| Research BackgroundFood allergy(FA)has become an important public health problem that affects both children and adults.In recent decades,the incidence of food allergy has been increasing at a high rate worldwide,especially in industrialized countries and regions.Theoretically,any food can be a potential allergen and cause allergic symptoms ranging from mild to severe.Food allergy can affect different organs and systems,most commonly the gastrointestinal tract,but also the skin,cardiovascular system,respiratory system,and even the whole body.Food allergy is an adverse immune reaction to a specific food.The study of its pathogenesis is of great significance for safe and effective clinical treatment.Food allergy can be divided into three categories:immunoglobulin E(IgE)-mediated,non-IgE-mediated,mixed IgE and non-IgEmediated.IgE-mediated food allergy is the most common,which is characterized by the production of food allergen-specific IgE,the polarization of Th2 cells,the excessive production of Th2 cytokines,and the release of inflammatory mediators by effector cells such as mast cells.However,the mechanisms underlying why some people are susceptible to certain food allergens remain unclear.Recently,an increasing number of studies have shown that allergic constitution is related to barrier epithelial dysfunction.Barrier epithelial cells recognize pathogen-associated molecular patterns(PAMPs)through pattern-recognition receptors(PRRs)and generate epithelial cell-derived cytokines(ECDCs)in response to environmental danger signals.As the central regulator of food allergy,intestinal epithelial cells(IECs)not only constitute the first barrier against food antigens but also initiate,regulate and aggravate allergic reactions by producing ECDCs including thymic stromal lymphopoietin(TSLP),interleukin-25(IL-25),and interleukin-33(IL-33).ECDCs can act on a variety of innate and adaptive immune cells,including dendritic cells(DCs),T cells,type 2 innate lymphoid cells,mast cells,eosinophils and so on.As critical proallergic factors,ECDCs control the fate of downstream allergic responses.The abnormal production of ECDCs is an essential initiator and amplifier in the pathogenetic process of food allergy.Therefore,specific therapies targeting ECDCs are emerging as new strategies for the treatment of food allergy.However,most of the current studies focus on the regulatory role of ECDCs in the downstream allergic response;the generation of ECDCs and their underlying mechanisms remain unclear.Oxytocin(OT),which was the first peptide hormone to be biochemically described and synthesized,is produced mainly by magnocellular neurons in the hypothalamus and released into the systemic circulation to exert multiple functions.The classic functions of OT are the stimulation of uterine contractions during childbirth and milk ejection during breastfeeding.With further research,OT has been found to play multiple functions under various physiological and pathological conditions,including regulating maternal and social behaviors,the autonomic nervous system,and the immune system.Furthermore,the utilization of OT or its analogs has been identified as a potential clinical intervention and has made significant progress in clinical trials for the treatment of autism and schizophrenia.Recently,OT has been proposed to be used as a "natural medicine" with anti-inflammatory and antioxidative effects and is considered to have a certain potential in the treatment of immune diseases.However,most of the relevant studies are focused on infectious diseases and mental disturbances related to immune disorders.More studies are needed to explore the role of OT in autoimmune diseases,hypersensitivity,immune deficiency and transplant rejection.Studies from our research group and other investigators have confirmed that OT is expressed in enteric neurons and that oxytocin receptor(OTR)is expressed in the intestinal epithelium.Previously,our research group found that OT can regulate the functions of macrophages and mast cells in experimental colitis.However,the role of the OT/OTR signaling system in food allergy,especially in the secretion of ECDCs during allergy,is still unclear.Through the use of clinical samples,animal models and cell experiments,this study systematically explored the role and underlying mechanism of OT/OTR signaling in food allergy and ECDCs production,with the aim of providing a novel strategy and theoretical basis for the treatment of food allergy.Part 1 The regulatory effect of oxytocin in food allergyObjectivePrevious studies have shown that OT has anti-inflammatory effects,but its function in food allergy remains unclear.In this part of the study,clinical samples and animal models were used to explore whether OT is involved in the regulation of food allergy and the secretion of ECDCs.Methods1.The collection of clinical samples.13 children with food allergy and their 12 agematched healthy controls,12 adults with food allergy and their 11 age-matched healthy controls were selected as the research subjects.Plasma was collected from the subjects in all groups for follow-up detection.2.The establishment of an animal model of food allergy.On days 0 and 14,BALB/c mice were sensitized by two intraperitoneal injections of ovalbumin(OVA)and aluminum potassium sulfate adjuvant mixture.Beginning on day 28,the OVA solution was prepared and gavaged every other day.The incidence of diarrhea,fecal characteristics,and allergic symptoms were observed and recorded.The mice of each group were sacrificed after the final intragastric administration,and blood and intestinal tissue were collected for follow-up detection.3.OT treatment in the OVA-induced food allergy model.Mice were induced by OVA.Beginning on day 0,mice were intraperitoneally injected with 1 mg/kg OT once a day.The incidence of diarrhea,fecal characteristics,and allergic symptoms were observed and recorded.The mice of each group were sacrificed after the final intragastric administration,and blood and intestinal tissue were collected for follow-up detection.4.Hematoxylin-eosin staining was used to observe the pathological changes in jejunal morphology and inflammatory cell infiltration.5.Toluidine blue staining was used to observe the number,morphology and degranulation of mast cells in the jejunum.6.The target proteins in human and mouse samples were detected by ELISA.These measurements included the OT concentration in human plasma,the OT concentration in the serum and jejunum tissue homogenate of mice,the levels of antibody and mouse mast cell protease-1(mMCP-1)in the serum of mice,the levels of Th2 cytokines and histamine in the jejunum tissue homogenate of mice,and the ECDCs levels in the jejunal mucosa of mice.7.Western blot was used to detect OTR expression in the jejunal mucosa of mice.Results1.Plasma OT concentration is abnormally elevated in patients with food allergyTo explore whether OT is involved in the regulation of food allergy,the plasma from children and adults with food allergy and age-matched healthy controls were collected and detected.Compared to those of healthy controls,the plasma OT concentrations in both children and adults with food allergy were significantly elevated,suggesting that OT is involved in regulating food allergy.2.The expression of OT and OTR is abnormally elevated in OVA-allergic miceWe established an animal model induced by OVA.The serum and intestinal tissues of OVA-allergic mice and their control group were collected and detected.The OT levels in the serum and jejunum of OVA-allergic mice were significantly increased.The expression of OTR in the intestinal epithelium of the jejunum was increased.These results indicate that OT/OTR signaling is upregulated in OVA-allergic mice.3.OT administration alleviates the allergic symptoms induced by OVAAbnormal upregulation of OT/OTR signaling in clinical samples and animal models suggests that OT is involved in the regulation of food allergy.Therefore,we further explored the function of OT in food allergy by injecting OVA-allergic mice with 1 mg/kg OT.The results showed that continuous OT administration significantly reduced the incidence of allergic diarrhea and relieved the allergic symptoms induced by OVA.Histological evaluation showed that continuous OT administration significantly alleviated jejunal damage and reduced the inflammatory cell infiltration induced by OVA.These results indicate that OT treatment may ameliorate the allergic symptoms induced by OVA.4.OT administration attenuates the allergic response in OVA-allergic miceThe antibody levels,Th2 cytokine levels and mast cell status were detected to evaluate the severity of the allergic response.The results showed that continuous OT administration reduced the levels of IgE and OVA-IgE in serum and decreased the production of IL-4,IL-5,IL-9 and IL-13 in the jejunum of OVA-allergic mice.The numbers of mast cells and the release of mMCP-1 and histamine were also decreased by OT administration.These results indicate that OT treatment may alleviate the allergic response induced by OVA.5.OT administration inhibits the production of ECDCs in OVA-allergic micePrevious studies have shown that ECDCs are key upstream mediators in initiating and aggravating allergic responses.The jejunal mucosa of each group was isolated and collected,and the expression of ECDCs was detected.The results showed that continuous OT administration significantly reduced the production of TSLP,IL-25,and IL-33 in the jejunal mucosa of OVA-allergic mice.We speculated that the inhibitory effect of OT on the allergic response might be mediated by its regulation of the secretion of ECDCs,which will be explored further.Part 2 The regulation of oxytocin on the expression of epithelial cell-derived cytokines and its underlying mechanismObjectiveOT can inhibit the secretion of ECDCs,but the mechanism is not clear.Through cell experiments and animal models,this part of the study explored the function and underlying mechanism of OT/OTR signaling in regulating the ECDCs expression in IECs,with the aim of providing potential new targets for the treatment of food allergy.Methods1.The human intestinal cell line HT-29 was obtained by serial subcultivation in vitro.2.The expression of OTR and β-arrestin2 in HT-29 cells was downregulated by the small interfering RNA technique.3.The establishment of an animal model of food allergy is the same as in Part 1.4.The levels of TSLP,IL-25 and IL-33 in HT-29 cell lysates were detected by ELISA.5.The levels of NF-κB phosphorylation,IκB-α protein and β-arrestin2 protein in HT-29 cell lysates and jejunal mucosal homogenates of mice were detected by Western blot.Results1.The production of TSLP,IL-25,and IL-33 is stimulated by LPSTo further study the effect of OT on the production of ECDCs in the intestinal epithelium,we selected human intestinal epithelial HT-29 cells for in vitro experiments.OVA contains a small amount of LPS,which is a common factor in stimulating barrier epithelial cells to produce ECDCs.Therefore,HT-29 cells were stimulated with 0.2,2,or 20 mg/ml OVA and 10,100,or 1000 ng/ml LPS to detect the expression of ECDCs in HT-29 cells.The results showed that both 2 or 20 mg/ml OVA and 100 or 1000 ng/ml LPS could stimulate the expression of TSLP,IL-25,and IL-33 in HT-29 cells.Therefore,100 ng/ml LPS was used in the follow-up experiments to simulate the effect of OVA.2.OT inhibits LPS-stimulated ECDCs expression through its receptor OTRFirst,we confirmed whether HT-29 cells expressed OTR.The results showed that HT-29 cells constitutively expressed OTR,as detected by Western blot.Then,we detected the changes of ECDCs in HT-29 cells after incubation with 10-6,10-7,10-8 or 10-9 M OT.The results showed that the expression of TSLP,IL-25 and IL-33 in LPS-stimulated HT-29 cells was significantly reduced by pretreatment with 10-7 M and 10-8 M OT.To identify the receptor that OT interacts with in this process,OTR siRNA was used to downregulate OTR expression in HT-29 cells,and its transfection efficiency was detected.Then,HT-29 cells were transfected with OTR siRNA and its control Scr siRNA,and the concentration of OT of each group was detected.After transfection with OTR siRNA,the inhibitory effects of 10-8 M OT pretreatment on the expression of TSLP,IL-25,and IL-33 in LPS-stimulated HT-29 cells were abolished.These results indicate that OT/OTR signaling exerts an inhibitory effect on TSLP,IL-25,and IL-33 expression.3.OT inhibits LPS-stimulated ECDCs expression through β-arrestin2β-arrestin2 is a key protein of the non-G-protein dependent pathway that mediates the intracellular signaling of G-protein-coupled receptor(GPCR)activation.The expression of βarrestin2 in HT-29 cells was downregulated by LPS stimulation.After incubation with 10-8 M OT,the LPS-induced downregulation of β-arrestin2 protein was significantly alleviated.To further clarify the role of β-arrestin2 in OT/OTR regulation of ECDCs expression,the expression of β-arrestin2 in HT-29 cells was downregulated by βarr2 siRNA and its transfection efficiency was detected.Then,HT-29 cells were transfected with Parr2 siRNA and its control Scr siRNA,and the concentration of OT of each group was detected.After transfection with Parr2 siRNA,the inhibitory effects of 10-8 M OT pretreatment on LPS-stimulated TSLP,IL-25,and IL-33 expression in HT-29 cells were abolished.These results indicate that β-arrestin2 is an important mediator of the regulation of TSLP,IL-25 and IL-33 expression by OT/OTR signaling in HT-29 cells.4.OT/OTR signaling inhibits the LPS-stimulated ECDCs expression by suppressing the NF-κB pathwayPrevious studies have shown that ECDCs expression is regulated by the NF-κB pathway.These results showed that LPS induced the phosphorylation of NF-κB and the degradation of IκB-α in HT-29 cells.After blocking the NF-κB pathway with Bay 11-7082,the expression of TSLP,IL-25 and IL-33 were no longer affected by LPS.These findings suggest that LPS stimulated ECDCs expression through the NF-κB pathway.Then,we found that incubation with 10-8 M OT inhibited LPS-stimulated NF-κB phosphorylation and IκB-α degradation in HT-29 cells.After transfection with OTR siRNA in HT-29 cells,the above effects of 10-8 M OT were abolished.After transfection with βarr2 siRNA in HT-29 cells,the inhibitory role of 10-8 M OT on NF-κB phosphorylation was also abolished.These results suggest that OT/OTR signaling may inhibit ECDCs expression through the NF-κB pathway,in which β-arrestin2 plays an important role.5.OT administration suppresses the NF-κB pathway and upregulates β-arrestin2 expression in OVA-allergic miceWe further validated the above pathway in the OVA-induced food allergy model.The jejunal mucosa of each group was isolated and collected,and the levels of β-arrestin2 protein and NF-κB pathway-related protein were detected.In OVA-allergic mice,NF-κB phosphorylation and IκB-α degradation were upregulated,and β-arrestin2 expression was downregulated.The above changes were significantly inhibited by continuous OT administration,which was consistent with the results in HT-29 cells.Part 3 The role of the endogenous oxytocin signaling system in food allergy and its underlying mechanismObjectiveOur previous studies demonstrated that OT alleviated food allergy and inhibited ECDCs expression through the OTR-β-arrestin2-NF-κB pathway.This part of the study explored the role and underlying mechanism of OT/OTR signaling in food allergy through the use of OTR knockout mice,providing new ideas and theoretical bases for the treatment of food allergy.Methods1.OTR knockout mice were established.OTR-/-mice were obtained by crossing OTRfl/fl mice and ubc-Cre mice.Littermates(OTRfl/fl mice)were used as controls.2.OVA-induced OTR-/-mice were prepared in the same way as in Part 1.3.Jejunal tissues from OTR-/-mice and OTRfl/fl mice were stained with hematoxylin-eosin and toluidine blue.Pathological changes in the jejunal morphology,inflammatory cell infiltration,and the number and degranulation of mast cells were observed.4.Target proteins in OTR-/-mice and OTRfl/fl mice were detected by ELISA.These proteins included the levels of antibody and mMCP-1 in serum,the levels of Th2 cytokines and histamine in the jejunum tissue homogenate,and the levels of ECDCs in the jejunal mucosa.5.The levels of NF-κB phosphorylation,IκB-α protein and β-arrestin2 protein in jejunal mucosa of OTR-/-mice and OTRfl/fl mice were detected by Western blot.Results1.OTR-/-mice develop food allergy induced by OVATo explore the role of OT/OTR signaling in the pathogenesis of food allergy,OTR-/-mice were established.Compared to their littermates,OVA-induced OTR-/-mice exhibited a significant increase in the occurrence of allergic diarrhea.OVA-induced OTR-/-mice displayed severe liquid stool and allergic symptoms.OVA-induced OTR-/-mice exhibited severe jejunal tissue damage and increased inflammatory cell infiltration.These results suggest that endogenous OT has a protective effect on food allergy.2.OTR-/-mice induced by OVA show an active allergic responseTo assess the severity of allergic responses,antibody levels,Th2 cytokine levels and mast cell status were detected.Compared to their littermates,OVA-induced OTR-/-mice exhibited significantly increased levels of IgE and OVA-IgE in the serum and increased production of IL4,IL-5,IL-9 and IL-13 in the jejunum.The numbers of mast cells and the release of mMCP-1 and histamine were also increased in OVA-induced OTR-/-mice.These results suggest that endogenous OT/OTR signaling inhibits the allergic response induced by OVA.3.OTR-/-mice induced by OVA express abnormally elevated levels of ECDCsTo further explore the role of OT/OTR signaling in food allergy,the jejunal mucosa of OTR-/-mice and their littermates was isolated and collected and the expression of ECDCs was detected.Compared to their littermates,OVA-induced OTR-/-mice exhibited significantly increased levels of TSLP,IL-25,and IL-33 in the jejunal epithelium.These results suggest that OT/OTR signaling may inhibit the production of ECDCs in vivo.4.OTR-/-mice induced by OVA exhibit abnormal activation of the NF-κB pathway and downregulation of β-arrestin2To further explore the mechanism of OT/OTR signaling in vivo,the jejunal mucosa of OTR-/-mice and their littermates was isolated and collected,and the levels of β-arrestin2 protein and NF-κB pathway-related protein were detected.Compared to their littermates,OVA-induced OTR-/-mice exhibited significantly activated IκB-α degradation,upregulated NF-κB phosphorylation,and downregulated β-arrestin2 expression.These results suggest that OT/OTR signaling may inhibit the NF-κB pathway and upregulate β-arrestin2 expression in the intestinal epithelium.Research Conclusions1.The OT/OTR signaling system has an antiallergic effect.In animal models,OT administration may significantly alleviate allergic reactions and suppress intestinal inflammation and ECDCs secretion.Moreover,OTR-/-mice develop more severe allergic reactions and exhibit increased intestinal inflammation and ECDCs secretion.These findings suggest that both exogenous and endogenous OT have antiallergic effects.2.The OT/OTR signaling inhibits ECDCs expression.Both in vitro and in vivo experiments suggest that OT relies on OTRs to inhibit TSLP,IL-25 and IL-33 expression in IECs via the inhibition of the NF-κB pathway and the upregulation of β-arrestin2.3.Plasma OT is elevated in patients with food allergy,and the expression of OT and OTR is upregulated in an animal model of food allergy.These findings suggest that OT may be used as one of the biomarkers for the occurrence of food allergy. |
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