The Regulatory Mechanism Of Lipopolysaccharide On The Barrier Function Of The Intestinal Mucus Layer In The Co-culture Of Caco-2/HT-29 Cells

Endotoxin is a component of the outer membrane of all Gram-negative bacteria,it is considered to be a class of nonspecific biomolecules released after bacterial cell death.The biochemical characterization of endotoxin is Lipopolysaccharide(LPS).There are trillions of symbiotic bacteria in the intestinal tract of mammals,intestinal is the largest reservoir of LPS in the animal body.Damage to the intestinal mucosa allows LPS molecules to enter the bloodstream,causing endotoxemia.The intestinal mucus layer separates commensal bacteria and endotoxins from internal tissues to resist the invasion of exogenous bacteria and intestinal microorganisms,and plays an important role in maintaining intestinal microecological balance.However,how does LPS affect the structure of the intestinal mucosal barrier,and the possible mechanism of mucin MUC2,an important component of intestinal mucus layer,is still unclear.In this paper,Caco-2/HT-29 co-cultured cells were used as an in vitro test model,and techniques such as CCK8,Alcian/PAS staining,immunofluorescence,si RNA,RNA-seq,ELISA,and fluorescence quantitative PCR were used,elucidated the effect of LPS on intestinal epithelial mucus layer and intercellular tight protein,and in the absence of MUC2,LPS effects on the intestinal epithelial barrier and its mechanisms.The results obtained are as follows:1.The expression levels of three genes MUC2,MUC5 AC and ALPi in 3:1(Caco-2:HT-29)and 9:1(Caco-2:HT-29)co-cultured cells were detected by q PCR,it was proved that 3:1(Caco-2:HT-29)expresses higher mucin m RNA than 9:1(Caco-2:HT-29),which is a better ratio.CCK8 cell proliferation toxicity test confirmed that 400 μ g/m L was the most suitable challenge dose,the 3:1(Caco-2:HT-29)co-cultured cells were treated with medium supplemented with400μg/m L LPS for 12 h,24h,36 h,and 48 h,and then stained with Alcian blue and PAS,respectively,the results showed that the co-cultured cells in the control group secreted the most acidic mucin and mucopolysaccharide at 24 h,and LPS promoted the expression of acidic mucin and mucopolysaccharide.Immunofluorescence staining showed that LPS damaged tight junction protein Occludin at 24 h,36h and 48 h.The results of q PCR and ELISA showed that LPS could significantly increase(P<0.05)the expression of MUC2 m RNA and protein at 24 h.2.The expression of MUC2 gene in Caco-2/HT-29 co-cultured cells was silenced by si RNA technology.The negative control si RNA labeled with FAM fluorescent label was observed to have high transfection efficiency under fluorescence microscope,q PCR detected that the expression of MUC2 gene was extremely significantly reduced after transfection compared with the control group(P<0.01).RNA-seq results showed that compared with LPS(-),LPS(+)group had less differential gene numbers and was enriched to less GO functional annotations and KEGG signaling pathway,regardless of whether the LPS(+)+si RNA group was compared with the LPS(-)group,or the LPS(+)+si RNA group was compared with the LPS(+)group,the number of differential genes,enriched GO functional annotations and KEGG signaling pathway numbers increased significantly.It showed that LPS had almost no effect on the intestinal epithelium when the intestinal mucus was intact,but when MUC2(the main component of intestinal mucin)was absent,the effect of LPS on the intestinal epithelium was aggravated.3.Through the data mining of the KEGG signaling pathway enrichment analysis in the LPS(+)group and the LPS(+)+si RNA group,this experiment found that in the case of MUC2 silencing,the focal adhesion and ECM receptor interaction signaling pathways were significantly different,the focal adhesion pathway was enriched with38 differentially expressed genes and 5 new genes,and the ECM-receptor interaction pathway was enriched with 28 differentially expressed genes and 1 new gene.Key factors in the differential signaling pathway were verified by q PCR,and the results were consistent with RNA-seq.In this experiment,the m RNA expression of intercellular connection factors Claudin-1,ZO-1,JAMA,desmosome,Occludin and E-cadherin were also detected in LPS(+)group and LPS(+)+si RNA group,the expression levels of Claudin-1,ZO-1,JAMA,desmosome,Occludin and E-cadherin in LPS(+)+si RNA group were significantly decreased(P<0.01),indicating that LPS enhanced the damage of intercellular connections under the reduced expression of MUC2,LPS disrupted tight junctions、adhesion junctions and desmosomes.In conclusion,LPS promotes the secretion of MUC2 from the intestinal goblet cells,protecting the intestine from LPS damage.After silencing the expression of the major intestinal mucin MUC2 gene by si RNA,LPS caused a large number of gene expression differences,and the effect of LPS on the intestinal epithelium was aggravated,among them,the expression of some genes in the focal adhesion and ECM receptor interaction pathway was significantly reduced.