| Objective: Epilepsy is a chronic neurological disorder consisting of different symptoms and signs, which is characterized by paroxysmal, te mporal and stubborn onset. Epidemiological study has shown that the incidence of epilepsy is about 5‰-7‰. At present,China has about 6.5to 9.1 million patients with epilepsy, among which the teenager and t he senile occupy the two peak ages of onset. Currently,the pathogenesi s of epilepsy is still not entirely clear,but some important aspects of t he process have been known by the reseachers. Now there are a few pathologic mechanisms considered taking part in the onset of epilepsy involving excitatory amino acid toxicity theory, ATP depletion, calciu m overload, oxidative stress, proinflammatory cytokines damage and ap optosis.Apoptosis is a programmed cell death controlled by the gene to maintain the homeostasis. Apoptosis is not a passive process, but a initiative process, which involves a series of genetic activation, expres sion and regulation and so on.Cell apoptosis will happen after the occ urrance of epilepsy.Studies have shown apoptosis is related with imbal ances and disorders of apoptosis pathway.Bcl-2 family is the main reg ulatory cytokine in mitochondrial apoptosis pathway.Bcl-2,as a apoptoti c inhibitor,has a strong anti-apoptotic function to extend the lifespan of the cell.On contrary,bax is an important proapoptotic protein.The ra tio between bcl-2 and bax decides the survival of cell. Magnolol, a hy droxylated biphenyl compound isolated from the Chinese herb Hou p’u of Magnolia officinalis, is widely used in the Chinese traditional med icine. Studies have shown that it has the ability of scavenging free rad ical, inhibition of neutrophil infiltration, anti-inflammation, inhibiting pl atelet aggregation, suppressing pathogenic microorganisms, sedation, muscle relaxants and anti-apoptosis and other pharmacological effects. Ma gnolol, as a potential neuroprotective agent,is drawing more and more attention regarding to its therapeutic effects about cerebrovascular dise ase, epilepsy, cognitive disorders and other central nervous system dis ease.Studies have shown that magnolol has anti-apoptotic effects on car cinoma and central nervous system disease. In this study, adult male Sprague-Dawley(SD) rats are administered pentylenetetrazol(PTZ) by intraperitoneal injection to establish a stable animal model of chronic e pilepsy. Magnolol is administered before the injection of PTZ to obse rve the epileptic severity and duration and behavior of rats in each gr oup.HE staining is used to observe the morphology of hippocampus an d Transmission Electron Microscopy(TEM) to observe the ultrastructure changes of neurons.Western Blot is used to observe the bcl-2 and ba x protein expression in rat hippocampal tissue. In conclusion,this study is to investigate the protective effects of magnolol on PTZ-kindled S D rats and related mechanism.Methods:Adult male cleaning SD rats,7-8 weeks old and weighing 180-220 g were randomly divided into five groups: normal control group(NC group), epilepsy group(PTZ group), low dose Magnolol group(L group), middle dose magnolol group(M group) and high dose Magn olol group(H group).Each group had 15 SD rats(If rat was dead durin g the experiment,another one was supplemented timely). Since the first day of the experiment, drugs were administered by intraperitoneal injecti on at 9:00 am every day. NC Group: rats were injected with propylene glycol(1ml / kg) intraperitoneally everyday.After observing for 30 min, s aline(3.5 ml / kg)was administered intraperitoneally, continually observin g for one hour(a total of 42 days). PTZ Group: rats were injected wit h propylene glycol(1ml / kg) intraperitonealiy everyday.After observing f or 30 min, PTZ(3.5 ml / kg)was administered intraperitoneally, continuall y observing for one hour(a total of 42 days). L Group: rats was inject ed with magnolol(2.5mg / kg) intraperitoneally every day.After observing for 30 min, PTZ(3.5 ml / kg)was administered intraperitoneally, contin ually observing for one hour(a total of 42 days). M Group: rats were injected with magnolol(5mg / kg) intraperitoneally every day.After obser ving for 30 min, PTZ(3.5ml / kg)was administered intraperitoneally, conti nually observing for one hour(a total of 42 days).H Group: rats were i njected with magnolol(10mg / kg) intraperitoneally every day.After obser ving for 30 min, PTZ(3.5 ml / kg)was administered intraperitoneally, con tinually observing for one hour(a total of 42 days).According to the R acine criterion,the epileptic grades and duration were observed every da y.In 24 hours after the end of the experiment,six rats were perfused an d decapitated to observe the hippocampal neuron damage by HE stainin g. Three rats were used to observe the neuronal morphology and ultrast ructural changes with TEM in the CA1 region of the hippocampus.The lastsix rats were used to study the bcl-2 and bax protein expression of the hippocampus with Western Blot method.Results: 1 Results of behavior NC group:The rats behaved normally and had a regular dietary.The weight was increased gradually and the hair was shiny.No seizures were found in normal control group. PTZ group:No significant change of the body weight or a slightly reduction was happened. With the time passed by,the food intake of the rats gradually reduced and the rats became inactive to stimulation. From the fourth to seventh day,epileptic symptoms were observed including nod,washing faces,drifting tails,unilateral or bilateral forelimb clonus.Then, from the tenth to fifteenth day, the rats developed into generalized tonic- clonic seizures with standing and falling down, and ultimately achieved Ⅳ-Ⅴ grade seizures according to the Racine criterion. If three consecutive seizures of grade ⅣorⅤwere observed,the rat was considered to be kindled successfully.In the end of the experiment, fourteen rats were kindled successfully and the kindled rate was 93.3%. L group:A slight increase of the rat weight was observed.Dietary and the activities were moderate.The strength grades were mainly between Ⅱ-Ⅲ grade and the attack of Ⅳ-Ⅴgrade could not happen consecutively. Five rats were successfully ignited,so the kindled rate was 33.3%. M group:The weight was increased and dietary and behavior were closely normal. The strength grade was reduced and the latency(20th-28 th day) was extended compared with the PTZ group and L group, mainly between Ⅱ-Ⅲ grade.Only one rat was kindled,so the kindled rate was 6.7%. H group: The rats in this group had a poor health conditions including loss of weight, loss of appetite, reduced activity and low spirits. During the experiment three rats died(due to non-epileptic seizures), and the same amount of rats were supplemented. The kindled rate was 13.3% calculated without involving the dead rats.2 Results of HE staining NC group: the hippocampal neurons had normal shape and arrangement.The number of the cells were normal,an d no neuron loss were observed.The ratio of cytoplasm and nucleus wa s normal. The nucleolus was visible,cytoplasm transparent.The chromatin was well-distributed. PTZ group:the neurons of hippocampus arranged in a mess.The boundry of the cell was unclear and the neurons lost evid ently. Eosinophilic neurons were observed charactered with cell shrinkag e, chromatin condensation and nuclear condensation. L/M group:the neur ons morphology were nearly normal and arranged in a good manner. T he number of neurons were larger than that of the PTZ group, only a small fraction of eosinophilic neurons could be observed occasionally. H group: The distribution of hippocampal neurons was out of order.The boundry was ill-defined.Neuron loss was clearly but better than that of the PTZ group.3 Results of Western blot test The bcl-2 expression level of the PTZ group was significantly lower compared with NC group, L group, M group and H group(P<0.05). Oppositely, the bax expression level of PTZ group was significantly higher than the rest groups(P<0.05). The expression level of bcl-2 in M group was significantly increased campared with L and H group(P<0.05),but the expression level of bax decreased campared with L and H group(P<0.05).4 Results of TEM NC group: The neurons had whole shape and the structure was clear.The nuclei was in round shape.The chromatin distributed well. Mitochondria, endoplasmic reticulum, Golgi were abundant. Mitochondria were round or oval,and crest were clearly visible. Neurofilament microtubules in axons were also clearly visible. PTZ group: Hippocampal neurons structure were damage, with nuclear swelling and irregular, reduced nuclear chromatin and some nuclear condensation. Vacuolization of mitochondria and cristae fracture were obvious. Neurofilament microtubule structure was not clear. L/M group: The hippocampal neurons had complete, and normal morphology,so as to the nucleus. The chromatin was distributed evenly. The morphology of mitochondria, endoplasmic reticulum were closely normal. H group: Hippocampal neurons were in irregular shape. Nucleus swelling and loose, nuclear chromatin condensation,mitochondrial swelling, cristae fracture and swelling of the endoplasmic reticulum were all observed.Conclusions:1 Magnolol effectively suppress PTZ-induced epileptic onset of SD rats, and reduce the level of seizures. 2 Epilepsy can induce cell apoptosis, reduce the anti-apoptotic protein bcl-2 expression in the hippocampus, and increase the pro-apoptotic protein bax expression. 3 Magnolol can increase the expression of bcl-2 but reduce the expression of Bax, suggesting that Magnolol can exert neuroprotective effect through upregulating the expression of bcl-2,and reducing the expression of bax. 4 The neuroprotective effect of nagnolol is in a dose-dependent manner. Over doses magnolol may cause severe side effects caused cell damage. the results suggest that magnolol in the middle dose has a more significant neuroprotection effect.. |
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