Effert And Mechanism Of The Lactobacillus GG Alleviate Necrotizing Enterocolitis

BackgroundNecrotizing enterocolitis(NEC)is a severe inflammatory bowel disease in the neonatal period,predominantly affecting preterm or low birth weight infants,characterized by high incidence,high mortality,complex pathogenesis,and long-term sequelae.NEC occurs postnatally,with bacterial communities rapidly proliferating in the neonate’s intestine.Its nonspecific early symptoms include feeding intolerance,abdominal distension,and/or bloody stools,typically progressing rapidly to intestinal perforation,systemic hypotension,and sepsis,requiring immediate medical and surgical intervention.Pathological features primarily display various inflammatory cell infiltrations in the mucosa,intestinal mucosal and submucosal hemorrhage and edema,with dark red mucosa,and as the disease worsens,superficial mucosal necrosis or full-thickness intestinal wall necrosis occurs.The current pathogenesis of NEC remains unclear,but host-microbial interactions play an important role in the onset and progression of NEC.In these interactions,microbial dysbiosis leads to changes in barrier permeability,loss of integrity,intestinal inflammation,and bowel wall necrosis.Therefore,investigating the interrelationship between the intestinal microenvironment and NEC is crucial.This study is divided into two parts.The first part validates human NEC and rat NEC models,observes pathological sections,and verifies changes in rat permeability and tight junction proteins(TJs).The second part introduces Lactobacillus rhamnosus(LGG)and evaluates its effects on NEC by observing TJs,intestinal permeability,intestinal microbiota,and the Wnt signaling pathway.Part Ⅰ:Establishment of NEC Rat Model and Investigation of Changes in Intestinal Barrier FunctionObjective1.Investigate gross specimens and HE staining scores of NEC patients and rat NEC models,validating NEC model establishment.2.Utilize in vivo intestinal permeability experiments to explore changes in intestinal permeability in normal control rats and NEC rats.3.Investigate the expression of the main components of TJs,namely claudin-1,occludin,and ZO-1,in normal control rats and NEC rats.Method1.Human tissue sections were obtained from pathological sections of the small intestine perforation site in NEC patients at Qilu Hospital of Shandong University,while control group sections were from patients with intestinal atresia.Animal tissue sections were obtained from NEC rats and normal control newborn rats’ small intestine tissues.HE staining and NEC scoring was used as evaluation criteria.2.Four hours before euthanasia,rats were orally administered fluorescein isothiocyanatedextran 4(FD4).Blood samples were collected from the orbital vein 4 hours later,and FD4 levels in rat serum were measured.3.Small intestine tissues were collected at the time of euthanasia,and the expression levels of claudin-1,occludin,and ZO-1 in NEC rats and normal control newborn rats were measured using the WB method.Result1.HE staining revealed intact villi,well-defined layers,and columnar villi protruding into the intestinal lumen in both human and rat normal groups,with little or no inflammatory cell infiltration.In NEC patients and NEC rat groups,hemorrhage,edema,dark red mucosa,discontinuous villi,and extensive inflammatory cell infiltration were observed in the mucosal and submucosal layers.2.In vivo intestinal permeability experiments were performed on normal newborn rats and NEC rats.ELISA results indicated significantly elevated FD4 levels in the serum of NEC rats compared to the control group(P<0.01).3.WB analysis was performed on small intestinal tissues of normal newborn rats and NEC rats,revealing that the expression levels of the main components of TJs,namely claudin1,occludin,and ZO-1,were significantly reduced in NEC rats compared to the control group(P<0.01).Conclusion1.A successful NEC rat model can be created by combining multiple factors,such as a high osmotic diet,hypoxia,and cold stimulation.2.In the model,the rats’ intestinal barrier function is weakened,intestinal wall permeability is increased,and tight junction proteins are decreased.Part Ⅱ:LGG Improves Intestinal Barrier Function through the Wnt Signaling Pathway Acting on ISCsObjectiveBased on previous research,this study aims to clarify the effects of Lactobacillus rhamnosus on NEC and intestinal repair mechanisms through various approaches,including histopathological scoring of intestinal tissues,intestinal stem cell(ISC)differentiation expression,Wnt/β-catenin signaling pathway,and changes in intestinal permeability.Method1.Establish the NEC model using the methods from Part 1 and record the mortality rate during modeli Employ immunofluorescence IF staining to detect the fluorescence staining and intensity of LGR5 and BMI1 in each group.2.At the time of euthanasia,retain small intestine tissues from each group for HE staining.Use HE staining as the NEC scoring criterion.3.Use the WB method to detect the expression of various proteins,including claudin-1,occludin,ZO-1,LGR5,BMI1,Wnt3a,Wnt7b,and β-catenin,in the small intestinal tissues of each group.4.IF staining to detect the fluorescence staining and intensity of LGR5 and BMI1 in each group.5.Retain colon feces at the time of euthanasia,and use 16S rDNA high-throughput sequencing to detect intestinal microbiota composition.6.Measure the levels of IL-6 and TNF-α in the serum of the three rat groups using the ELISA method.Result1.Compared to the control group,the NEC group showed poorer general conditions,increased intestinal permeability,higher mortality rate during modeling,and decreased levels of various proteins,including LGR5,BMI1,Wnt3a,and Wnt7b(P<0.01).Additionally,serum levels of inflammatory factors IL-6 and TNF-α were elevated(P<0.01).2.After adding LGG,the survival rate of the LGG+NEC group newborn rats increased compared to the NEC group(P>0.05).HE staining pathological scores improved(P<0.05),and the expression levels of various proteins,including claudin-1,occludin,ZO-1,LGR5,BMI1,Wnt3a,Wnt7b,and β-catenin,were elevated(P<0.05 or P<0.01).Intestinal permeability in the rats was ameliorated(P<0.01).3.16S rDNA high-throughput sequencing of the intestinal microbiota revealed that the intestinal microbial composition in the NEC+LGG group rats was reshaped compared to the NEC and control group rats.The expression of serum inflammatory factors IL-6 and TNF-αwas reduced(P<0.01).Conclusion1.LGG can activate the Wnt signaling pathway,promote ISC proliferation,and facilitate the recovery of small intestinal villi.2.LGG reshapes the intestinal microbiota,improves intestinal barrier function,and reduces NEC damage.3.LGG can serve as a supplementary and alternative functional food for the prevention and treatment of NEC.