The Western And Traditional Chinese Medicine Study Of Intestinal Epithetlial Cells Injuried By Heat Stress

ObjectivesInvestigating the effect of gradient heat stress (HS) on intestinal epithetlial cells and cell connections through the in-depth verification on the cellular and molecular levels in vitro. In order to provide the experimental basis to clarify the possible mechanisms of intestinal mechanical barrier damage in heat stroke.Methods1.the effect of heat stress on intestinal epithetlial cells morphology and cytoskeletonIntestinal epithelial cell line (IEC-6) cells came from mormal rat were cultured in vitro, according to the different degrees of HS to divide the experimental groups:Exposure to HS(37℃,39℃,41℃,43℃)for1h. then observed the cell morphology under an inverted microscope; Coomassie brilliant blue R250staining test to observed the changes in cell morphology and cytoskeletal protein in different degrees of HS under an oil microscope, and photography.2. The effect of heat stress on the viability of intestinal epithelial cellsIntestinal epithelial cell line (IEC-6) cells came from mormal rat were cultured in vitro, according to the different degrees of HS to divide the experimental groups:Exposure to HS(37℃,39℃,41℃,43℃)for1h,3h,5h,7h. then re-placed in the incubator under the standard37℃,5%C02for2h after add the CCK-8into the culture supematant in corresponding time point, read absorbance value (A) values at450nm by enzyme-linked immunoassay analyzer and calculated cell activity.3.The effect of heat stress on the activity of DAO in intestinal epithelial cells Intestinal epithelial cell line (IEC-6) cells came from mormal rat were cultured in vitro, according to the different degrees of HS to divide the experimental groups:Exposure to HS(37℃,39℃,41℃,43℃)for lh, until24h later to collect the culture supernatant to detected the DAO activity by ELISA.4. The effect of heat stress on the paracellular permeabilityIntestinal epithelial cell line (IEC-6) cells came from mormal rat were cultured in vitro, according to the different degrees of HS to divide the experimental groups:Exposure to HS(37℃,39℃,41℃,43℃)for lh, then the transepithelial electrical resistance (the TEER) was detected by the Millicell-ERS system.5. The effect of heat stress on the tight junctions of intestinal epithelial cellsIntestinal epithelial cell line (IEC-6) cells came from mormal rat were cultured in vitro, according to the different degrees of HS to divide the experimental groups:Exposure to HS (37℃,39℃,41℃,43℃) for lh, after24hours collected the cell protine to detected the expression levels of Occludin protein by Western blotting.Results1.The effect of heat stress on intestinal epithetlial cells’morphology and cytoskeletonUnder light microscope, Compared to the normal control group, cells of each heat stress groups had become rounded, pseudopod was shorter and cell space increased. with the increasing of temperature, this phenomenon is more obvious. To43℃, most of the cells were necrosis. Coomassie brilliant blue R250staining test showed cytoskeleton gradually became shorter and disorders, appeared stress fibers, rearranged in a one-way distribution of nonpolar. The higher the temperature, the changes more obvious.2. The effect of heat stress on the viability of intestinal epithelial cellsHS could result in the cell vitality declined.Compared with the normal control group, the cell vitality of each HS groups were significantly decreased(P<0.01). and the decrease showed time-and thermal-dependent.3. The effect of heat stress on the activity of DAO in intestinal epithelial cellsThere were significiant differences in the vitality of DAO in the cell culture supernatant between the four groups(P<0.01); but there were no significantly differences between39℃group and normal control group(P>0.05);41℃and43℃groups were significantly higher compared to the normal control group (P<0.05).4.The effect of heat stress on the paracellular permeabilityCompared with the normal control group, the numerical value of TEER were significantly decreased (P<0.05) and there were significantly different among each of heat stress groups(P<0.01). Indicating that the decreasing of TEER level can cause increased intestinal permeability, and was temperature dependent.5.The effect of heat stress on the tight junctions of intestinal epithelial cellsExpression level of Occludin protein in heat stress groups were higher than that of normal control group (P<0.05).41℃and43℃groups were significantly higher compared to the39℃group (P<0.05).ConelusionHeat stress has cytotoxic effect on intestinal epithelial cells and causes the changment in cell morphology、the decline of cell viability、the destruction of the cell structure. At the same time, Heat stress can lead to the permeability of the intestinal mucosal barrier increasing mainly through the rearrangement of the cytoskeleton and the TEER value decreased. However, in our study, we found heat stress can lead to the level of Occludin protein which as a key members in cell tight junctions has rised,we speculate that Occludin protein in this process may play a role in repair.