Electroacupuncture And NBD Peptides Regulate Ischemic Region C-rel Expression In Rat Model Of Focal Cerebral Ischemia/Reperfusion

BackgroudThere are many effective methods to treat clinical cerebral ischemia,including improve the cerebral circulation, protect the cerebral,anti-encephaledema, reduce intracranial pressure and so on. Whilethrombolytic therapy was considered to be the most efficient measure inmoderm times of treating cerebral ischemia. Within time window,thrombolytic/reperfusion can rescure the ischemia penumbra. But theexistence of ischemia/reperfusion (I/R) injury limits the application ofthrombolytic therapy in clinical. So how to improve the effect ofischemia/reperfusion has be the matter of most concern. Inflammation runthrough the period after I/R as an important kind of pathological process,whose form is associated with secondary injury of nerve, vascularendothelial cell (VEC) and blood brain barrier (BBB). It exists a seriousof brain tissue damage cell death process and complex pathological mechanism and so on. Nuclear factor κB (NF-κB) family is important inregulating the immuno-inflammatory response after I/R and c-rel is onemember of this family. The I/R can upregulate the expression of c-relprotein, someone scholars considers the upregulated c-rel may protectcerebral, some other scholars thinks the upregulated c-rel andoveractivation of c-rel may aggravate the injuty of inflammation. So furtherexploration of the influnce c-rel protein to inflammation after I/R isnecessary. NEMO Binding Domain(NBD) peptide is a kind of specificinhibitor of NF-κB whose permeability is accepted. It can prevent IκappaBkinase (IKK) to degradation IκappaB (IκB) so that to restrain the activationof NF-κB subunits. NBD peptide can reduce the injury of cerebralhemisphere after I/R in rats. The selective protection of NBD peptide isavailable to explor the function of c-rel in inflammation after I/R. OurTraditional Chinese Medicine--acupuncture has its definite curative effectto the brain stroke. Animal experiments show that acupuncture can confrontinjuty of brain ischemia, improve the blood circalation of brain ischemiaregion, protect nerve cell, better the electrical activity of nerve cell and soon. Electro acupuncture (EA) can regulate the inflammation after cerebralI/R though control the NF-кB.Objective1. To investigate the expression of c-rel cyteblast and cytoplasm indifferent time point of inflammation after focal cerebral I/R and its effect to inflammation;2. To investigate the mechanism of NBD peptides affecting the c-relexpression of cyteblast and cytoplasm in different time point ofinflammation after focal cerebral I/R;3. Compared with NBD peptides, we investigate the mechanism of EAaffecting the c-rel expression of cyteblast and cytoplasm in different timepoint of inflammation after focal cerebral I/R.MethodsWe replicated the right middle cerebral artery occlusion2h/reperfusion modal (MCAO/R) of Sprague–Dawley (SD) rats. Rats wererandomly divided into sham-operated group (sham group), MCAO/R group(model group), MCAO/R+EA group (EA group) and NBD+MCAO/Rgroup (NBD group) and each group was divided into reperfusion6,12,24and48h subgroups. NBD peptide was injected into the right lateralventricle. Take the “Baihui”(DU20) and “Siguan”(Hegu LI4, TaichongLR3) as the acupuncture pionts. Neurobehavioral evaluation, pathologicalchanges and the expression of c-rel, IkappaBα (IκBα), IL-1α and IL-1βprotein in the cortical ischemic region were measured. Also thecombination capacity of c-rel and DNA was detected.Results1. Neurobehavioral evaluationOur data indicate that the neurological deficit score of model group, EA group and NBD group gradually decreased, the neurological functionof NBD group recovered (P<0.05), but no significant difference was foundbetween model group and EA group (P>0.05) at each time point afterreperfusion. NBD improves behavioral recovery at the acute period ofinflammation after I/R, the effect of EA is not much obvious at our selectedtimepoints.2. Haematoxylin-eosin (HE) stainingHE stained assayed the histological alterations of cortex area afterfocal MCAO/R in rats. The sham group showed a normal cellularmorphology with distinct karyon and intact nucleolus. The cellular swelling,karyopyknosis and disordered cells after focal cerebral I/R in rats werecaused by cerebral hypoperfusion. An increase of necrosis anddegeneration accompany gliacyte hyperplasia were discovered in6h afterreperfusion and evident at24h. A decrease of cellular swelling,karyopyknosis and disordered cells was detected in EA group and NBDgroup.3. Immunofluorescence (IF)IF showed that c-rel (red) and p65(green) were coexpressed in thecortex area after focal cerebral I/R in rats, especially in ischemia butnon-infarction zone of cortex. Red of c-rel sparkled highly in cytoplasm at6h and decreasingly with time went on. In model group, EA group and NBDgroup, red of c-rel sparkled in cyteblast, peaked at24h. But the EA group and NBD group were gloomier than the model group. Green of p65sparkledin cyteblast at12h and obviously at48h.4. Western blotIn model group, EA group and NBD group, c-rel expression in cyteblastand cytoplasm were higher than that in the sham group (P<0.01). In EAgroup and NBD group, c-rel expression in cyteblast were obviously belowthe model group (P <0.05) and peaked at24h after reperfusion. In EA group,c-rel expression in cytoplasm was clearly higher than that in the model groupat every timepoint after reperfusion, so were the6h,12h and24h of NBDgroup (P<0.01). In model group, EA group and NBD group, IκBα expressionin cytoplasm at24h after reperfusion was lower than that in the sham group(P<0.01). In EA group and NBD group, IκBα expression was obviouslyovertoped the model group (P<0.01). But no significant defference wasfound between the EA group and NBD group (P>0.05).5. Quatitative RT-PCRIt is about the expression of c-rel mRNA at12h after reperfusion. InEA group and NBD group, the c-rel mRNA expression were lower thanthat in the model group (P<0.01), especially in the NBD group.6. EMSAEMSA showed that c-rel binding DNA activity was at a lower level inEA group and NBD group compared with model group (P<0.01). Themodel group peaked at24h but no significant difference was found at other timepoints. The EA and NBD significantly decreased the activity of c-relbinding DNA with time went on (P<0.01).7. ELISAIL-1α: In the model group, the IL-1α can be detected at6h after I/R,peaking time is24h. No significant difference of IL-1α levels was foundamong the model group, EA group and NBD group at6h (P>0.05). Withtime went on, IL-1α level of EA group and NBD group were lower thanthat in the model group (P<0.01). IL-1β: The IL-1β can be detected clearlyat12h after I/R. With time went on, it was increased significantly until48hafter I/R. IL-1β level of EA group and NBD group were lower than that inthe model group (P<0.01)Conclusions1. We datected that at the early stage of inflammation of focal cerebralI/R, the over activation of c-rel can aggravate the inflammatoryinjury.2. NBD peptides can upregulate IκBα, downregulate the activation ofc-rel and its binding activity after focal cerebral I/R, thereby reducethe severity of inflammation, improve of inflammation, improvethe movement of suffered limbs in the acute period.3. Similar to NBD peptides, EA can upregulate IκBα, downregulate theactivation of c-rel and its binding activity after focal cerebral I/R,thereby reduce the severity of inflammation in the acute period. 4. The effect of EA is little weaker than NBD peptides in ourobservation period. EA may through regulate c-rel to reduce theinflammatory injury in the acute period after focal cerebral I/R.