The Protective Effect And Anti-leakage Mechanism Of Sodium Aescinate On Rat Blood-Brain Barrier Exposed To Acute Hypobaric Hypoxia

Background and objectives:High altitude cerebral edema (HACE) is a kind of specific acute severe mountainsickness (ASMS) for people, who ascending quickly to a high altitude environment. Althoughthe low partial pressure of oxygen at high altitudes is reported to be an important initiatingagent for HACE, the concrete mechanism underlying HACE is as yet undefined up to now.Additionally, HACE is usually characterized by delitescence in onset, rapid progression,easily complicated by brain crisis, and lacking of specific biologic indicators for earlyprediction and diagnosis for it. Therefore, it endangers in life-threatening situations to personwho rapidly entering altitude.At present, the theory of favored by most researchers is that changes of the blood-brainbarrier (BBB) permeability closely related to the occurrence, development and prognosis ofHACE. During the formation of various brain edema caused by destruction of BBB, thedegradation and (or) loss of major structural protein molecules in tight junctions (TJs), suchas Occludin, Zo-1and Claudin-5, and the passive activation of transcription factor NF-κB, arebelieved to play an important role in the disruption of BBB. Previous studies indicated thatacute cerebral hypoxia, ischemia or ischemia/reperfusion injury, could cause changes ofOccludin, Zo-1, Claudin-5in protein and transcription level directly or indirectly, thus leadingto the disruption of “articulation” state, and eventually increase the permeability of BBB. Atthe same time, nuclear factor κB (NF-κB) that localized in cytoplasm can be activated by thestress state, it can continue to activate MMPs system which would lead the destruction ofvascular basement membrane and formation of vasogenic cerebral edema. Furthermore, NF-κB can also produce a series of inflammatory response, which would lead to theunbalance of energy metabolism and injury of brain cell，and further aggravate the cerebraledema. Therefore, we hypothesize that Occludin, Zo-1, Claudin-5and NF-κB may participatein the occurrence of HACE in an anoxic environment, and represent important moleculartargets in the formation, development and turnover of it.Aescine posses many significant biological properties: such as anti-inflammation,anti-leakage, anti-edema, anti-oxidation and maintaining the normal vascular permeability.Thus, it had already been widely used in the prevention and treatment of various cerebraledema in plain areas, and its therapic effect is reliable. a. Previous research mainly focused onthe pharmacological activity of aescine in cerebrovascular disease under normoxia condition,however, very little is konwn about its pharmacological mechanism underlying brain edemain plateau condition. Therefore, observeing the pharmacological effects of aescine on HACEin plateau condition and exploreing its relative mechanism is of great significant work.Just because of above–mentioned background, we established rat model of experimentalHACE via acute hypobaric hypoxia exposure complicated with exhaustive exercise method.Based on this platform, we observed the protective effects of sodium aescinate against HACE,expounded emphatically the expression changes of Occludin, Zo-1, Claudin-5and NF-κB inbrain and their relationship with pathological changes of HACE. These results are helpful toelucidate the mechanism underlying the protective effects of sodium aescinate on HACE, andprovide theoretical basis for clinical therapy, and particularly for exploreing more novel andefficient therapeutic target dugs for HACE treatment.Methods:1. Establishment of an animal model of HACE in rat: Adult male SD rats were dividedinto plain control group (PC) and altitude hypoxic group (AH) randomly, with20in eachgroup. After treadmill training in2days in plain area, AH group rats were exposed into ahypobaric room stimulated elevation of4000m above sea level, meanwhile carried outexhaustive exercise for2days. Then changed level to8000m quickly and followed by3daysof simple hypobaric hypoxia exposure. Hypobaric room temperature was about18℃,light/dark regime was24h and rat were free to eat and drink. While no intervention for PCgroup with a normal mode of life in cages in the plain area.Finally, samples of AH group were collected under stimulated altitude of5000m and that of PC group in plain region. Then measure water content and evans blue content in brain,observe pathological and ultrastructural changes of brain, study lanthanum nitrate particledistribution in brain, and use them to evaluate animal model comprehensively.2. Study on brain protective mechanism of sodium aescinate on experimental HACEmode in rat: Adult male SD rats were randomly divided into plain control group (PC), plaindrug group (PD), altitude hypoxic group (AH) and hypoxia treated group (HT), with25ineach group. Animal model of HACE was established as above. In AH and HT group, hypoxiatreatment were the same as above in hypobaric room. In PC and PD group, rats were fed inroutine way in plain. The rats of HT group was begun to receive injection of sodium aescinate(5mg/kg.bw,1intraperitoneal injection/day) at the end of exhaustive exercise in elevation of4000m, and then put them into stimulated elevation of8000m. At day1and day2withstimulated elevation8000m, change the elevation to5000m and the rats in HT group gaveinjection of sodium aescinate by same method, then return altitude to8000m quickly. At thesame time, rats of PD group with sodium aescinate in the same way, AH and PC group wereinjected with0.9%sodium chloride (as control). After experiment finished, brain watercontent, brain evans blue content, pathological and ultrastructural changes of brain andlanthanum nitrate particle distribution in brain were assayed, expression of occludin, zo-1,claudin-5gene and Occludin, NF-κB P65proten in frontal and parietal cortex were detectedrespectively.Results:1．Compared with PC group, the content of brain water and evans blue were obviouslyincreased in AH group(P＜0.01)；Results under light microscope: we saw that hippocampalformation of AH group damaged seriously, pyramidal cells number decreased, disarrangedand lost obviously; Under transmission electron microscope (TEM): the structure of brainchanged into a loose state significantly, tight junctions widened, plenty of lanthanum nitrateparticle leaked out from blood capillary inside and deposited into cortex by opening tightjunctions, some of neurons and glial cells were swollen, nerve fibers showed light swelling.2．The content of brain water and evans blue in HT group were significantly lower thanthose in AH group(P＜0.05); Results under light microscope: Compared with AH group,hippocampal formation of HT group was minimal abnormal, pyramidal cells numberdecreased, disarranged and lost, but to a lesser degree; Under TEM：HT group had a light loose state, most of lanthanum nitrate particle located in endovascular while a little of whichexuded to extracapillary and brain tissue space，tissue showed mild edema. Compared withPC group, the expression levels of occludin, zo-1and claudin-5mRNA were lower noticeablyin AH group(P＜0.01，P＜0.05， P＞0.05), and the levels of them in HT group wereobviously higher than those in AH group(P＜0.05). Occludin protein expression ascended inHT group while declined in AH group significantly compared with PC group, and there weredistinct difference between AH and HT group, also AH and PC group(P＜0.05). But contraryresult appeared with NF-κB P65protein expression, there was statistically significant betweenAH and PC group(P＜0.05) while no difference among four groups (P＞0.05).Conclusions:1．Acute hypobaric hypoxia exposure complicated with exhaustive exercise couldsuccessfully established animal model of HACE, and this work might afford an opportunity toresearch the mechanism of HACE formation and related drugs function.2．After acute hypobaric hypoxia exposure, BBB of SD rats damaged severely, tightjunctions opening state and permeability increased, and then could result in the formation ofbrain edema. Pyramidal cells number lost obviously, some of neurons and glial cells wereswollen, nerve system damaged.3. A rapidly decrease the expression levels of occludin, zo-1, claudin-5mRNA andOccludin protein, and increase of NF-κB P65protein may be closely related to the HACEoccurrence and development. Occludin, Zo-1, Claudin-5and NF-κB in brain may beattractive drug targets for prevention and cure of HACE.4. The neuroprotective mechanism of sodium aescinate may be associated withup-regulation expression of Occludin, Zo-1, Claudin-5and down-regulation expression ofNF-κB P65, reduce BBB permeability and neurons damage, attenuate cerebral edema inHACE induced by acute hypoxia exposure.