Study On The Efficacy And Mechanism Of Anti-fatigue Nutrients In Regulating The Emotional Cognition Of Rats With Liver Depression And Spleen Deficiency Syndrome Due To Long-distance Voyage Fatigu

ObjectiveTo explore the effect and related mechanism of anti-fatigue nutrition on long voyage fatigue.From the perspective of symptoms and syndromes,brain tissue metabolism,and molecular expression of transmitter pathways,Micro PET-CT,molecular biological experimental methods,UHPLC-MS,literature mining and other technical methods were used comprehensively.To investigate the effects and mechanisms of anti-fatigue nutrition on fatigue and emotional cognitive impairment in rats with long voyage fatigue model,we observed the changes of brain glucose metabolism,GABA/Glu pathway related transmitter receptors and syndrome characteristics from macro to micro,and provided evidence for the study of long voyage fatigue and the application of anti-fatigue nutrition.MethodsLiterature research:1.Systematic retrieval of literatures on the application of TCM compound liver-soothing and spleen-strengthening method,extraction of prescription information and data mining,clustering analysis,factor analysis and other methods to explore the core drugs of liver-soothing and spleen-strengthening and prescription law,providing theoretical guidance for clinical practice.2.Systematic retrieval of relevant literature on syndrome model evaluation of Liver depression and spleen deficiency was conducted to summarize and sort out the disease category,macroscopic indications and application of quantitative indicators of characterization,so as to provide guidance for syndrome evaluation of long-haul fatigue model rats.Experimental study:1.Chemical basis of anti-fatigue nutrition was identified by UHPLC-MS.2.By comparing the indexes related to water maze,open field test and gripping force experiment and serum ALT,AST,BUN,CK and LDH levels of anti-fatigue nutrition(liver-relieving and spleen-strengthening group),Sijunzi Decoction(spleen-strengthening group),Sini SAN(liver-relieving group)and Coenzyme Q10(Western medicine group)in the intervention of long-haul fatigue model rats.To observe the effects of anti-fatigue nutrition on body fatigue,negative emotion and cognitive impairment in rats with long voyage fatigue.3.The syndrome characteristics of rats with long voyage fatigue were observed and evaluated by means of drug counter syndrome of liver-relieving group and spleen-strengthening group and observation scale evaluation of liver depression and spleen deficiency syndrome in rats.4.Small animal positron emission tomography imaging(Micro PET-CT)was used to observe and locate clusters and brain regions with different signals of glucose metabolism in the long-flight fatigue model and the blank group.5.Serum GABA and Glu were detected by ELISA.Histopathological changes of hippocampus and cortex were observed by HE staining.The protein and mRNA expressions of GABAARαl,GABAARγ2,GABABR1,GAD65,GAT-1 and NMDAR2B in hippocampus and prefrontal cortex were observed by WB and PCR.The positive expressions of GABABR and NMDAR2B were observed by immunohistochemistry.To investigate the effect of anti-fatigue nutrition on GABA/Glu pathway in rats with long voyage fatigue.ResultsLiterature research results:1.Data mining results showed that among 154 TCM prescriptions applying liver-soothing and spleen-strengthening method,a total of 103 Chinese medicines were related to homologous drugs of medicine and food and Chinese medicines that could be used in health products,with a total frequency of 1307 times;The medicinal properties of prescription drugs were mainly flat and warm.The most frequently used medicinal flavor was sweet Chinese medicine;The return meridian is mainly spleen and stomach meridian.The efficacy of the drug mainly focuses on antiperspirant,dampness and water,spleen and qi.Systematic cluster analysis and factor analysis showed that the main components of anti-fatigue nutrition were angelica,Fructus aurantii,Astragalus membranaceus and hawthorn,which belonged to different efficacy categories.2.A total of 134 literature related to the evaluation of the syndrome model of Liver depression and spleen deficiency were included in the literature study,including 26 types of quantitative evaluation indexes,among which weight change,food intake,syndrome integral evaluation scale and visceral sensitivity evaluation were widely used.A total of 26 macro indicators were involved in the evaluation of the syndrome model of liver depression and spleen deficiency,with a total application frequency of 546.Among them,macroscopical characterization indexes are widely used,including fur condition,stool condition,mental condition,activity state,reaction to stimulus,etc.Experimental results:1.After detection and identification,the main chemical components of anti-fatigue nutrition agent contain maulanin,citric acid,schisandraine,glucoside,ferulic acid,naringin,nehesperidin.2.The results showed that the serum AST,BUN,LAC and LDH levels were increased in rats with long voyage fatigue(P<0.01,P<0.001),and the serum AST and LDH levels were down-regulated by anti-fatigue nutrition(P<0.001).The value of grip strength in the model group was decreased(P<0.001),while that in the anti-fatigue nutrition group was increased(P<0.05).Open field test showed that long voyage fatigue model rats,stay in the middle of the total time,movement distance and central wear number percentage,total number,number of upright,standing time,the number of modification,and modification time were significantly lower(P<0.001,P<0.05),compared with model group,the anti-fatigue nutrients group rats upright,the indexes of standing time,number of modifications and modification time were significantly increased(P<0.001,P<0.01,P<0.05),and the center lattice retention time,total distance,percentage of center lattice penetration times and total lattice penetration times were increased(P<0.01,P<0.001).In the water maze experiment,compared with the control group,the escape latency in the target quadrant of the long endurance fatigue model rats was significantly prolonged(P<0.05),and the swimming time,The times of reaching the platform and crossing the quadrant in the target quadrant of the platform were significantly reduced(P<0.001).The indexes of escape latency,target quadrant swimming time,platform times and cross quadrant times in the anti-fatigue nutrition group were significantly improved(P<0.001,P<0.01,P<0.05).In the index of target quadrant swimming time,the improvement effect of liver-nourishing group was better than that of liver-nourishing group and western medicine group.3.Long voyage fatigue syndromes in rats to observe,to change the observation scale hint at long voyage fatigue rats sleep lazy lie and thin pond stool frequency increased,fur yellow,dry,scattered impurity,irritability and easily capture resistance strong,anxiety reactions to noise sensitivity is abate,upright times is significantly reduced,Chi-square test of the frequency of the items mentioned above indicated that there were statistically significant differences among each group(P<0.001).It was found that Sijunzi Decoction(spleen-invigorating group)and Sini Powder(liver-soothing group)could alleviate long voyage fatigue to varying degrees,and the occurrence of long voyage fatigue may be related to the dysfunction of liver and spleen.4.Micro PET-CT study showed that there were 8 clusters with significantly different signals in the blank group and the long voyage fatigue model group,and the brain regions with different metabolism were mainly involved in the thalamic nucleus group,hippocampus,amygdala,cerebellum,hypothalamus,cortex and other 25 brain regions.The brain regions with high frequency were hippocampus,posterior cerebellar lobe,posterior splenic cortex,anterior cerebellar lobe and corpus callosum.5.HE staining showed that glial cells were damaged in hippocampus and prefrontal cortex.WB and PCR results showed that the balance of Glu and GABA in rats with long voyage fatigue was abnormal.Compared with blank group,the protein and mRNA expressions of GABAARal,GABAARγ2 and GABABR1 in hippocampus and prefrontal lobe of rats with long voyage fatigue model group were significantly increased(P<0.001,P<0.01,P<0.05),GAT-1 and NR2B protein and mRN A expression decreased(P<0.001,P<0.05).Anti-fatigue nutrition group can down-regulate the protein expression of GABAARγ2(P<0.01)and GABABR1(P<0.001)and GAD67(P<0.05)in hippocampus and prefrontal lobe of long voyage fatigue model rats.Down-regulated the mRNA expression.of GABABR and Gababrl in hippocampus(P<0.001)and GABAARy2 in prefrontal cortex(P<0.01),and up-regulated the mRNA expression of GAD67 in prefrontal cortex(P<0.001).The expression of GABA/Glu pathway related receptor protein arid mRNA in SNS group,SJZT group and CoQ10 group was consistent with the trend of anti-fatigue nutrition.In addition,the SNS group could down-regulate the expression of GAD67 protein in hippocampus.The mRNA expression of GABAARal and GABAARγ2 and the mRNA expression of NMDAR2B in the prefrontal cortex were down-regulated(P<0.05).The protein and gene levels of NMDAR2B in the positive drug group were different from those in the model group(P<0.01,P<0.05).The improvement of Glu receptor protein NMDAR2B and GABA transporter GAT-1 was not obvious in most treatment groups.ConclusionLong voyage fatigue model rats increased body fatigue,negative emotions and the decline in cognitive function,the metabolism of glucose uptake difference brain regions involved in hippocampus and the amygdala,cerebellum,hypothalamus,cortex,thalamus,in areas such as the nuclear group of the mechanism of the excessive activation of GABA can system and Glu can inhibit of the system,and its performance in accordance with liver depression syndrome characteristics of spleen deficiency.Anti-fatigue nutrition treatment can effectively improve the body fatigue,cognitive function and emotional state of rats with long haul fatigue,and also can regulate the changes of glutamate and y-aminobutyric acid pathway related receptor transmitters in long voyage fatigue state.The main components of anti-fatigue nutrition include maulanin,citric acid,schisandraine,glucoside and so on.