The Mechanism Study Of Probiotic Intervention In Food Allergy Correlated With Caesarean Section

Background:Food allergy,which is also called allergic reaction of the digestive system,is an adverse reaction to ingested food protein mediated by the gut-associated lymphoid tissues(GALT).The major route of exposure to food allergens occurs via the gastrointestinal tract.Symptoms of allergic responses to ingested foods include nausea,vomiting,diarrhea,stomach discomfort,food refusal,early satiety,dysphagia,food impaction,hematochezia,and dysmotility(regurgitation and constipation).GALT constantly samples foreign antigens and has developed to distinguish between potentially harmful pathogens and innocuous antigens in order to build a protective immune response or to intentionally ignore benign antigens like food or commensal microorganisms.Oral tolerance is the default immunological response in the gut,characterized by active systemic unresponsiveness to ingested dietary antigens.Food allergy,on the other hand,occurs when oral tolerance fails.Food allergies are most common in infants and young children,with an estimated prevalence of 5%～10%in childhood,as a result of the immaturity of the gut barrier and the immune system in these age groups.And children with food allergies face food and social restrictions,which may lead to impaired quality of life and even threats to life.Thus,the problem of food allergy needs to be solved.Strict avoidance of allergens is the most effective means to deal with food allergies,but allergens are unpredictable and unknown,so it is urgent for clinicians to develop effective treatment methods.Epinephrine,antihistamine,and other relief are often used after the occurrence of food allergies.However,currently available drugs only control the symptoms of food allergies but do not address the underlying immune disorder.Therefore,an in-depth understanding of the molecular mechanism of food allergy and the development of new therapeutic methods from the perspective of immunity have become the focus and hotspot of food allergy research.In recent years,the disruption in the diversity and/or function of intestinal microbiota,referred to dysbiosis,in the development of food allergies has attracted increasing attention.Evidence exists for altered gut microbiota in children with food allergy compared with that of healthy children,and the regulation of oral tolerance by gut bacteria and the transmission of food allergy by fecal transfer have been further confirmed in germ-free mice.Although germ-free animals are ideal models for studying the role of introduced microbiota in the course of food allergy,the lack of a complete micro-ecosystem of their own makes it uncertain whether dysbiosis will produce long-term allergy-related effects in a more clinical setting.The fast growth of intestinal microbiota in babies occurs throughout the first few years of life.The number of immune cells formed within a certain window period will impact the host’s lifetime immunity and susceptibility to illness,thanks to the influence of gut microbiota.Recent decades have witnessed an increasing prevalence of delivery by caesarean section(CS)across the globe.CS leads to a distinct pattern of microbiota colonization in newborns,exhibiting a lower relative abundance of Lactobacillus,Prevotella,and Bifidobacterium compared to babies born by vaginal delivery(VD).Furthermore,a growing body of work has implicated CS in food allergy and several other immune-mediated diseases,including asthma,inflammatory bowel disease,eczema,atopic dermatitis,and immune deficiencies.However,CS-induced changes in the gut microbiota have been neglected in clinical and preclinical research on food allergy.In this work,we employed an OVA(ovalbumin)-induced allergic model to investigate the impact of CS on experimental food allergy and gut microbiota in rats and the role of probiotic intervention for microbiota differences between these two delivery modes in reversing such effects.This study shows a relationship between birth mode and the severity of food allergy from the perspective of intestinal microbiota.Part I Effects of caesarean section on ovalbumin-induced food allergyAims:1.To analyze the influence of delivery modes on food allergy in rats.2.To detect the expression of T helper 2(Th2)cell cytokines and tight junction proteins in the ileum of rats with different delivery modes.Methods:1.The VD-OVA group and the CS-OVA group were given 1 mg OVA for 49 days from the 21st day,while VD-PBS group and the CS-PBS group were given a constant volume of solvent daily.On Day 70,rats in each group were challenged by gavage of 100 mg OVA.2.The rectal temperature of the allergic group and the control group was measured immediately and 25 min after challenge respectively,and the temperature difference was calculated.The diarrhea of each group was observed and the stool score was made.3.Blood was collected from the tail vein of rats,and the levels of OVA-specific immunoglobulin E(OVA-sIgE),OVA-sIgG,and histamine in serum were detected by Enzyme-linked immunosorbent assays(ELISA).4.The rats were sacrificed 8 h after challenge and ileal tissues were collected.The levels of histamine were detected by ELISA.The levels of Th2 cytokines interleukin-4(IL-4)and IL-10 in tissues were detected by ELISA and Quantitative Real-Time Polymerase chain reaction(qRT-PCR).Western blot and Immunohistochemistry(IHC)staining were used to detect the expression of tight junction proteins ZO-1,occludin,and claudin-1.Results:1.Compared with VD-OVA group,CS-OVA group had decreased rectal temperature(P<0.01),more severe diarrhea(P<0.01),higher OVA-sIgE and OVA-sIgG levels(P<0.05),and higher histamine levels in tissue and serum(P<0.05).2.IHC staining and Western blot results showed that OVA sensitization caused the reduced levels of tight junction proteins ZO-1,occludin,and claudin-1 in CS rats,but no such phenomenon occurred in VD rats.3.qRT-PCR and ELISA showed that the expression of Th2 cytokines IL-4 and IL-10 in ileum of CS-OVA rats was significantly higher than that of VD-OVA rats(P<0.05).Conclusion:1.Rats delivered by CS were found to be more responsive to OVA sensitization than vaginally born ones and have more severe symptoms.2.Abnormal Th2 responses and intestinal barrier damage are characteristics of CS-mediated food allergies.Part II Effects of different delivery modes on intestinal microbiota of ratsAims:1.To evaluate the effects of different delivery modes on intestinal microbiota of rats.2.To screen the characteristic bacteria related to the immune reaction in food allergy.Methods:1.Intestinal contents or fecal samples of rats in each group were collected on Day 7,21,and 70 for 16S Ribosomal RNA(rRNA)high-throughput sequencing to assess the microflora differences between VD and CS rats from a diversity,β diversity,composition,and LEfSe analysis.2.Spearman correlation analysis was performed on protein levels of IL-4 and IL-10 in the top 30 genera on Day 70.Results:1.Compared with VD rats,the Shannon,Ace,and Chao indexes of CS rats were significantly increased on Day 7(P<0.001),and β diversity was significantly different on Day 7(P<0.01)and Day 21(P<0.01).OVA sensitization had a greater impact on the intestinal microbiota of CS rats on Day 70.2.Community composition analysis showed that Firmicutes were dominant in both CS and VD rats.On Day 7,the relative abundance of Firmicutes in CS rats was significantly lower than that in VD rats(P<0.001).And on Day 21,the abundance of Actinomycetes in CS group was significantly lower than that in VD group(P<0.05).At the genus level,Lactobacillus(Day 7 and Day 21)and Bifidobacterium(Day 21)in CS rats were significantly lower than those in VD.3.Ruminococcus,norank_f_Muribaculaceae,and unclassifed_f_Prevotellaceae,the abundance of which had significant positive correlations with IL-4 and IL-10 protein levels,were enriched in CS_OVA rats on Day 70.Conclusion:1.The microbiota structure of CS and VD rats was significantly different.2.The reduction of Lactobacillus and Bifidobacteria in the early life of CS rats was consistent with that of CS infants,indicating that the CS rat model can be used to study the effect of probiotic intervention on CS-mediated intestinal microbiota difference.3.Genera highly associated with allergic Th2 cytokines were enriched in the gut of CS rats.Part III Probiotic intervention alleviate food allergy symptoms correlated with caesarean section and its mechanismsAims:1.To clarify the effect of probiotics intervention on CS-related food allergies.2.To explore the mechanisms of probiotics in regulating CS-related food allergies.Methods:1.From birth to Day 21,CS rats in the experimental group were intragastrically fed with a probiotic mixture daily,while rats in the control group were intragastrically fed with a constant volume of solvent every day.All rats were set to OVA modeling for 49 days later.2.Stool samples were collected on Day 21 and Day 70 for 16S rRNA high-throughput sequencing and analysis.3.After OVA modeling,symptoms were evaluated in the experimental and control groups.The levels of antibodies,histamine,tight junction protein,and Th2 cytokine were measured.Results:1.Probiotic intervention changed α diversity(P<0.05)and β diversity(P<0.01)of CS rats on Day 21.The relative abundance of Ruminococcus(P<0.05),norank_f_Muribaculaceae(P<0.05),and unclassifed f Prevotellaceae(P=0.2956)decreased after OVA sensitization.2.Food allergy symptoms were relieved,and levels of antibodies and histamine(P<0.01)decreased significantly in the experimental group compared with that of the control group.3.Probiotic intervention restored the barrier proteins ZO-1,occludin,and claudin-1(P<0.01),and reduced the expression of Th2 cytokines IL-4 and IL-10(P<0.05).Conclusion:1.Probiotic intervention can partially offset the impact of CS on microbiota,alleviate food allergy symptoms,providing a basis for probiotics intervention in CS infants.2.Probiotic intervention can alleviate food allergy by inhibiting harmful bacteria in the intestine,regulating Th2 response,and enhancing the expression of intestinal tight junction proteins.