Protective Effects Of 6-shogaol On Tight Junctions Of Caco-2 Cells Against Palmitic Acid-induced Damages Via MiR-216a-5p

Tight junctions(TJs)among intestinal epithelial cells are the major components of the intestinal mechanical barrier,and maintaining their structural and functional integrity are vital for intestinal homeostasis and prevention of intestinal disorders-related diseases.However,TJ-associated proteins are highly susceptible to external stimuli,among which high-fat diet is one of the main causes.Studies have shown that excessive intestinal inflammatory response triggered by high-fat diet is an important mechanism underlying intestinal TJ damage.Therefore,screening natural active ingredients that are able to inhibit excessive intestinal inflammatory response is of significance for the intervention of intestinal barrier dysfunctionrelated diseases.Our previous study found that 6-shogaol,the active ingredient in ginger,has strong in vitro anti-inflammatory activity.On this basis,in this study,palmitic acid was used to stimulate human colon cancer epithelial Caco-2 cells to construct a TJ injury model under the high-fat diet mode.The protective effect of 6-shogaol on Caco-2 cells against palmitic acid-induced TJ damage was studied by analyzing cell integrity,permeability,expression and localization of TJ proteins,inflammatory factors and related regulatory signaling pathways.Combined with bioinformatics analysis and cell transfection inhibition experiments,the protective mechanism of 6-shogaol was discussed from the perspective of micro RNA regulation.The main research contents and results are as follows:(1)Toxicity of 6-shogaol towards Caco-2 cells and choose of its concentration.After 21 days of Caco-2 cell culture,both TEER value and cell permeability index(Papp)were determined,indicating that the single layer cell model was successfully constructed.After treatment with 6-shogaol and palmitic acid,the cell survival rate was analyzed by MTT method.The results showed that the survival rate of the cells was higher than 97% when the concentrations of 6-shogaol and palmitic acid were 0.625-2.5 μM and 100-400 μM,respectively,indicating that neither 6-shogaol nor palmitic acid had toxic effect on the cells.Therefore,0.625,1.25,2.5 μM of 6-shogaol and 400 μM palmitic acid were selected for the subsequent experiments.(2)Protection of 6-shogaol against palmitic acid-induced TJ damage of Caco-2 cell.By measuring TEER and Papp values,it was found that 400 μM palmitic acid significantly reduced the monolayer integrity of Caco-2 cells(TEER decreased by 48%)and increased the cell bypass permeability(Papp increased by 4.1 times).Compared with palmitic acid injury group,6-shogaol significantly(P < 0.05)restored monolayer integrity and reduced monolayer permeability in a dose-dependent manner.q RT-PCR and WB were used to detect the effect of6-shogaol on the m RNA and protein expression of TJ in Caco-2 cells.The results showed that compared with the control group,palmitic acid treatment significantly down-regulated m RNA and protein expression of TJ-associated proteins,i.e.,Claudin-1,Occludin and ZO-1.6-Shogaol significantly(P < 0.05)restored their m RNA and protein expression levels,and 2.5μM of 6-shogaol treatment group presented the most significant effect.Furthermore,the cell immunofluorescence experiment showed that the palmitic acid treatment showed low expression of Claudin-1,Occludin and ZO-1,but their expression was recovered when 2.5μM of 6-shogaol was added.Moreover,6-shogaol treatment significantly inhibited palmitic acid-induced activation of myosin light chain kinase(MLCK).(3)6-Shogaol alleviates palmitic acid-induced inflammation by inhibiting TLR4/My D88/NF-κB signaling pathway.The effects of 6-shogaol on the levels of major inflammatory cytokines(IL-6,IL-1β and TNF-α)were detected by q RT-PCR and ELISA.Compared with the control group,palmitic acid significantly increased the levels of these inflammatory cytokines,while 6-shogaol significantly decreased their levels.The effect of 6-shogaol on TLR4/ My D88/NF-κB signaling pathway was determined by q RT-PCR and WB.The results showed that compared with the control group,the palmitic acid treatment group had higher expression levels of TLR4 and My D88 and phosphorylation levels of NF-κB.However,6-shogaol treatment significantly inhibited the activation of TLR4/My D88/NF-κB signaling pathway.(4)Screening and functional analysis of TLR4-targeting mi RNAs.Eight candidate mi RNAs that targeted TLR4 were screened using bioinformatics prediction tools.Further PCR experiments showed that mi RNA-216a-5p was upregulated most by 6-shogaol.To further analyze the role of mi RNA-216a-5p in the 6-shogaol’s protective effects,mi RNA-216a-5p inhibitor was pre-transfected into cells,and results showed that the 6-shogaol’s protective effect disappeared or was significantly suppressed,as evidenced by the decreased monolayer integrity,increased permeability,downregulated TJ proteins(Claudin-1,Occludin and ZO-1),activated TLR4/My D88/NF-κB and MLCK signaling pathways,and increased pro-inflammatory cytokines IL-6,IL-1β and TNF-α.These results suggest that the protective effect of 6-shogaol on palmitic acid-induced intestinal TJ injury is attributed to the inhibition of excessive cellular inflammatory response and mi RNA-216a-5p.