The Effect And Mechanism Of L-Theanine On Glutamine Metabolism And Immune Function In SD Rats

Glutamine,as a conditionally essential amino acid,plays an important role in the body’s immune system.Glutamine is normally supplied by the body itself without exogenous supplementation,but when the body suffers from stress or trauma,the immune system and organs such as the liver,kidney and intestine have an increased demand for glutamine and require exogenous supplementation.Supplementation of exogenous glutamine in organisms with severe metabolic stress can alleviate the glutamine deficiency in the body and meet the metabolic demands of the immune system and gastrointestinal tract.L-theanine is a characteristic amino acid in tea,and the molecular structure and physiological function are all quite similar to glutamine,which has many health effects such as regulating body nutrient metabolism,improving immune function,antioxidation,and improving cognition.It has been speculated that L-theanine acts on immune function by regulating glutamine metabolism in the body,but few related studies have been reported.In this study,low(300 mg/kg·d^-1),medium(600 mg/kg·d^-1)and high(900 mg/kg·d^-1)doses of L-theanine,L-glutamine and their combinations(L-theanine 300 mg/kg·d^-1+L-glutamine 600 mg/kg·d^-1,L-theanine 450mg/kg·d^-1+L-glutamine 450 mg/kg·d^-1,L-theanine 600 mg/kg·d^-1+L-glutamine 300 mg/kg·d^-1)were fed to normal or E44813-stressed 8-week-old SD male rats for 21 days,and the effects of L-theanine on glutamine metabolism and immune function in normal and E44813-stressed rats were investigated.Based on the investigation of the regulatory effects of L-theanine on glutamine metabolism and immune function,these groups were selected from the normal and E44813 groups,and the four groups with better effects in the normal and E44813stressed groups by L-theanine intervention.Blot,computer simulated molecular docking and cellular level targets were used to verify the predicted mechanisms,and then the regulatory effects and mechanisms of L-theanine on glutamine metabolism and immune function in SD rats were investigated.The main findings and conclusions are as follows:（1）Effect of L-theanine on glutamine metabolism in normal rats.Compared to the normal group,the medium dose of L-theanine significantly increased the ratio of intestinal to plasma glutamine content and the length of intestinal villi and crypt depth（p<0.05）;glutamine synthetase activity in skeletal muscle was increased in each dose group of L-theanine（p<0.05）,and there was a tendency to decrease glutamine synthetase protein expression,and the change in intestinal glutaminase activity was not significant,and glutamine protein expression was decreased（p<0.05）;these data indicated that L-theanine has the effect of increasing the activity of glutamine synthetase in skeletal muscle and intestinal glutaminase unit protein,and the effect was better with medium dose of L-theanine.（2）Effects of L-theanine on immune function and glutamine metabolism in intestinal stress rats.The levels of intestinal inflammatory factors IL-1β,IL-6 and TNF-αwere significantly higher in SD rats treated with E44813 stress once for 7 days than their levels in the normal group（p<0.05）,indicating that the E44813 intestinal stressed rat was successfully modeled.In the E44813-stressed organism,L-Theanine intervention could promot the growth of intestinal villi length and crypt depth（p<0.05）,increase the activities of catalase and superoxide dismutase in serum（p<0.05）,decrease the content of malondialdehyde,a product of membrane lipid peroxidation（p<0.05）,and down-regulate the expression of pro-inflammatory factors TNF-αand IL-1βin the intestine（p<0.05）;compared with exogenous supplementation of L-glutamine in E44813 stressed organisms,exogenous supplementation of the same dose of L-theanine was more effective in increasing superoxide dismutase activity and down-regulating IL-1βexpression;meanwhile,the optimal dose of L-theanine was 600mg/kg·d^-1,and the same effect of L-theanine was achieved at 900 mg/kg·d^-1 of L-glutamine;compared with high dose of L-glutamine alone,the combination of L-theanine and L-glutamine showed a stronger modulatory effect on the relief of inflammation in the organism（p<0.05）,the effect of L-theanine in alleviating inflammation was superior to that of glutamine in oxidatively stressed organisms,and to some extent,it could act as a substitute for glutamine,suggesting that the pathway of action of L-theanine on immune function in oxidatively stressed organisms may not through glutamine metabolism.Based on the above results,the normal group,medium-dose L-theanine group,E44813-stressed group and E44813+medium-dose L-theanine group were selected for histological analysis.（3）The protein pathway of L-theanine regulating glutamine metabolism and immune function in E44813-stressed rats was predicted based on metabolomic and proteomic.Compared with the normal group,the differential metabolites after L-theanine intervention were mainly enriched in pathways such as protein digestion and absorption,amino acid metabolism and pyrimidine metabolism,and the differential proteins were mainly enriched in pathways such as pyruvate metabolism,glycolysis,nitrogen metabolism,carbon metabolism,amino acid metabolism,purine metabolism and the ECM receptor signaling pathway;Compared with the E44813-stressed group,the differential metabolites in the E44813-stressed body with L-theanine intervention were mainly enriched in pathways such as steroid hormone biosynthesis and amino acid metabolism,the differential proteins were mainly enriched in pathways such as MAPK signaling pathway,PI3K-Akt signaling pathway,PPAR signaling pathway,ECM receptor interaction pathway,VEGF signaling pathway,TNF signaling pathway,GABAergic synapse,Glutamatergic synapses and pathways such as oxidative phosphorylation.Among the proteomics results of the two comparison groups:E44813-stressed group vs.normal group and E44813+L-theanine group vs.E44813-stressed group,three differential proteins with opposite trend of expression changes were picked out:Atp2b4,Pdcd4 and Tspan8,and the differential metabolites associated with them were 2-AG.Combining the above results with the literature,the predicted pathway of L-theanine regulating the immune function in E44813-stressed body:L-theanine inhibits the stimulation of VEGFR2 by VEGF through m Glu R3,downregulates the expression of PLC to inhibit the conversion of pi to 2-AG,prevents the signaling of CB1 receptor,upregulates the expression of PDCD4 by COX-2 inhibition,and finally regulates the body immune function by downregulating TNF-αand upregulating the expression of IL-10;The predicted pathway of L-theanine regulating glutamine metabolism in the E44813-stressed organism:L-theanine inhibits signaling through the CB1 receptor via the same pathway described above,relieves the inhibition of ERK1/2 phosphorylation by CB1,and finally affects glutamine metabolism by inhibiting the expression of GS proteins.（4）Validation of the predicted pathways of L-theanine regulating glutamine metabolism and immune function in normal and E44813-stressed rats.The results of Western blot showed that treatment of normal organism with E44813 stress increased the expression of VEGFR2（p<0.05）,decreased the expression of COX-2（p<0.05）,increased the expression of Pdcd4（p<0.01）,and decreased the expression of p-ERK1/2（p<0.01）;After L-theanine intervention in E44813 stressed organisms,there was a trend towards lower VEGFR2 expression,but the effect was not significant,increased COX-2 expression（p<0.01）,decreased Pdcd4 expression（p<0.05）and increased p-ERK1/2 expression（p<0.05）,the levels trends of p-ERK1/2,COX-2 and Pdcd4 were all consistent with the predicted mechanistic pathways;after L-theanine intervention in normal organisms,there were no significant changes in the expression of CB1and COX-2,Pdcd4 expression decreased（p<0.01）and p-ERK1/2 expression decreased（p<0.01）,which were largely consistent with the predicted pathways.The results of molecular docking showed that the binding free energy of L-theanine to m Glu R and CB1 were-15.6kcal/mol and-39.2 kcal/mol,respectively,the binding possibility of L-theanine to CB1 was greater than L-theanine to m Glu R3.The results of cellular assays showed that COX-2expression and IL-10 levels increased in normal cells after L-theanine or Rimonabant hydrochloride intake（p<0.05）,which is consistent with the immune predicted pathway of L-theanine-CB1-COX2-Pdcd4-NFκB-IL10;COX-2 expression and IL-10 levels were increased（p<0.05）and TNF-αlevels were decreased（p<0.05）after the action of L-theanine or Rimonabant hydrochloride on LPS-stressed cells,consistent with the predicted immune pathway of L-theanine-CB1-COX2-Pdcd4-NFκB-IL10,TNFα;The expression of ERK1/2was essentially unchanged,the proportion of p-ERK1/2 to total ERK1/2 decreased（p<0.05）and the expression of GS increased（p<0.05）after L-theanine or Rimonabant hydrochlorid intake in normal cells,consistent with the predicted pathway of L-theanine-CB1-Gβy-p ERK1/2-GS;After the action of L-theanine or Rimonabant hydrochloride on LPS stressed cells,ERK1/2 expression were basically unchanged,the proportion of p ERK1/2 to total ERK1/2 increased（p<0.05）and GS expression decreased（p<0.05）,consistent with the L-theanine-CB1-p ERK1/2-GS pathway.In conclusion,in normal organisms,L-theanine competitive binding to CB1,inhibits the ERK1/2 phosphorylation pathway via Gβy,affecting the expression of GS,a key enzyme in glutamine metabolism,and thus regulating glutamine metabolism;at the same time,after L-theanine antagonized CB1 activity,it relieved the inhibition of COX-2 expression by CB1,down-regulated the expression of Pdcd4 and NFκB,and finally improved the immune function of the body by enhancing the expression of anti-inflammatory factor IL-10.In E44813-stressed organisms,L-theanine affected the glutamine key enzyme GS by inhibiting the activity of CB1and promoting the nuclear transfer of p ERK1/2,which in turn regulated glutamine metabolism;meanwhile,after L-theanine antagonizes CB1 activity,it upregulates the expression of anti-inflammatory factor IL-10 and downregulates the expression of pro-inflammatory factor TNFαthrough the same COX2-Pdcd4-NFκB pathway as in the normal organism,ultimately achieving the effect of improving the immune function of the organism.