulation of endogenous antioxidant enzyme activities.