The Effects Of Total Flavones Of Rhododendra On Rat Hippocampal Neuron Induced By Anoxia-reoxygenation And Its Action On BK_Ca Channel

Objective:To observe the protective effect of total flavones of rhododendra (TFR) againstprimary cultured hippocampal neuronal injury induced by anoxia-reoxygenation, andthe role of large conductance calcium-activated potassium channels (BKCa) in thiseffect.Methods:1. After the primary hippocampal neurons were cultured for6~8days, theimmunofluorescence of microtubule associated protein-2(MAP-2) was used to identifyneurons.2. The cultured primary hippocampal neurons were randomly divided into normalgroup, model group and TFR preconditioning group with different concentration. Thehippocampal neurons were exposed to anoxia environment (cultured in94%N2+5%CO2+1%O2) then reoxygenated for24h (cultured in aerobic environment).（1）At24h after reoxygenation, neuronal viability was assessed by MTTreduction assays, a spectrophotometric method.（2）At24h after reoxygenation, the content of malondialdehyde (MDA) andactivities of lactate dehydrogenase (LDH) and neuronspecific enolase (NSE) in culturesupernate were detected by spectrophotometric methods.（3）The apoptosis rate of hippocampal neurons after anoxia-reoxygenation wasidentified morphologically with Hoechst33258dye by fluorescence microscope. （4）The BKCachannels protein expression were detected by Western blot analysison hippocampal neurons after anoxia-reoxygenation.3. The BKCachannel currents were recorded using the whole-cell patch clampconfiguration on cultured hippocampal neurons in rats, and effect of different doses ofTFR on BKCachannel was observed.Results:1. Following immuno-fluorescence, MAP-2protein that is selectively distributed indendrites and cell bodies, was present in cultured hippocampal neuron. This resultdemonstrate that the cultured hippocampal cell is hippocampal neuron.2. The analysis of cell viability by the MTT method revealed that the absorbance ofhippocampal neuron was decreased significantly after anoxia-reoxygenation. TFRshowed a neuronal protective effect when used in the culture medium. TFR protectedneuronal death in a concentration-dependent manner, when3.7~300mg/L TFR wereapplied, compared with the model group.3. The LDH and NSE activity and the content of MDA in supernate were increasedsignificantly at24h after reoxygenation. TFR (3.7,11.1,33.3,100,300mg/L) significantlyinhibited the increases of LDH and NSE activity and the content of MDA whencompared to the model group.4. At24h after reoxygenation, the nuclei of cultured neuron was stained withHoechst33258. Exposure of neuronal cultures to anoxia-reoxygenation significantlyinduce neuronal apoptosis. Application of TFR (33.3,100,300mg/L) could significantlyinhibit the percent of apoptotic nuclei on hippocampal neurons.5. After reoxygention the protein expression of BKCachannels did not change, andTFR (100,300mg/L) has no significant effect on the protein expression of BKCaon thehippocampal neurons.6. The current of BKCachannels were recorded with whole-cell patch clamp oncultured hippocampal neurons, which was outward current and characteristic of high amplitude, quick activation and almost never inactivation. The current could be blockedby IBTX (100nmol/L), a specific BKCachannel blocker. The current of BKCachannelswere increased significantly by TFR in a concentration-dependent manner at the dosesof33.3~300mg/L. Moreover, the enhancement of the current of BKCachannels by TFRcould be blocked by IBTX (100nmol/L).Conclusions:1. TFR has a certain protective effect on primary cultured hippocampal neuronalinjury induced by anoxia-reoxygenation.2. TFR can increase the open probability of BKCachannel, and this might be one ofthe possible mechanisms which TFR have protective effect on brain ischemic injury.