The Study Of Brain Function And Mechanism Of Enteromorpha Clathrate Polysaccharides On The Rats With Sepsis Associated Encephalopathy

Sepsis-associated encephalopathy(SAE)is a major nervous system dysfunction in sepsis patients,and the mortality is increased with severity of SAE.Cognitive dysfunction caused by SAE is not related to the severity,while lowers the quality of patients' life.Therefore,it is of desperate need to explore the pathogenesis of SAE and develop the corresponding drugs to reduce the severity of SAE,finally diminishing the death rate of SAE.Oxidative stress was known to be closely associated with the pathogenesis of SAE.Excession of oxygen free radicals and/or antioxidant deficiency to different brain regions was reported to trigger evident morphological dysfunction and cognitive deficits.And inhibition of oxidative stress by drugs could indeed reverse cognitive decline in sepsis-associated encephalopathy rats.Further more,high levels of inducible nitric oxide synthase(i NOS)could also promote the generation of SAE.Brain-derived neurotrophic factor(BDNF)is one of the most important members in neurotropic family and is widely distributed in the brain.A previous investigation illustrated that BDNF played a crucial role in neuronal survival and synaptic transmission.In a mouse model of SAE,it was found that activation of BDNF by valproic acid could remarkably improve cognitive deficits,suggesting that BDNF might be served as the useful therapeutic target for alleviation of SAE.Enteromorpha clathrate polysaccharide(ECP)is purified from Enteromorpha Prolifera.It has anti-inflammatory,anti-oxidative and anti-tumor potentials.Previous study has observed that ECP could decrease leukocyte and neutrophil leukocyte in septic rat and alleviate inflammatory reaction;while it can increase lymphocyte and CD 4+ T lymphocyte and improve immune function.However,whether ECP can improve cognitive performance in septic rat remains elusive.Therefore,our present work aimed to evaluate the brain protective effect of ECP on SAE and further figure out the potential mechanisms.Objective: This experiment is aiming to the effect of ECP to the cognitive function of sepsis rats,and to explore the mechanism of brain protection,providing a theoretical basis for clinical application.Methods:(1)Grouping and establishing animal models The animals were categorized into three groups: CON group(sham operation plus normal saline),SAE group(CLP plus normal saline),and SAE+ECP group(CLP plus 100mg/kg ECP).We choose cecal ligation and puncture(CLP)method to establish the SAE model.Take the SD rat,anaesthetize it by injecting 2% pentobarbital sodium(0.3 m L/100 g)in the abdominal cavity,shave its belly and disinfect with iodophors.Then cut along the medioventralline about 2cm to expose the cecum,and ligate it at the far-end of the ileocecalvalve.Then,puncture the cecum twice with a needle,gently squeeze it to allow the secretion of a small amount of intestinal contents.Next,return the cecum to the abdominal cavity and close the abdominal cavity.In the sham-operation group,the laparotomy is given to the rat,and its cecum is put outside for 1 minute without cecalpuncture or ligation,then the cecum is put back into the abdominal cavity and the cavity is closed.Each rat used in the operation is given hypodermic injections with normal saline(30 m L/kg)and ceftriaxone(30 mg/kg)every 6 hours after operations.Prepare the model of rats with sepsis,and evaluate the rats' neurological behavior after 24 hours,so those who own a score of nerve reflex below 6 points are the SAE model.We would give up the rest ones not conforming to the standards,and prepare new models of rats with sepsis.(2)General conditions in rats The mental state,fever,diarrhea,vertical hair,skin and mucous bleeding were observed before or after operation,as well as the basic vital signs,such as US temperature,heart rate and blood pressure were measured at 6 h,12 h and 24 h after CLP operation.The weight of rats during 7 day after operation was evaluated.(3)Behavioral experiments Open field test,Morris water maze task and Y Maze experiment were conducted to measure the general locomotor activity,learning and memory ability of rats in each group.(4)Oxidative stress indicators in hippocampus The level of serum ammonia was measured by an autoanalyzer.Content of NO was determined with nitric acid deoxidize enzyme method;Activity of i NOS was determined with L-arginine oxidation method;Content of MDA was determined by thiobarbituric acid;Activity of total SOD was determined by xanthine oxidase technique;Activity of CAT and GSH –Px were also measured.(5)BDNF and p-Trk B expression in hippocampus Animals were sacrificed and their brains were harvested 24 hours later.Samples were selected,and the expressions of BDNF and p-Trk B protein in the hippocampus were measured by Western blot.Results:(1)General state of rat Normal group rats are generally in good condition,the basic vital signs such as heart rate,arterial pressure and body temperature were steady.The septic rats appeared crouching,pilomotor,hemiplegia or seizures.Significant decreased mean arterial pressure and body weight,increased heart rate suggested the successful induction of sepsis in the present study,while there is no significantly change in the US temperature after sepsis.(2)Behavioral experiments In Open Field test,it was noteworthy that there were no significant differences for the total distance in the open field among groups(P>0.05),suggesting that the general locomotor activity was not affected by sham operation,CLP or ECP treatment.Cognitive function was assessed by Morris Water Maze test and Y maze experiments.Septic mice treated with ECP exhibited reduced escape latency(P<0.01)and mean path length(P<0.01)while enhanced percentage of time spent in target quadrant(P<0.01)and number of times of crossing the platform(P<0.01).The swimming speed was similar among different groups(P<0.01).Y maze test disclosed that ECP significantly diminished the number of learning trials in SAE-induced mice(P<0.01).All results demonstrated that the learning and memory ability was injured in SAE rats,and ECP can significantly improve cognitive performance in septic rat.(3)Oxidative stress indicators in hippocampus No significance was observed for the serum ammonial levels among different groups(P>0.05),suggesting that ECP improves cognitive function was not by increasing serum ammonial level.Compared with normal control rats,the content of NO in hippocampus of SAE groups and SAE+ECP groups increased significantly(P<0.01);compared with SAE groups,NO content decreased in SAE+ECP group(P<0.01).Compared with normal control rats,the activity of i NOS in hippocampus of SAE groups and SAE+ECP groups increased significantly(P<0.01);compared with SAE groups,i NOS activity decreased in SAE+ECP group(P<0.01).Compared with normal control rats,the content of MDA in hippocampus of SAE groups and SAE+ECP groups increased significantly(P<0.01);compared with SAE groups,MDA content decreased in SAE+ECP group(P<0.01).Compared with normal control rats,the activity of CAT in hippocampus of SAE groups and SAE+ECP groups decreased significantly(P<0.01);compared with SAE groups,CAT activity increased in SAE+ECP group(P<0.01).Compared with normal control rats,the activity of SOD in hippocampus of SAE groups and SAE+ECP groups decreased significantly(P<0.01);compared with SAE groups,SOD activity increased in SAE+ECP group(P<0.01).Compared with normal control rats,the activity of GSH-Px in hippocampus of SAE groups and SAE+ECP groups decreased significantly(P<0.01);compared with SAE groups,GSH-Px activity increased in SAE+ECP group(P<0.01).All these results indicated the increased oxidative stress in hippocampus of septic mice.However,these indices was markedly reversed in SAE-induced mice when treatment with ECP.(4)BDNF and p-Trk B expression in hippocampus Compared with normal control rats,the expression of BDNF protein in hippocampus of SAE groups and SAE+ECP groups decreased significantly(P<0.01);compared with SAE groups,BDNF expression increased in SAE+ECP group(P<0.01).Compared with normal control rats,the expression of p-Trk B protein in hippocampus of SAE groups and SAE+ECP groups decreased significantly(P<0.01);compared with SAE groups,p-Trk B expression increased in SAE+ECP group(P<0.01).These results indicated the downregulation of BDNF and p-Trk B protein in hippocampus were induced SAE,and ECP can promote their expression.Conclusion:(1)CLP models simulate the typical septic clinical manifestations;SAE model is established successfully by evaluating behavior trials and can be used in SAE mechanism study.ECP has a protective effect on the cognitive function of SAE rats.(2)ECP can decrease the level of hippocampal oxidative stress injury induced sepsis and might exert its protective effect by inhibiting oxidative stress damage.(3)Sepsis-associated encephalopathy can cause a decrease in BDNF expression and Trk B phosphorylation,and ECP might exert its protective effect by promoting BDNF-Trk B pathway.