Study On The Mechanism Of Sinapic Acid On The Repair Of Tight Junction Injury Of Intestinal Epithelial Cells Induced By LPS

Objective: To investigate the anti-inflammatory effect in vitro of sinapic acid in TLR4/NF-?B/MLCK-p MLC pathway and its protective effect on the high permeability of LPS-induced Caco-2 cells.Methods: Caco-2 cells induced inflammation under the action of LPS to establish a cellular inflammation model,and the concentration gradients of 5 ?mol/L? 10 ?mol/L and 15 ?mol/L sinapic acid were respectively treated and cultured for 24 h for follow-up experiments.Cell viability was determined by MTT assay.Cellular immunofluorescence was used to detect the nuclear transport of NF-?B p65 and the distribution of Claudin-1 and ZO-1.The m RNA expressions of TLR4?NF-?B p65?IL-8?IL-1??Occludin?Claudin-1 and ZO-1 were detected by q-PCR.The protein expressions of major intercellular Occludin?Claudin-1?ZO-1 and major signaling factors including TLR4?My D88?p-NF-?B?NF-?B?I?B?p-I?B?IKK??p-IKK??MLCK?MLC were detected by Western-blot.Results: The results of MTT assay showed that the cell survival rate decreased for 24 hours after LPS treatment compared with the control group.5 ?mol/L?10 ?mol/L?15 ?mol/L of sinapic acid can promote cellproliferation in 24 hours,but with the extension of the action time,cell proliferation was inhibited.The results of cell immunofluorescence detection showed that NF-?B p65 in the control group was present in the cytoplasm,and NF-?B p65 in the model group was transferred to the nucleus.However,in the sinapic acid treatment group,the nuclear transfer of NF-?B p65 was reduced,and the change was most significant in the treatment group with 15 ?mol/L of sinapic acid.The results of q-PCR showed that,compared with the control group,the m RNA expression levels of Occludin?Claudin-1 and ZO-1 in the model group were decreased(P<0.05),and the m RNA expression levels of TLR4?NF-?B p65?IL-8 and IL-1? were increased(P<0.05).Compared with the model group,the m RNA expression levels of Occludin?Claudin-1 and ZO-1 in sinapic acid treatment group were increased,and the increase effect was most significant in the treatment group with 15 ?mol/L of sinapic acid(P<0.05).While,the m RNA expression levels of TLR4 ?NF-?B p65?IL-8 and IL-1? were decreased(P<0.05),and the changes in expression levels had dose-effect.The results of Western-blot showed that,compared with the control group,protein expression levels of TLR4?My D88?p-NF-?B?p-I?B?p-IKK??MLCK and MLC were increased in the model group(P<0.05).Compared with the model group,the protein expression levels of TLR4?My D88?p-NF-?B?p-I?B?p-IKK??MLCK and MLC in sinapic acid treatment group were decreased(P<0.05).Theexpression of Occludin ? Claudin-1 and ZO-1 in the model group,compared with the control group,were decreased(P<0.05).After treatment with sinapic acid,the protein expression levels increased compared with the model group,and the effect of 15 ?mol/L sinapic acid treatment was the most significant(P<0.05).Similarly,in the detection of Claudin-1 and ZO-1 proteins distribution by cellular immunofluorescence,we found that treatment with sinapic acid can prevent the uneven distribution of fluorescent signals of cell tight junction protein to some extent and reverse the redistribution of Claudin-1 and ZO-1 proteins in LPS-induced Caco-2 cells.Conclusions: Sinapic acid has a strong anti-inflammatory activity.By regulating TLR4/NF-?B/MLCK-p MLC pathway,sinapic acid can reduce the expression of inflammatory factors,up-regulate the m RNA and protein expression of tight junction related proteins in Caco-2 cells,and improve the occurrence of LPS-induced high intercellular permeability in Caco-2 cells.