Influence Of Nano-TiO₂ On The Expression Of Intestinal Epithelial Tight Junction Proteins Occludin And ZO-1and Effect Related Signaling Pathway

As the nanotechnology improved,there is a wide variety of nanomaterials in the market.Titanium dioxide nanoparticles?nano-TiO₂?have applied in food additives,food packaging materials,pharmaceuticals and cosmetics due to the special physicochemical properties.Oral-intaked nano-TiO₂ can reach the intestinal tract in human body and interact with the intestinal cells.Intestinal epithelial cells are important parts of physical intestinal barrier in gut.A series of tight junction proteins between intestinal epithelial cells constitute continuous circuits.This tightly connected structure maintains people's health through preventing the body from pathogenic harmful substances in intestine.However,few researches elucidate the relationship between nano-TiO₂ and tight junction proteins in the intestinal barrier.Therefore,exploring the effects of nano-TiO₂ on tight junction proteins in intestinal epithelial cells is of great significance for the appropriate application of nano-TiO₂ in reality.Based on the universality of nano-TiO₂ materials and the importance of intestinal tight junction proteins in human body,intestinal epithelial cells were used to analyze the effect of nano-TiO₂ on the expression of tight junction proteins.At the same time,the level of inflammatory factors in intestinal epithelial cells treated by nano-TiO₂was measured and the mechanism related to this interaction was studied.Evaluation of the safe application of nano-TiO₂ can be achieved after the experiments and analysis.Our research has been divided into following three parts:Part I Characterization of nano-TiO₂ObjectiveTo analyze the size,polymer dispersity index?PDI?and zeta potential of nano-TiO₂ according to the characterization of the nano-TiO₂.MethodsThe shape of nano-TiO₂ was observed by transmission electron microscope?TEM?;the average hydrated particle size,PDI and the Zeta potential of nano-TiO₂were measured by dynamic light scattering method?DLS?.ResultsThe TEM indicated spherical shape of nano-TiO₂.The average hydrated particle size of dispersed nano-TiO₂ in the medium was 64.59±2.05 nm,PDI was 0.23±0.01,and the Zeta potential was-11.4±0.8 mV.Part II Effects of nano-TiO₂ on the expression of tight junction proteins Occludin and ZO-1 and the level of inflammatory factors in IEC-6 cellsObjectiveTo investigate the expression of tight junction proteins Occludin and ZO-1 and the inflammation in IEC-6 cells after administration of nano-TiO₂.Methods1.IEC-6 cells were treated with different concentrations of nano-TiO₂?5.0,10.0,15.0,20.0,40.0,60.0?g/mL?for 24 h.Control group was treated with concentration of 0.0?g/mL nano-TiO₂.Cell viability was assessed by thiazolyl blue?3-?4,5-dimethyl-2-thiazolyl?-2,5-diphenyl-2-H-tetrazolium bromide,MTT?under the different concentrations of nano-TiO₂.2.The tight junction proteins?Occludin and ZO-1?in control and experimental groups?5.0,10.0,15.0,20.0?g/mL?of nano-TiO₂ were tested by Western blot.3.The level of inflammatory cytokines including IL-6,IL-1?,TNF-?and iNOS were examined in control and other groups treated with different doses?5.0,10.0,15.0,20.0?g/mL?of nano-TiO₂.Results1.There was a negative correlation between the viability of IEC-6 cells and doses of nano-TiO₂?r=-0.767,P<0.001?.Viability of IEC-6 cells decreased significantly under the treatments of 40.0?g/mL and 60.0?g/mL nano-TiO₂?P<0.05?.2.WB tests indicated the expression and gray values of Occludin and ZO-1 in IEC-6 cells went down as the concentration of nano-TiO₂ raised(r_occludin=-0.742,P<0.01;r_zo-1=-0.744,P<0.01).Compared with the control group,the expression of Occludin protein decreased in each nano-TiO₂-treated group,and the expression of ZO-1 protein reduced in the concentration of 20.0?g/mL nano-TiO₂ group?P<0.05?.3.Compared with the control,treatments of nano-TiO₂ caused significant secretions of IL-6,IL-1?,TNF-?and iNOS in IEC-6 cells in a dose-dependent manner(r _IL-1?=0.870,P<0.01;r_IL-6=0.938,P<0.01;r _TNF-?=0.982,P<0.01;r_iNOS=0.982,P<0.01).Part III The roles of MAPK and JAK2/STAT3 signaling pathways both in the level of inflammatory cytokines and the expression of tight junction proteins Occludin and ZO-1 in IEC-6 cells after administration of nano-TiO₂ObjectiveTo explore the roles of MAPK and JAK2/STAT3 signaling pathways in nano-TiO₂-produced inflammation in IEC-6 cells and in the low expression of tight junction proteins Occludin and ZO-1 in IEC-6 cells with treatment of nano-TiO₂.To illuminate the possible reasons of the decreasing level of tight junction proteins in IEC-6 cells resulted from nano-TiO₂.Methods1.Western blot was used to measure the expression of p-ERK,ERK,p-p38,p38,p-JNK,JNK,p-JAK2,JAK2,p-STAT3 and STAT3 in control and nano-TiO₂ groups?5.0,10.0,15.0,20.0?g/mL?.2.The experimental groups were divided into control group,U0126?10?M?inhibitor group,SB203580?10?M?inhibitor group,SP600125?20?M?inhibitor group,AG490?20?M?inhibitor group,nano-TiO₂ group,U0126+nano-TiO₂co-treatment group,SB203580+nano-TiO₂ co-treatment group,SP600125+nano-TiO₂co-treatment group and AG490+nano-TiO₂ co-treatment group.ELISA test was used to identify the inflammatory factors in each group.3.Western blot test was used to determine the level of p-ERK and ERK in control group,U0126?10?M?inhibitor group,nano-TiO₂ group and U0126+nano-TiO₂co-treatment group.The expression of p-p38 and p38 was identified in control group,SB203580?10?M?inhibitor group,nano-TiO₂ group and SB203580+nano-TiO₂co-treatment group.The expression of p-JNK and JNK was identified in control group,SP600125?20?M?inhibitor group,nano-TiO₂ group and SP600125+nano-TiO₂co-treatment group.The level of p-JAK2,JAK2,p-STAT3 and STAT3 was tested in control group,AG490?20?M?inhibitor group,nano-TiO₂ group and AG490+nano-TiO₂ co-treatment group respectivily by Western blot assay.4.Western blot assay was adopted to gain information about the expression of Occludin and ZO-1 in above-mentioned groups.Results1.When the concentrations of nano-TiO₂ given to IEC-6 cells increased,phosphorylation level of ERK,p38,JNK,JAK2 and STAT3 proteins and values of p-ERK/ERK,p-p38/p38,p-JNK/JNK,p-JAK2/JAK2 and p-STAT3/STAT3 went rise as well?P<0.05?.2.Compared with the nano-TiO₂ group,inhibitors of SB203580,U0126,SP600125and AG490 indeed reduced the level of inflammatory factors in nano-TiO₂-treated IEC-6 cells?P<0.05?.3.Compared with the nano-TiO₂ group,inhibitors of SB203580,U0126,SP600125 and AG490 inhibited phosphorylation level of ERK,p38,JNK and JAK2/STAT3 proteins,respectively?P<0.05?.4.Compared with the nano-TiO₂ group,addition of inhibitors U0126?10?M?,SB203580?10?M?,SP600125?20?M?and AG490?20?M?into IEC-6 cells before administration of nano-TiO₂ had effective function to promote the expression of Occludin and ZO-1?P<0.05?.ConclusionNano-TiO₂ in IEC-6 cells induced low level of tight junction proteins Occludin and ZO-1 between epithelia and significant secretion of inflammatory factors.MAPK and JAK2/STAT3 signaling pathways played important roles in inflammation and low level of tight junction proteins both triggered by nano-TiO_2.The low expression of Occludin and ZO-1 in IEC-6 cells may result from nano-TiO₂ was related to inflammation caused by nano-TiO₂.