Protection of Cactus Polysaccharide against H_2O_2-induced damage in the rat cerebral cortex and hippocampus Differences in time of administration

BACKGROUND： Pharmacological research has shown that cactus polysaccharide （CP） has anti-oxidant, anti-inflammatory, anti-tumor, anti-aging, and immune-stimulating activities. It may also provide protective effects against oxidative stress injuries in the rat brain. OBJECTIVE： To validate the effects of CP on H2O2-induced oxidative stress injuries in the rat cerebral cortex and hippocampal slices 30 minutes prior to injury, as well as 30 minutes and 2.5 hours after injury. DESIGN： A randomized controlled experiment. SETTINGS： Department of Pharmacology, Tongji Medical College, Huazhong University of Science and Technology; Department of Pharmacology, College of Medical Science, Yangtze University. MATERIALS： A total of 50 male Sprague Dawley （SD） rats, normal grade and weighing 200 300 g, were provided by the Laboratory Animal Center of Tongji Medical College, Huazhong University of Science and Technology. The protocol was performed in accordance with ethical guidelines for the use and care of animals. Cactus polysaccharide, a dried needle crystal, was extracted from Opuntia milpa alta at the Chemistry and Environment Engineering School of Yangtze University. The following chemicals and instruments were used： 2,3,5-triphenyl tetrazolium chloride （Sigma, St Louis, Missouri, USA）; lactate dehydrogenase （LDH）, superoxide dismutase （SOD）, glutathione （GSH）, and total antioxidant competence （T-AOC） assays （Jiancheng-Bioeng Institute, Nanjing）; McIllwain tissue chopper （Mickle Laboratory Engineering, USA）; and ELISA reader and Magellan software （TECAN, Austria）. METHODS： This experiment was performed at the Department of Pharmacology, Medical College of Yangtze University, between March and June 2006. All rats were sacrificed after anesthesia. The cerebral cortex and hippocampus were dissected. Several cerebral cortex and hippocampus slices were selected as controls, while other sections were co-incubated with H2O2 for 30 minutes to induce an oxidative stress injury. The experimental slices were randomly divided into 3 groups： model group, low-dose group, and high-dose group. Slices from the low- and high-dose group were incubated with CP 30 minutes prior to injury, as well as 30 minutes and 2.5 hours after injury. The number of cerebral cortex and hippocampus slices at each time point was greater than 10 and 7, respectively. The control group slices were incubated with artificial cerebrospinal fluid （aCSF） for 3 hours. The model group slices were incubated with aCSF for 30 minutes, followed by 30 minutes of aCSF with H2O2 incubation to establish the oxidative stress injury model. Thereafter, the slices were re-incubated with aCSF for 2 hours. The slices in the low- and high-dose group were co-incubated with 0.33 mg/L CP or 1.67 mg/L CP, respectively. CP was applied either for 30 minutes prior to the H2O2 treatment, co-incubated with H2O2 for 30 minutes, or applied 2 hours after H2O2 treatment. MAIN OUTCOME MEASURES： Brain slice activity was determined by TTC staining. Biochemical markers, such as LDH, SOD, GSH, and T-AOC, were analyzed in clear supernatant liquid to study the pharmacological mechanisms. RESULTS： （1） Effects of CP on H2O2-injured brain slices： TTC absorption level at 490 nm decreased with a 30-minute CP administration before and after injury. The TTC absorption values in the low- and high-dose group were significantly lower than the control group （P 〈 0.05 - 0.01 ）. CP administration prior to insult had greater neuroprotective effects on brain slices than administration during the insult. CP Administration after insult had no obvious protective effects on the brain slices. There was no significant difference in TTC absorption levels between administration after 2.5 hours and the model group （P 〉 0.05）. （g） Effects of CP on substances released from H2O2-induced injury slices were as follows： following incubation with 2 mmol/L H2O2 for 30 minutes, LDH release was significantly increased from the rat cortical and hippocampal brain slices. T-AOC and GSH were decreased, which is in accordance with the TTC assay for evaluating the degree of slice injury. Prior administration of 0.33 mg/L CP and 1.67 mg/L CP decreased LDH release and enhanced T-AOC and GSH release in a dose-dependent manner. After H2O2-induced damage for 30 minutes, the release of SOD increased to some extent. SOD levels were further raised by pre-incubation with 0.33 mg/L CP or 1.67 mg/L CP, compared to the model group （P 〈 0.05）. CONCLUSION： CP can protect rat cerebral cortex and hippocampal slices from H2O2-induced injury, This protective effect is dose-dependent. CP administration before injury is more effective than during or after injury.