Regulatory Effect And Mechanism Of L-theanine On Intestinal Mucosal Immunity In Ovalbumin-sensitized Model Mice

The incidence of food allergy disease has continued to rise in the past two decades,which has seriously affected the quality of life of patients and has become a problem that cannot be ignored.It is essentially an adverse reaction caused by excessive specific immunity induced by the intestinal mucosal immune system against food proteins and other allergens.As a characteristic amino acid of tea with various biological activities,L-theanine can not only regulate the internal immune homeostasis of the body,but also has a good immune regulation effect in some in vitro experimental studies.The goal of this study was to construct a BALB/c mouse model sensitized to ovalbumin,to investigate whether L-theanine can alleviate ovalbumin-induced allergic reaction by regulating intestinal mucosal immunity,and further explore its mechanism of action.First,the research conducted scientific experiments in three aspects:allergen dose,model animal gender,and challenge frequency,and optimized the conditions for building allergy models by using the detection of allergic symptoms and related serum indicators.After that,based on the successful establishment of the allergy model,6-week-old BALB/c female mice were intervened by three different doses of L-theanine at low(100mg·kg^-1),medium(200mg·kg^-1),and high(400mg·kg^-1)for 41 days.Food allergy symptoms,inflammatory mediators,specific antibodies,cytokine levels and intestinal pathological changes were evaluated.At the same time,16S r RNA was used to detect the effect of L-theanine on the intestinal microbial composition of allergic mice.In addition,flow cytometry,Western blot and other methods,in order to study the immunoregulatory effect and mechanism of L-theanine on the intestinal mucosa of ovalbumin-sensitized model mice,in order to prevent and treat food allergies,maintain intestinal health balance,and promote the multi-component of L-theanine.Chemical utilization provides relevant theoretical basis.The main findings and conclusions are as follows:（1）Method for successful construction of ovalbumin-sensitized BALB/c mouse model.On the basis of previous studies,an allergy model was established in 6-week-old BALB/c mice by intraperitoneal injection combined with gavage challenge,and three aspects of sex selection,administration dose and challenge frequency were studied in order to construct sensitization Allergy model with good effect and high success rate.By observing allergy symptoms in model mice and detecting OVA-specific IgE levels in serum,female mice were finally selected,the allergen dose was 50μg OVA,and the number of challenges was four for subsequent experiments.（2）The regulatory effect of L-theanine on intestinal immunity in ovalbumin-sensitized mice.Based on the successful construction of the allergic model,L-theanine intervention experiment was conducted,and it was found that L-theanine could alleviate allergic symptoms in mice,reduce intestinal mast cell degranulation and intestinal inflammatory cell infiltration,and reduce intestinal content.The levels of secretory OVA-specific IgA and serum OVA-specific IgE and other antibodies indicated that L-theanine could reduce the humoral immunity associated with allergic reactions.At the same time,L-theanine can significantly reduce the release of allergic mediators such as HIS and m MCP-1 in the serum of mice（P<0.05）,and inhibit the weight loss and immune organ index increase caused by allergic reactions.In addition,the medium and high dose L-theanine groups significantly increased the relative abundance of Lactobacillus in the gut,the high dose group significantly increased the relative abundance of Oscillospira,the low,medium and high doses both groups could significantly reduce the relative abundance of intestinal harmful bacteria like Helicobacter（P<0.05）.（3）The regulatory mechanism of L-theanine on intestinal mucosal immunity in ovalbumin-sensitized BALB/c mice.Compared with normal mice,antigen-presenting cells DC and effector cells MC cells were highly expressed in the intestine when allergic reactions occurred,and the expression levels of each dose of L-theanine group were significantly decreased（P<0.05）.By detecting the changes of T cell subtypes Th1,Th2,Treg,Th17 cells in mesenteric lymph nodes and the corresponding cytokine levels in intestinal tissue,it was confirmed that when food allergic reaction occurs,T cell differentiation changes significantly and Th1/Th2 shifts.The intestinal immune tolerance function of mice was dysfunctional,and the number of Treg cells was significantly reduced（P<0.05）.L-theanine can relatively weaken the Th2 response,improve the Th1/Th2 imbalance,and enhance the Treg response.At the same time,L-theanine nutritional intervention could significantly reduce the overexpression of inflammatory factors such as TNF-αand IL-6 in the intestinal tract of OVA-sensitized mice（P<0.05）.On this basis,the expressions of T-bet,GATA-3,Foxp3 and RORγt related regulatory proteins of T cell differentiation in the intestinal tissue of mice were detected,and the results showed that L-theanine intervention increased intestinal T-The protein expressions of bet and Foxp3 decreased the expression of GATA-3 and RORγt proteins.At the same time,each dose of L-theanine could significantly down-regulate the expression of TLRs/MyD88/NF-κB signaling pathway key proteins TLR2,TLR4,MyD88,and inhibit the phosphorylation of NF-k Bp65 protein（P<0.05）.In summary,L-theanine can affect the process of immune response by reducing the expression of DC and MC in the intestinal lymph nodes of allergic mice,reduce the generation of specific antibodies and the release of allergic mediators;it can regulate the key transcription factor T-bet/GATA-3 promotes the balance of Th1/Th2 cells,regulates the key transcription factor Foxp3/RORγt to promote the balance of Treg/Th17 cells,affects the functional differentiation of T lymphocytes and the expression of related cytokines,and enhances intestinal immune tolerance,weakens intestinal humoral and cellular immune responses,and inhibits the occurrence of allergic reactions;It can inhibit the phosphorylation of NF-k Bp65by down-regulating the expression of key regulatory proteins TLR2,TLR4,and MyD88 on the TLRs/MyD88/NF-κB signaling pathway,block the inflammatory response caused by excessive specific immunity and relieve intestinal tissue damage caused by allergic reactions.