The Study On The Protective Effects Of Estradiol Benzoate On Focal Cerebral Ischemia Reperfusion Injury In Rats

Backgroud and ObjectiveFor the high incidence, mortality and disability rate, ischemia cerebralvascular disease (ICVD) are seriously endangering human's health. With the increasing of old people the incidence of ICVD is continuously rising too. The mechanism of neuronal injury and death in ICVD has been the emphasis of fundamental and clinical study.After cerebral ischemia cell death has two ways: necrosis or apoptosis. Apoptotic is mainly occurring in the area around ischemic focus which related to the expansion of ischemic focus, and occupies important position in the cerebral ischemic injury. Due to the expression of apoptotic related genes, apoptosis is modulated by many exterior and interior factors such as intracellular Ca²⁺ overloading, oxygen free radical, excitatory amine acids or mediator of inflammation et al. Recently the relationship between mediator of inflammation and cell apoptosis is increasingly emphasized. Many scholars explore the core target of anti-inflammation factors after cerebral ischemia reperfusion. Recent studies reveal that nuclear factor-KappaB(NF-κB) closely related to inflammation reaction. NF-κB is a kind of protein which generally resides in central nervous system and can combine withκB sequence of genic enhancer ofκlight chain immune globulin. There are totally five members in NF-κB family: RelA(p65), RelB, c-Rel, NF-κB1(p50/pl05), NF-κB2(p52/p100). P65 contains transcriptase active domain, which can enhance expression and transcription of target genes. P65 is generally and massively expressed throughout cells, and is regarded as the major target to study. NF-κB directly regulates the expression of many kinds of mediators which participating inflammatory reaction after cerebral ischemia reperfusion, such as TNF-a IL-1βIL-6 ICAM-1 VCAM-1, iNOS COX-2, et al , consequently it is regarded as important link of inflammation injury after cerebral ischemia reperfusion. Nitrogen monoxidum(NO), a gas material which acts as messenger and neuro-transmitter within cells, reveals dual effects: neuro-protection and neuro-damage in cerebral ischemia reperfusion injury. No transmits physio-information in lower density and reveals severe neuro-damage in higher density. After cerebral ischemia, by delaminating and decreasing RNA reducing enzyme, No can destroy the structure of DNA, inhibit reproduction and reparation of DNA, depredates cell matrix and ATP of injured DNA activated polymerase, quickly consumes of energy and in the end causes cell apoptosis .Studies display that the density of No is very low at the early period of ischemia and increases obviously in the later subacute stage. iNOS is the major rate-limiting enzyme which mediates the synthesis of No in this stage . iNOS is No-Ca²⁺ /CaM dependent enzyme , which does not express in physiological conditions , and injuries can generate continuously large amount of No until substrate is exhausted , and due to the long half-life of iNOS mRNA it can continuously transcript iNOS , so iNOS is the major cause of excessive NO.Estrogen is a kind of steroid hormone ,β-estradiol is the major component of estrogen , its biological efficacy is most strongest. According to epidemiological investigation, the incidence rate of stroke in pre-menopausal females is lower than males of the same age, but obviously increases after menopause. Estrogen replacement therapy lowers the relative risk of catching a stroke and the mortality related to stroke. But the mechanisms of the protective effects of estrogen on cerebral ischemia is still unclear up to now, and the reports of effects of estrogen on the expression of NF-κB, iNOS and cell apoptosis in cerebral ischemia reperfusion is very few . In this experiment, in order to observe the expression of NF-κB, iNOS and cell apoptosis around the ischemic focus, rat cerebral ischemic reperfusion injury model was established with suture emboli method to approach the mechanisms of cerebral ischemia reperfusion injury and effects of estrodial benzoate, and provide theoretical evidence for clinical therapy for ischemic cerebralvascular disease.Materials and methods(1) 24 healthy male SD rats weighing from 260- 300 grams were randomly divided into three groups (8 rats each group): sham-operation group, control group (ischemia 2 hours / reperfusion 24hours); estrogen administration group (ischemia 2 hours / reperfusion 24hours plus estrodial benzoate );(2) Medication: estrogen administration was intraperitoneally injected with estrodial benzoate (specification: 2mg/1mls), dose: 200ug/kg, once a day for 7 days; sham-operation group and control group was injected with the same amount of sodium chloride;(3) Rat models of cerebral ischemia reperfusion were established with suture emboli method after drug delivery. Sham-operation group received the operations without ischemia. Horner's sign in left side and paralysis of the right front limbs were observed to judge the success of model .at the same time the neurological function deficiency assessment was made too . After ischemia 2h reperfusion 24h all rats were anesthetized and perfused with 200ml normal saline and subsequently with 4 % paraformaldehye in 0.1M phosphylate buffer .Brains were removed, postifixed, embedded, sectioned and processed for HE staining or NF-κB, iNOS immunohistochemistry staining and TUNEL detection;(4) Statistic software SPSS11.0 for windows was exploited to analysis data. All thedata were expressed by ( x±s) , Deviation and difference were compared using the ANOVA, t-test between two groups. The differential significance is judged byα=0.05.Results(1) In control group and estrogen administration group showed Horner's sign in left side and paralysis of the right front limbs after focal cerebral ischemia which indicating the succession of Middle cerebral artery occlusion model, and rats in shame-operation group showed only Homer's sign in left side;(2) The neurological function deficiency assessment(1.63±0. 52) of estrogen administration group was lower than control group(2.25±0.46)(P<0.05).The experiment shows that estrogen can decrease neurological function deficiency of rats suffered with cerebral ischemia reperfusion;(3) Brain tissue slice of H.E staining shows there was no infarction in shame-operation group. There were many trachychromatic neuronal cellulars in control group, cellulars were off position and pintsized, acidophilia of kytoplasm increased, there were dead neurons in core area , outline of neurons was uneasy to identified ,cellular structure disappeared , kytoplasm degenerated like cavity .many glial cells hyperplasyed in the infarction area . In the rounding zone of necrosis, cellular obviously pyknosised and anachromasised , acidophilia of kytoplasm increased obviously .There were no obvious infarction area in estrogen administration group , the shapes and structure of neurons were relatively normal, edema in brain interstitial space was light, which shows that estrogen can ameliorate neurological pathological changes in cerebral ischemia/reperfusion rat brain and has protective functions to the lesions of brain;(4) The results of immunohistochemistry of NF-κB : The number of NF-κB positive cells in each group : 2.25±0.64 in shame-operation group ; 32.08±4.32 in control group ; 23.73±4.16 in estrogen administration group .The expression of NF-κB in control group is higher than that in shame-operation group(P<0.01) . The expression of NF-κB in estrogen administration group is lower then that in control group(P<0.01).The experiment shows that estrogen can inhibit the expression of NF-κB in cerebral ischemia/reperfusion rat brain;(5) The results of immunohistochemistry of iNOS : The number of NF-κB positive cells in each group : 2.15±0.69 in shame-operation group ; 26.34±3.73 in control group ; 17.85±3.07 in estrogen administration group . The expression of iNOS in control group is higher than that in shame-operation group (P<0.01). The expression of iNOS in estrogen administration group is lower then that in control group (P<0.01). The experiment shows that estrogen can inhibit the expression of iNOS in cerebral ischemia/reperfusion rat brain;(6) The results of TUNEL: No apoptotic cells were observed in shame-operation group ; The number of TUNEL positive cells is 38.58±4.05 in control group;and that is 28.34±3.33 in estrogen administration .The above result indicated that estrogen can reduce neuronal apoptosis after cerebral ischemia reperfusion.Conclusions(1) NF-κB and iNOS participate physiopathologic process in cerebral ischemia reperfusion;(2) Estrodial benzoate could lighten neurological functional deficiency and phato-structural injury of cerebral ischemia/reperfusion, the mechanism maybe through efficiently reducing neuronal apoptosis by suppressing the expression of NF-κB and iNOS after cerebral ischemia reperfusion.