Baicalin Protects Against TNF-?-induced Injury By Regulating MicroRNA In IEC-6 Cells

Background and objectiveThe objective of this study was to investigate the mechanism of the protective effect of baicalin on TNF-?-induced injury in intestinal epithelial cells.Metabolic inflammation is a low degree,chronic systemic inflammation triggered by ingestion of nutrients and metabolic excess,which lead to increase of inflammatory molecules such as tumor necrosis factor(TNF),interleukin 6(IL-6)and C-reactionprotein(CRP).Apart from saturated fatty acids and other endogenous damage,exogenous toxins such as gram-negative bacterial cell wall lipopolysaccharide(LPS),are also related with metabolic inflammation.Intestinal endotoxin activated TNF-a-mediated defects in tight junctions of enterocytes,leading to intestinal barrier dysfunction,increased intestinal permeability,thereby increasing endotoxin levels in blood circulation and intestinal.Recent studies have shown that atherosclerosis,nonalcoholic fatty liver disease,diabetes mellitus and obesity possess the common characteristics of metabolic inflammation.Chinese medicine baicalin has been confirmed to have extensive anti-inflammatory effect.MicroRNAs are a class of small,noncoding RNAs that regulate gene expression and play an important role in a series of processes such as cell proliferation,differentiation,apoptosis and metastasis.We have shown earlier that baicalin reduced the level of LPS induced by high fat diet in mice,reversed the down-regulation of the TJ protein ZO-1 and decreased TNF-? level after LPS stimulation in I EC-6 cells,thereby maintaining the integrity of endothelial barriers.However,whether baicalin directly mediates through the regulation of TNF-?-related pathway on the cell function is to be further studied.In this study,bioinformatics analysis technique was used to explore the mechanism of baicalin on intestinal permeability,and to provide a theoretical basis for the application of traditional Chinese medicine on anti-metabolic inflammation.Methods1.Protective effects of baicalin on TNF-?-induced IEC-6 cellsThe effect of baicalin on the proliferation of TNF-?-induced IEC-6 cells was evaluated using CCK-8 assay.The effect of baicalin on the permeability of TNF-?-induced IEC-6 cells was detected by cell resistance meter.FITC Annexin V and PI staining were used to detect the apoptotic rate by flow cytometry.2.Bioinformatics analysis of differences in the expression of miRNA in IEC-6 cells induced by TNF-? and baicalinIEC-6 cells were divided into TNF-? group and TNF-? + baicalin group.TNF-? group was treated with 50 ng/mL TNF-? for 24 h,TNF-?+ baicalin group was pretreated with baicalin(40 ?g/mL)for 1 h before exposure to 50 ng/mL TNF-? for 24 h.RNA was extracted for microRNA sequencing.The differentially expressed miRNA target genes were predicted by bioinformatics analysis,the GO function and KEGG pathway enrichment analysis of differentially expressed miRNA target genes were performed by DIANA TOOLS mirPath v.3.0 software.3.Baicalin protects against TNF-?-induced injury by down-regulating miR-191a in IEC-6 cells(1)Effect of baicalin on expression of tight junction protein in IEC-6 cells induced by TNF-?IEC-6 cells were divided into three groups,normal group,TNF-? model group,TNF-? + baicalin group.According to the results of bioinformatics analysis,the differentially expressed miRNAs were predicted to be closely linked to proteins Z0-1,Claudin-Claudin-2 and Claudin-8,the expression of TJ proteins was assayed by Western Blot.The distribution of Z0-1 cells was detected by immunofluorescence.(2)Effects of baicalin on TNF-?-induced Z0-1 expression by regulating miR-191aMiR-191a mimics and inhibitors were transfected into IEC-6 cells for 24 h.On the other hand,IEC-6 cells were divided into normal control group,baicalin group,TNF-? group,TNF-? +baicalin group,TNF-? +miR-191a inhibitor group,TNF-? +baicalin+miR-191a inhibitor group,TNF-? + baicalin+miRNA inhibitor negative control group,TNF-? + miR-191a mimic group,TNF-a +baicalin+miR-191a mimetic group,TNF-? +baicalin+miRNA mimics negative control group,the expression of miR-191a and target gene ZO-1 was detected by real-time PCR and Western Blot.(3)Effects of baicalin on TNF-?-induced activity and migration injury in IEC-6 cells through miR-191a Cells were divided into normal control group,baicalin group,TNF-?group,TNF-a +baicalin group,TNF-a +miR-191a inhibitor group,TNF-?+baicalin +miR-191a inhibitor group,TNF-?+ baicalin +miRNA inhibitor negative control group.Cell viability was measured by CCK-8 method,and the cell migration ability was measured by wound scratch healing assay.Results1.Different concentrations of baicalin(10,20,40 ?g/mL)did not show any inhibitory effects on the proliferation of IEC-6 cells compared with the normal control group,viability of cells exposed to increasing concentrations of TNF-?(5,10,30,50 ng/mL)for 24 h was 98.26±0.73,94.22±2.03,84.95±1.20,and 74.94±1.78%of the control group respectively(Fig.1B).In cells pretreated with baicalin(10,20,40 ?g/mL)the viability rose to 82.60±2.56,83.94±2.51 and 92.25±1.63%respectively.40 ?g/mL baicalin pretreatment inhibited TNF-?-induced high permeability and decrease the apoptosis rate in IEC-6 cells.2.MiRNA expression analysis between baicalin pretreatment group and TNF-a group showed that there were 40 differentially expressed miRNAs.Compared with TNF-a group,7 miRNAs were significantly up-regulated and 33 miRNAs were down-regulated after baicalin pretreatment,KEGG pathway analysis showed that the target gene regulated by baicalin was closely related to the pathway of cell adhesion,cell gap junction pathway,cell adhesion molecule pathway,FoxO signal pathway,microRNA on tumor pathway and phosphoinositol metabolism pathway.The enrichment of GO gene in the intersection of miRNAs suggested that baicalin was probably involved in regulation of cell structure,biological process,molecular function,nucleus and protein-related celll function.3.Compared with normal group,TNF-? treatment significantly decreased the expression of ZO-1 and increased expression of Claudin-2(P<0.05),while the expression of' Claudin-1 and Claudin-8 showed no significant difference.Compared with TNF-? group,the expression of Claudin-1 and Claudin-8 did not significantly affected by baicalin pretreatment,however,baicalin pretreatment significantly reversed the effect of TNF-? on the expression of ZO-1 and Claudin-2(P<0.05).Immunofluorescence staining showed that the overall level of ZO-1 was reduced with uneven distribution in cells treated with TNF-? only,whereas in cells pretreated with baicalin before TNF-? treatment the ZO-1 protein was continuous and homogeneous.Transfection with miR-191a mimic resulted in an obvious increase in miR-191a level and a.concomitant decrease in ZO-1 level when compared with the negative control.Conversely,transfection with the inhibitor showed an inverse correlation between the expression of miR-191a and ZO-1 protein level in IEC-6 cells(P<0.05).There was no significant effect of baicalin alone on the expression of ZO-1 in IEC-6 cells.Compared with normal control,the expression of miR-191a significantly increased and ZO-1 significantly decreased after treatment with TNF-? for 24 h,baicalin pretreatment significantly decreased miR-191a]evel,reversed the effect of TNF-? on ZO-1,both at the mRNA and protein levels(P<0.01).Compared with TNF-?+ baicalin group,miR-191a inhibitor significantly enhanced ZO-1 protein expression that was restored by baicalin.In contrast.,miR-191a mimic significantly suppressed the baicalin mediated reversion of ZO-1 inhibition(P<0.05)4.Scratch experiments showed that haicali;alono(40 ?g/mL)had no effect on the cell migration rate,the motility of IEC-6 cells was significantly inhibited after stimulation with TNF-a,when compared to this,the migration ability of the cells pretreated with baicalin was obviously enhanced.Moreover,transfected with miR-191a inhibitor before baicalin and TNF-? treatment resulted in obvious increase in the migration of IEC-6 cells(P<0.05).Conclusion1.TNF-? inhibits cell migration ability of IEC-6 cells partly by increasing the level of miR-191a,decreasing the expression of target gene tight junction protein Z0-1.TNF-? inhibits proliferation,promotes cell apoptosis and increases cell permeability,leading to defects in intestinal mucosa.2.Baicalin inhibits TNF-?-induced apoptosis and cell viability injury in IEC-6 cells.Baicalin exerts a protective effect against TNF-?-induced injury by down-regulating miR-191a that targets the tight junction protein Z0-1 in IEC-6 cells,increases the ability of cell migration,and reduce the expression of Claudin-2,protect the normal permeability of intestinal epithelial cells.