Composition And Mechanism Of Nutrient And Foodborne Functional Compound Formulations For Improving Cognitive Function In Heat Stress Mice

BackgroundWorking in high temperatures can lead to cognitive decline,people working in high temperatures showe reduced reaction time,memory and concentration.Studies on animal models also found that heat stress rats and mice showed decreased learning ability,long-term and short-term memory,and slower reaction speed.The main reason for this phenomenon are the damage of some organelles of hippocampal neurons and the increased expression of apoptosis-related proteins,which lead to increased neuronal apoptosis.Mitochondrial damage plays an important role.Heat stress leads to depolarization of mitochondrial membrane potential,aggravation of oxidative stress,dysfunction of respiratory complex,release of cytochrome C into cytoplasm,which ultimately leads to neuronal dysfunction and apoptosis.The nutritional metabolism of high temperature environment is different from that of normal temperature environment.In high temperature environment,the loss of B vitamins,vitamin C,calcium and zinc increase.The plasma lipid peroxide metabolism significantly increases in heat stress mice.Some nutrients and foodborne functional compounds have been found to have a role in improving cognitive function in high temperature environment,so additional nutritional supplements can be used to help solve this problem,and the formulation for a variety of nutrients and foodborne functional compounds is better than a single ingredient.Therefore,our study aimed to explore formulations made up of nutrients and foodborne functional compounds that can improve the cognitive performance of heat stress mice,and to investigate the mechanism of the mitochondrial nutrient formulation.Objectives1.To investigate the effects of heat stress on cognitive function of mice.2.To screen nutrients and foodborne functional compounds that can improve the cognitive function of mice after heat stress,then make them into formulations and verify the effect.3.To research the possible mechanisms of the combination of mitochondrial nutrient formulation.Methods1.Animal grouping and interventionHeat stress mice:Male SPF C57 BL/6 mice,after 5 days of adaptation,the mice were divided into Control group,Heat Stress 1 group and Heat Stress 2 group.Control:without heat exposure;Heat Stress 1:42.5±0.5℃,RH 60±10%,1 h/day,lasted for 7days;Heat Stress2:41±0.5℃,RH 60±10%,1 h/day,lasted for 7days.The behavioral experiment was carried out 1 h after heat exposure.Nutrients and foodborne functional compounds intervention:After adaptation,the mice were divided into Control group,Heat Stress group and different intervention group.Control:normal saline,21 days;Heat Stress:normal saline,21 days;HT-ac:50 mg/kg/d,21 days;HBET:33.7 mg/kg/d,21 days;L-tyrosine:300 mg/kg/d,14 days;B vitamins:Vitamin B₁/B₂/B₆ 5.5 mg/kg/d,pantothenic acid 13.75 mg/kg/d,B₁₂ 0.00925 mg/kg/d,14days;Quercetin:16 mg/kg/d,14 days;Resveratrol:50 mg/kg/d,14 days;Tea polyphenols:250 mg/kg/d,14 days;Genistein:230 mg/kg/d,14 days;Baicalein:300 mg/kg/d,14 days;Luteolin:285 mg/kg/d,14 days;Procyanidins:300 mg/kg/d,14 days;Naringin:100 mg/kg/d,14 days.Heat exposure was carried out on the last 7 days of gavage:42.5±0.5℃,RH60±10%,1 h/day.The behavioral experiment was carried out 1 h after heat exposure.Nutrient and foodborne functional compound formulations intervention:After adaptation,the mice were divided into different intervention group.High dose phytochemical formulation（Phy-H）group:procyanidin 300 mg/kg/d,naringin 100 mg/kg/d,quercetin 16 mg/kg/d,21 days;Low dose phytochemical formulation（Phy-L）group:procyanidin 100 mg/kg/d,naringin 40 mg/kg/d,quercetin 8 mg/kg/d;Nutritional fortifier（Nut）group:L-tyrosine 300 mg/kg/d,vitamin B₁/B₂/B₆ 5.5 mg/kg/d,pantothenic acid 13.75mg/kg/d,B₁₂ 0.00925μg/kg/d,21 days;Mitochondrial nutrient（HT-ac+HBET）group:HT-ac 50 mg/kg/d,HBET 33.7 mg/kg/d,21 days.Heat exposure was carried out on the last 7days of gavage:42.5±0.5℃,RH 60±10%,1 h/day.The behavioral experiment was carried out 1 h after heat exposure.2.Behavioral experimentsMorris water maze:The experiment was divided into two stages.Orientation navigation test:mice were placed randomly into the pool facing the pool wall from four entry points every day,and the latency and swimming distance was recorded,this training repeated4 times every day,and the average value was used as the result of the day.Spatial probe test:the platform was removed after the orientation navigation test,mice were place into the point farthest from the platform,the percentage of time/distance of the platform quadrant and the times of crossing the platform within 60 seconds were recorded.Shuttle Box Test:One experiment cycle was divided into acousto-optic stimulation,acousto-optic stimulation and resting phase.The active escape was to flee to the safe area during the acousto-optic stimulation period,and the passive escape was to flee to the safe area during the acousto-optic stimulation period.The experiment was divided into two stages.In the training stage,the number of cycles was set to 20 times,so that the mice could gradually form the memory of active escape.24 h after the training,the experiment was conducted,the number of cycles was set to 30 times,and the active escape times were recorded.Turn bar test:Set the speed and time parameters,then put the mice on the roller.The mice were shocked when they fell down,to make them run as hard as they can on the roller.The experiment lasted for 4 days.The first three days were training,and the success standard1 min.In the experiment,the mice fell as the end,each mouse for 3 times experiment,take the average time as the final result.Jumping stage test:Put the mice on the grid and electrify them,they would jump on the insulated platform,and then possibly jumped onto the grille,where they would be shocked and then jumped back on the platform.The training lasted for 5 min,and the test was carried out 24 h after the training period and lasted for 3 min.3.Hippocampal observationHE staining:The brain was fixed with 4%paraformaldehyde,embedded in paraffin and sectionalized.Stain with hematoxylin-eosin.Inverted optical microscope was used to observe and collect images.Mmunofluorescence:The slices were placed in antigen repair solution,microwaved for 15 min,cooled,washed,and enclosed with BSA.The diluted anti-NEUN antibody was kept at 4℃overnight,then removed and washed the next day.The second antibody was added and incubated for 50 minutes,followed by DAPI dye for 10 minutes.After drying by shaking,the self-quenching agent was added for 5 min.After washing,anti-fluorescence quenching sealing tablets were used to seal the tablets,and photos were taken under a fluorescent microscope.Transmission electron microscopy:The hippocampus was cut into 1 mm²,fixed with fixative solution under electron microscope,fixed with osmium acid,dehydrated,permeated,and polymerized.Ultrathin sections were observed under transmission electron microscope and images were collected.4.Screening for foodborne functional compoundsRelated databases were searched to identify potential targets（Keywords:heat stress,high temperature environment,cognitive function,learning and memory,attention,etc）.After PPI network analysis,ranking was conducted according to the number of nodes and possible pathways were co-enriched through KEGG.On this basis,compounds matching screening was performed using Drugban and Funfood databases and potential foodborne functional compounds were screened from the database.5.Cells culture and interventionHT22 mouse hippocampal neurons were cultured according to the provided guidelines（high glucose DMEM+10%FBS,37℃and 5%CO₂）.After the degree of cell fusion was about 50%,replace the medium:Control:high glucose DMEM+10%FBS;Heat stress:high glucose DMEM+10%FBS;HT-ac:high glucose DMEM+10%FBS with 10μM HT-ac;HBET:high glucose DMEM+10%FBS with 25μM HBET;HT-ac+HBET:high glucose DMEM+10%FBS with 10μM HT-ac and 25μM HBET.After 24 h,all groups expect control group were treated with heat treatment:43℃,80 min.The next experiment was conducted after the intervention.6.Cell viability,mitochondrial membrane potential,ROS and ATPThe cell viability was detected by CCK8 kit.Mitochondria from hippocampal and HT22 cells were isolated and purified using tissue and cell mitochondrial isolation kit,and mitochondrial membrane potential was used using JC-1 kit.The level of ROS in HT22 cells was detected by ROS kit.The level of ATP in HT22 cells was detected by ATP kit.7.Protein expression detectionTotal protein of mouse hippocampus and HT22 cells was extracted by RIPA lysate containing protease and phosphatase inhibitors.Protein concentration was detected by BCA kit.Add protein loading buffer,mix well,and get test samples after boiling and denaturation.The expression of protein was detected by western blot,and the images were scanned by Odyssey near infrared imager,and the gray level was analyzed by Image J software.8.RNA detectionTotal RNA from hippocampus and HT22 cells was extracted using RNAiso Plus and diluted with RNase-free dd H2O,followed by reverse transcription using a reverse transcription kit according to the manufacturer’s protocol.According to the manufacturer’s protocol,q PCR was performed using SYBR Green Master Mix（with ROX）dye.9.Statistical analysisAll data were statistically analyzed using Graph Pad Prism 8.0 software（Graph Pad Software,San Diego,CA,USA）.Data were presented as mean±standard deviation（SD）of at least three independent trials.All data were tested for normality and homogeneity of variance.Repeated measurement ANOVA was used for the data from the orientation navigation test,One-way ANOVA analyzed all other data,and Tukey multiple comparison method was used to compare different groups.P<0.05 were considered statistically significant.Results1.Heat stress induced cognitive decline and damage to hippocampal neurons in miceBehavioral experiment results showed that compared with the control group,the latency and distance of orientation navigation test increased in heat stress group（P<0.05;P<0.05）;the times of crossing the platform and the percentage of time and distance in the quadrant of the platform of spatial probe test decreased in heat stress group（P<0.01;P<0.05;P<0.05）;the active shuttle times of shuttle box tese decreased in heat stress group（P<0.05）;there was no change in the time of turn bar test（P>0.05）.HE staining showed that the number of neurons in hippocampal decreased and the number of cell layers decreased in heat stress mice.Neu N immunofluorescence showed that the number of mature neurons in the hippocampus was relatively less.2.Prediction and screening of nutrients and foodborne functional compoundsA total of 103 potential targets were identified through database screening and cross-comparison.46 possible pathways were enriched by KEGG analysis.compounds were sorted by the number of important targets in the protein PPI network,if the same,sorted by the number of all targets.After classifying compounds according to their structure,The results showed that flavonoids accounted for 40%,phenolic compounds accounted for 10%,terpenoids and alkaloids accounted for 10%each,amides,glycosides,alcohols,esters,amino acids accounted for 30%.Based on compound sequencing,we selected L-tyrosine,HT-ac,HBET,B vitamins(vitamins B₁,B₂,pantothenic acid,B₆,B₁₂),quercetin,resveratrol,tea polyphenols,genistein,baicalin,luteolin,procyanidin and ningalin as potential screening targets.The orientation navigation test showed that compared with the heat stress group,the latency of B vitamins group and naringin group improved（P<0.05;P<0.01）;the distance of L-tyrosine group,B vitamins group and naringin group improved（P<0.05;P<0.05;P<0.01）.The spatial probe test showed that compared with the heat stress group,the percentage of time and distance in the platform quadrant of HBET,L-tyrosine,B vitamins,quercetin,baicalein,procyanidin and naringin groups increased（P<0.01;P<0.01;P<0.01;P<0.01;P<0.05;P<0.05;P<0.01）;the platform crossing times of HT-ac,L-tyrosine,B vitamin,quercetin,resveratrol,baicalein,procyanidin and naringin groups increased（P<0.01;P<0.05;P<0.05;P<0.01;P<0.05;P<0.05;P<0.01;P<0.01）.Based on the results of behavioral experiments,we selected HT-ac,HBET,L-tyrosine,B vitamins,quercetin,procyanidins and naringin as components of formulation.3.Effect verification of formulationFour formulations:High dose phytochemical formulation（Phy-H,consisted of procyanidins,naringin and quercetin）;Low dose phytochemical formulation（Phy-L,consisted of procyanidins,naringin and quercetin）;Food nutrition fortifier（Nut,consisted of B vitamins and L-tyrosine）;Mitochondrial nutrient formulation（HT-ac+HBET,consisted of HT-ac and HBET）.Compared with the heat stress group,the orientation navigation test showed that the latency of HT-ac+HBET group and Nut group improved（P<0.05;P<0.05）;the distance of HT-ac+HBET group and Nut group improved（P<0.01;P<0.05）.The spatial probe test showed that compared with the heat stress group,the percentage of time and distance in the platform quadrant of HT-ac+HBET group,Phy-H group and Nut group increased（P<0.05;P<0.01;P<0.01）;the platform crossing times of HT-ac+HBET group,Phy-H group and Nut group increased（P<0.01;P<0.01;P<0.05）.The shuttle box tese showed that the active shuttle times of HT-ac+HBET and Phy-H group improved（P<0.01;P<0.05）.The jumping stage test showed that the error times of all formulation groups improved（P<0.01;P<0.05;P<0.01;P<0.001）.4.The combination of HT-ac and HBET improves neuronal morphology and mitochondrial functionHE staining showed that the morphology of hippocampal CA1 region of HT-ac+HBET group was more complete and neurons were arranged more orderly;Neu N immunofluorescence showed that the percentage of mature neurons of HT-ac+HBET group increased（P<0.05）.The transmission electron microscopy showed that the mitochondrial morphology of HT-ac+HBET group was basically intact,the defect of outer membrane and crest was relatively mild.The mitochondrial membrane potential increased significantly in HT-ac+HBET group（P<0.01）.CCK8 results showed that the cell viability of HT22 cells of HT-ac+HBET group was significantly improved（P<0.01）.JC-1,ATP and ROS tests showed that the mitochondrial membrane potential and ATP contents of HT-ac+HBET group increased（P<0.01;P<0.05）,while the ROS decreased（P<0.001）.5.The combination of HT-ac and HBET inhibites the activation of mitochondrial apoptosis pathwayThe q PCR and Western Blot of hippocampus showed that compared with the heat stress group,the expression of Bad,Bax and caspase-3 m RNA decreased（P<0.05;P<0.05;P<0.01）,the expression of bcl-2 m RNA increased（P<0.001）,the expression of Bax,caspase-3 and the content of Cyt C in cytoplasmic decreased（P<0.01;P<0.05;P<0.05）and the expression of Bcl-2 increased（P<0.01）in HT-ac+HBET group.The Western Blot of HT22 cells showed that compared with the heat stress group,the expression of Bax,cleaved-caspase-3 and the content of Cyt C in cytoplasmic decreased（P<0.05;P<0.01;P<0.001）,while the expression of Bcl-2 increased in HT-ac+HBET group（P<0.05）.6.The combination of HT-ac and HBET inhibites the downregulation of PKA/CREB/BDNF pathwayThe Western Blot of hippocampus showed that compared with the heat stress group,the expression of PKA,phosphorylated CREB and BDNF increased in HT-ac+HBET group（P<0.01;P<0.01;P<0.05）.The Western Blot of HT22 cells showed that compared with the heat stress group,the expression of PKA,phosphorylated CREB and BDNF increased in HT-ac+HBET group（P<0.05;P<0.05;P<0.01）.ConclusionOur study investigated the effects of heat stress on cognition of mice;predicted and screened for nutrients and foodborne functional compounds and made up three effective formulations;investigated the mechanism of mitochondrial nutrition formulation.The main conclusions are as follows:1.Heat stress reduced cognition and damaged hippocampal neurons and mitochondria in mice.2.Some formulations composed of nutrients and foodborne compounds（phytochemical formulation,nutrient fortifier formulation and mitochondrial nutrient formulation）could improve the cognition of heat stress mice.3.The combination of HT-ac and HBET may inhibit mitochondrial apoptosis by regulating the expression of Bax/Bcl-2 through the PKA-CREB-BDNF pathway,thus alleviating the damage of hippocampal neurons and improving the cognitive function of heat stress mice.