The Neuroprotective Effects Of Catalpol Pretreatment On Cerebral Ischemia/Reperfusion Injury

Background: To date there is still no effective drug that provides neuroprotection for cerebral ischemia/reperfusion (CI/R) injury. It is necessary to seek suitable neuroprotective drugs on ischemia-reperfusion, and clarify its mechanism. Recent studies have focused on the natural products that may ameliorate the brain insults due to ischemia-reperfusion. Catalpol is the main active component of Radix rehmanniae, studies have shown that catalpol has a neuroprotective effect, but the mechanism has not been fully elucidated.Objective:Neurobehavioral abilities were measured by strok index and Morris water maze to evaluate the neuroprotection effects of catalpol pretreatment. The neuroprotective mechanism of catalpol pretreatment on cerebral ischemia injury was explored by in vivo and in vitro experiment.Materials and methods:1. Neurobehavioral evaluation1.1 Gerbil model of CI/R was prepared by bilateral common carotid occlusion for 10 min followed by 6-hour reperfusion. Groups of gerbils (n= 10) were designed as sham-operated, vehicle-treated CI/R (CI/R control), catalpol-treated CI/R (5 mg/kg- C5,10 mg/kg-C10 and 20 mg/kg-C20, respectively). Catalpol treatment groups were injected intraperitoneally with catalpol daily,3 days and 30 min before operation. The neurologic deficit score was measured each hour for 6 h after CI/R, and they were calculated as the stroke index to evaluate the morbidity after reperfusion.1.2 Sprague-Dawley rat model of cerebral hypoperfusion (CH) was prepared by permanent occlusion of bilateral common carotid arteries. Groups were same above. Catalpol treatment groups were injected intraperitoneally with catalpol daily 3 days before operation and in 2 weeks after operation. The performance of rats,2 weeks after operation, was investigated by Morris water maze in order to evaluate the changes of learning and memory abilities.2. In vivoThe Superoxide dismutase (SOD) activity was measured using the Xanthine Oxidase method. The malondialdehyde (MDA) content was detected by the thiobarbituric acid method. The endothelin (ET) and calcitonin gene-related peptide (CGRP) contents in plasma were evaluated by radioimmunoassay method. The ATPase activity was measured using the phosphorus determination method. The glutamate (Glu) and aspirate (Asp) contents were determined using high-performance liquid chromatography method. The TNF-a and IL-1β contents were examined using ELISA kits.3. In vitroAstrocyte model of OGD/R was prepared by oxygen-glucose deprivation for 3-h followed by 24-hour reperfusion. Then astrocytes were divided into 5 groups, namely normal, model, catalpol low, medium and high dose groups (25,50 and 100μmol/l, respectively). Cell viability, SOD activity, MDA level and ATPase activity of OGD/R astrocytes, and LDH activity in OGD/R astrocytes medium were detected.Results:1 Neurobehavioral evaluation1.1 The CI/R model gerbils showed a significantly increased stroke index compared with sham-operated gerbils (P<0.01). Catalpol treament (C5、C10 and C20 groups) significantly improved the stroke index compared with CI/R control group (P<0.05).1.2 The model rats showed a significantly increased latency in the Morris water maze task compared with sham-operated rats (P<0.01). It was indicated that the spatial learning and memory abilities of CH rats were impaired significantly. CH rats administered catalpol showed significantly improved latency in Morris water maze compared to CH groups (P<0.05). It was suggested that catalpol treatment ameliorated the memory deficits of CH rats.2 In vivo2.1 Determination of SOD activity and MDA content in brain homogenateThe CI/R model gerbils showed a decreased SOD activity and increased MDA content compared with sham-operated gerbils (P<0.01). Each catalpol treatment group showed increased SOD activity, and decreased MDA content compared with CI/R group (P< 0.05). Catalpol treatment significantly increased at the doses of 10 and 20 mg/kg(P<0.01).2.2 Determination of ET and CGRP contents in plasmaThe CI/R model gerbils showed increased ET content (P＜0.01) and decreased CGRP content (P<0.05) compared with sham-operated gerbils. Each catalpol treatment group showed significantly decreased ET content (P<0.05), and had the tendency of increased CGRP content, but there was no significant difference with CI/R group (P>0.05).2.3 Determination of ATPase activityThe CI/R model gerbils showed a decreased ATPase activity compared with sham-operated gerbils (P<0.01). Catalpol markedly increased the activities of Na+-K+-ATPase at the doses of 10 and 20 mg/kg compared with CI/R group (P< 0.05). Each catalpol treatment group showed increased Ca2+-ATPase activity compared with CI/R group (P< 0.05). Each catalpol treatment group had no effects on Mg2+-ATPase (P>0.05).2.4 Determination of EAA contentsThe CI/R model gerbils showed increased Glu content (P＜0.01) and Asp content (P<0.05) compared with sham-operated gerbils. Each catalpol treatment group showed significantly decreased Glu content (P<0.05), however had no effects on Asp content (P>0.05).2.5 Determination of the brain TNF-a and IL-1 p levelsThe CI/R model gerbils showed increased TNF-a and IL-1β levels (P＜0.01) compared with sham-operated gerbils. The brain TNF-a and IL-1β levels in catalpol groups were significantly lower than the CI/R model group (P< 0.05). It suggeted that catalpol evidently inhibited the NF-κB and ICAM-1 activities in brain tissue of CI/R gerbils.3. In vitroCatalpol significantly increased the cell viability (P< 0.05), SOD activity (P< 0.01) and ATPase activity (P< 0.05) of cortical astrocytes subjected to OGD/R injury, compared to the model group. LDH activity in OGD/R astrocytes medium of catalpol groups was significantly lower than that of model group (P< 0.05). Catalpol evidently decreased MDA level of OGD/R astrocytes (P< 0.05).Conclusions:1. It was suggested that catalpol pretreatment significantly improved stroke index. Catalpol had evidently effect on CI/R gerbil.2. Catalpol pretreatment has an ameliorating effect on rats with spatial memory deficits caused by cerebral hypoperfusion.3. It was suggested that the efficacy of catalpol pretreatment on CI/R injury may be attributed to enhancement of SOD activities and inhibition of lipid peroxidation, enhancement of ATPase activities, decreased ET production and excitatory amino acid (EAA) toxicity, as well as inhibition of inflammatory response in brain tissue.4. The effectiveness of catalpol pretreatment in cortical astrocytes subjected to OGD/R injury appears to be associated with the improvement of cellular anti-oxidative ability and energy metabolism.