Exploring The Effect Of Hypoxia On The Colonic Mucosal Barrier Based On TLR4/NF-κB Signaling Pathway

Objective:The hypoxic environment at altitude can lead to a series of gastrointestinal discomfort symptoms and the occurrence and development of gastroenteritis,in which damage to the immune barrier of intestinal mucosal plays a key role.The TLR4/NF-κB signaling pathway is an important immune regulatory pathway in vivo.This study aimed to investigate the effect of hypoxia on the colonic mucosal barrier,and reveal the immunological mechanism of hypoxia affecting the colonic mucosal barrier through the TLR4/NF-κB signaling pathway.Methods:A hypoxia model simulating plateaus and a lipopolysaccharide(LPS)-induced inflammation model were established.The damage effects of hypoxia and LPS on mice colon were studied by measuring the length of the mice colon,analyzing the colon histomorphology with hematoxylin eosin(HE)staining,and detecting the expression levels of mice colonic tight junction proteins ZO-1 and Occludin with Western blot and immunohistochemistry(IHC)staining.At the cellular level,the human colorectal adenocarcinoma cell line Caco-2 cells with intestinal epithelial-like characteristics were intervened by hypoxia and LPS.The proliferation and apoptosis of Caco-2 cells were assessed by MTT cell proliferation assay,flow cytometry and the expression level of apoptotic protein Cleaved-Caspase 3with Western blot.In addition,the expression levels of tight junction protein were also detected by Western blot in Caco-2 cells.Based on the above,to study the regulation mechanism of hypoxia on colon innate immune response,the m RNA and protein levels of TLR4,MyD88,TRIF and NF-κB p65 were detected by quantitative real-time polymerase chain reaction(q RT-PCR)and Western blot.The m RNA levels of inflammatory factors TNF-α,IL-1β,IL-6 and IFN-γ were measured by q RT-PCR.Results:By studying the effects of hypoxia and LPS on the colon mucosal barrier of mice,it was found that hypoxia exposure had no significant effect on the length of the colon of mice,while the colon length of mice injected with LPS were significantly shortened.The mice exposed to hypoxia showed colon mucosal structure disorder and partial mucosal detachment,while mice injected with LPS exhibited inflammatory infiltration of the colonic mucosa.The expression levels of colonic tight junction proteins were significantly reduced in mice injected with LPS,indicating that LPS significantly disrupted the tight junction structure of mice colon.Hypoxia can significantly aggravate the damaging effect of LPS on the tight junction structure of the colon.By studying the effects of hypoxia and LPS on the expression of TLR4/NF-κB signaling pathway and its downstream inflammatory factors in mice colon,we found that LPS significantly up-regulated the expression levels of TLR4/NF-κB signaling pathway molecules and downstream inflammatory factors in mice colon.Hypoxia significantly inhibited the protein expression levels of mice colon TLR4/NF-κB signaling pathway molecules.Moreover,hypoxia exposure had a significant inhibitory effect on the LPS-induced activation of the TLR4/NF-κB signaling pathway and the expression of downstream inflammatory factors such as IFN-γ in mice colon.At the cellular level,hypoxia decreased cell viability and increased cell apoptosis of human Caco-2 cells.LPS intervention significantly increased the apoptosis rate of Caco-2 cells,and hypoxia exacerbated LPS-induced apoptosis in Caco-2 cells.LPS significantly down-regulated the expression levels of tight junction proteins in Caco-2cells,and hypoxia plus LPS further reduced the expression level of Occludin protein.LPS significantly up-regulated the expression levels of TLR4/NF-κB signaling pathway molecules and downstream inflammatory factors in Caco-2 cells.The expression levels of TLR4/NF-κB signaling pathway proteins were significantly reduced in Caco-2 cells after 48 h of hypoxia exposure.Hypoxia could significantly down-regulated LPSinduced activation of the TLR4/NF-κB signaling pathway and the expression of downstream inflammatory factors such as TNF-α and IFN-γ in Caco-2 cells.Conclusions:Hypoxia caused damage to the colonic mucosal barrier and promoted apoptosis of colonic epithelial cells.Hypoxia weakens the host colonic defense response by downregulating the colonic TLR4/NF-κB signaling pathway,which may lead to increased susceptibility and low clearance efficiency of the colon to pathogens.