Studies On The Preventive And Therapeutic Effect Of Tetrahydrocurcumin On High Altitude Cerebral Edema In Mice

Background:High-altitude military operations involve a rapid ascent that unacclimatized individuals,who are acute exposure to hypobaric hypoxia plateau environment,are at high risk of acute mountain sickness(AMS).The develop-the-west strategy and convenience of high-speed transportation as well as demands of tourism,exploration,business,etc.witnessed the increasing people,who came to plateau sections and the increasing AMS patients.Controlling the rate of ascent is a highly effective means of preventing AMS,however,due to military operations and disaster relief,people need to rush ascent into the plateau without acclimation.For instance,among the rescue workers in the 2010 Yushu earthquake relief operation,the incidence of AMS was up to 79.8%.High altitude cerebral edema(HACE)is considered as the end form of AMS,according to statistics,the incidence of HACE in the general population is only 0.4 ~ 3.4%,however,following the increasing rate of ascent the incidence can reach to 34%.HACE leads to severe neurological dysfunction,cerebral edema,hemorrhage,coma and even death,the features including insidious conditions,rapidly progressive and high mortality.To the clinical treatments of HACE,supplemental oxygen,acetazolamide,dexamethasone,and mannitol are often used with the symptomatic treatment.However,the lack of effective and specific preventive and therapeutic drugs has greatly restricted the combat effectiveness of our army and has become an important factor threatening the health of military personnel stationed in plateau areas.In view of this,is urgent to find and develop HACE protective drugs.Acute hypobaric hypoxia(AHH)exposure induce cerebral hypoxic injure and cerebral circulation disorders,especially the increase of oxidative stress,inflammatory factors,and blood-brain barrier(BBB)permeability which play important roles in the development of HACE.Previous studies have confirmed that hypoxia significantly induces the expression of VEGF in brain tissues,which disrupts the vascular endothelial cell integrity and dissolves the basement membrane of blood vessel,directly leading to the increased BBB permeability.Furthermore,in the case of hypoxia,the nuclear factor kappa-B(NF-κB)in the brain is activated,generating the inflammatory cascade and activating matrix metallopeptidase-9(MMP-9),which disrupts the basement membrane of vascular endothelial cells,causes BBB leakage,and promote the development of vasogenic brain edema.Therefore,we speculate that NF-κB,VEGF,and MMP-9 are important target molecules for the formation and development of HACE.The traditional Chinese medicine Curcumae Radix and Curcumae Longae Rhizoma are both derived from plants of genus Curcuma,Curcumae Radix is the dried root tubers of Curcuma longa,C.wenyujin,C.kwangsiensis and C.phaeocaulis,Curcumae Longae Rhizoma is the dried rhizomes of C.longa.They have similar medicinal effects.Curcumae Radix has the effects of activating qi,dissolving blood stasis,promoting blood circulation,and clearing heart.As a monarch herb,it is made into Xingnaojing injection with musk,borneol and zongzi.It is widely used in clinical treatment of AMS.Curcumae Longae Rhizoma is perfect for the syndromes of cold coagulation,qi stagnation and blood stasis.HACE patients mostly with qi deficiency,blood stasis and phlegm dampness,thus,we infer that Curcumae Longae Rhizoma has the potentially treatment effect to HACE.Curcumin,as the main active ingredient of traditional Chinese medicine Curcumae Radix and Curcumae Longae Rhizoma,has been proven to be useful in the treatment of HACE.However,due to its low bioavailability and rapid metabolism,few amount of the prototype drug are detected in the blood.As the main active metabolite of curcumin,tetrahydrocurcumin(THC)has the similar pharmacological effects,but it has stronger effect of anti-inflammatory,anti-oxidative and vascular protection activities,higher bioavailability and transmittance of BBB.THC may be one of the medicinal substance bases of Curcumae Radix and Curcumae Longae Rhizoma,and the essential reason for curcumin to treat HACE,but the therapeutic effect of THC on HACE has not been reported.Hence,it is meaningful to explore and study the preventive therapeutic effect of THC on HACE and its related mechanism.Objective:The purpose of this study was to observe the efficacy of THC in the prevention and treatment of HACE,and to preliminary study the related pharmacological mechanism by the experiments in vivo and in vitro.Methods:1.Establishment of HACE model and optimisation of THC prophylactic dose:C57BL/6 mice were placed in a decompression chamber with libitum access to feed and water,maintained the velocity of 15 m/s by controlling the decompression speed,stabilized at an altitude of 7,000 m for 24 h to establish the HACE model.Mice were randomly assigned into different prophylactic dose groups(10 mg/kg,20 mg/kg,40 mg/kg,80 mg/kg,and 160 mg/kg)with administration for 3 d before HACE modeling.After modeling,blood samples were collected from the retro-orbital plexus to determine the levels of TNF-α in the serum and several haematological parameters.Separated the brain on ice to determine the brain water content(BWC).Based on the obtained data to determine the optimal dose to be used for further experimentation.2.Research on the prevention and treatment mechanism of THC to experimental HACE mice:With Dexamethasone(DXMS)(6 mg/kg)and aescine(10 mg/kg)as the positive control drugs,the mice were placed in the decompression chamber for 24 h to duplicate HACE model,after 3 days of preventive administration.Counted the weight change of mice before and after modeling to calculate the percentage of weight loss;Separated the brain on ice to determine the BWC;Brain tissue homogenate was prepared to detect the contents of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)by Elisa method,detect the superoxide dismutase(SOD)activity value and content of malondialdehyde(MDA)by colorimetric method;After the brain tissue was fixed,performed coronal sections for haematoxylin-eosin(HE)staining to observe the pathological changes of brain tissue;Immunofluorescence staining was used to detect the expression and distribution of VEGF and MMP-9 proteins;After the cortex was fixed,the ultrastructural changes of cortical gliocytes were observed by transmission electron microscope;Western blot was applied to detect the content of VEGF,MMP-9 and NF-κB proteins in brain tissues.3.Research on the effect of different hypoxia durations on astrocytes:Different hypoxia exposure duration(0 h,1 h,3 h,6 h,12 h,and 24 h)were applied to a tri-gas incubator where the astrocytes were exposed to 4% O2 to simulate hypoxic injury.After that,IL-1β and TNF-α in the supernatant were measured by Elisa method;astrocytes climbing slice were fixed and subject to immunofluorescence staining to detect VEGF expression;Flow cytometry was used to measure the apoptosis rate in various periods of time.The effect of hypoxic duration on astrocytes was so comprehensively assessed.Comprehensively study the effect of hypoxia duration on astrocytes.4.Effect of THC intervention on the expression of VEGF and MMP-9 in astrocytes under hypoxia:The CCK8 detection method was used to detect the effect of THC,with different concentrations(1 μM,5 μM,10 μM,50 μM,100 μM,500 μM,1 m M,5 m M,and 10 m M),on astrocytes viability under normoxia or hypoxia to determination of effective dose;10 μM THC was selected to intervene in astrocyte hypoxia model,and detected the VEGF and MMP-9 expression of astrocytes by immunofluorescence staining.Results:1.AHH exposure for 24 h in a decompression chamber,simulating the hypobaric hypoxia environment at an altitude of 7,000 m,could significantly induce cerebral edema in C57BL/6 mice.Prophylactic administration of THC 40 mg/kg for 3 days significantly decreased the BWC and weight loss which were upregulated by AHH,at the same time,histopathology showed that the degree of gliocyte-edema,angioedema and mitochondrial damage in gliocytes were significantly reduced,inferred that the prevention of THC treatment could effectively relieve the edema induced by AHH in mice.Administration of THC significantly increased SOD activity,but no change was observed in the MDA levels,while both white blood cells and red blood cells in plasma showed a meter-dependent increase,suggesting that THC has a certain ability to improve the body’s ability to resist hypoxia.VEGF and MMP-9 expression was significantly increased in brain tissues after exposure to hypobaric hypoxia but down-regulated after THC intervention,suggesting that THC inhibits the increase of blood-brain barrier permeability by down-regulating VEGF and MMP-9 protein expression.In addition,THC significantly down-regulated the remarkably increased expression of TNF-α,IL-1β and NF-κB in brain stimulated by AHH,cluing the promoting effect of THC in prevention and treatment of cerebral edema through antiinflammatory actions2.Hypoxic stimulation significantly increased the expression of VEGF,TNF-α,IL-1β in astrocytes,and the release of inflammatory cytokines was prior to VEGF expression;Apoptosis rate detected by flow cytometry and cell activity detected by CCK-8 both showed that 24 h hypoxia had no significant effect on astrocyte apoptosis,and THC did not significantly increase the activity of astrocytes under hypoxia.We inferred that THC did not achieve HACE treatment by protecting astrocytes or inhibiting cell activation;VEGF and MMP-9 expression in astrocytes increased under hypoxic conditions and then declined after THC intervention,showing the important roles of astrocytes in vasogenic cerebral edema and that THC improved HACE through protection of blood brain barrier by down-regulating VEGF and MMP-9 expression.The expression of VEGF and MMP-9 in astrocytes was significantly increased under hypoxia,and the expression was significantly down-regulated after THC intervention,suggesting that astrocytes play an important role in vasogenic brain edema,and THC can protect BBB by down-regulating the expression of VEGF and MMP-9 to alleviate HACE.Conclusions:1.Prophylactic administration of THC can effectively prevent and treat AHHinduced vasogenic brain edema in mice,reduce the degree of pathological damages to brain tissue,inhibit the expression of inflammatory cytokines,promote the increase of SOD activity in the brain and inhibit oxidative stress injury.It has been confirmed that THC can improve brain edema injury by down-regulating the expression of NF-κB、VEGF and MMP-9.2.Under hypoxic conditions,inflammatory cytokines are preferentially expressed in astrocytes to VEGF.It is speculated hypoxia activates inflammatory pathways first.3.Under hypoxic conditions,astrocytes participate in and promote the development of HACE by releasing inflammatory factors,VEGF and MMP-9;THC can significantly down-regulate VEGF and MMP-9 protein expression in astrocytes under hypoxia.