Effect Of Glutamine On Tight Junction Protein Occludin During Ischemia-reperfusion Injury In Vitro

Aim:Abtain experimental model of ischemia-reperfusion injury in vitro. Detect the effect of glutamine on the activity of intestinal epithelial cells and the expression of tight junction protein occludin during ischemia-reperfusion injury in vitro. To detect that glutamine can protect the intestinal mucosa, and study the mechanisms of protecting intestinal mucosa by glutamine.Methods:Choose Caco-2, and abtain experimental model of ischemia-reperfusion injury in vitro by the method of hypoxic(90min)/re-oxygenation(30min), then culture cells in the culture medium with indicated concentration of glutamine. Measure the activity of cells by MTT and detect the expression of occludin by RT-PCR, western blot and immunofluorescence technique.Results:1. We successfully established the model of hypoxic/re-oxygenation in vitro.2. After modeling, we detected the activity of cells immediately, the relative expression ratio of mRNA, and the protein expression of occludin.①MTT:the value of OD decreases significantly(p<0.01).②RT-PCR:the he relative expression ratio of mRNA decreases significantly (p<0.05).③Western blot:the protein expression of occluding has no significant change(p>0.05).④The results of immunofluorescence showed that the fluorescence intensity and distribution had no obvious difference. Linear fluorescence occurred at the membranes of intercellular connections, and presented as honeycomb.3. After modeling, culture cells in the culture medium with indicated concentration of glutamine(Ommol/L,4mmol/L,8mmol/L,12mmol/L,16mmol/L). At the24th hour and the48th hour, detect the change of OD. Comparing with Ommol/L Gln groups, the activity of cells of4mmol/L and8mmol/L Gln groups increased obviously(p <0.05). But comparing with8mmol/L Gln groups, the activity of cells of12mmol/L and16mmol/L Gln groups decreased obviously (p<0.05). So, the two concentration group ruled out.4. After modeling, culture cells in the culture medium with indicated concentration of glutamine(Ommol/L,4mmol/L,8mmol/L), and had experiments at the two time points (24th hour and the48th hour).①RT-PCR:Gln significantly promote the mRNA relative expression of occludin protein (p<0.05). And there are a certain concentration correlation and time correlation.②Western blot:Gln significantly promote the expression of occludin protein (p<0.05). And there are a certain concentration correlation and time correlation.③Immunofluorescence technique:With the increasing of the concentration of Gln, occludin proteins fluorescence distribution was more continuous and stronger. And there are a certain concentration correlation and time correlation.Conclusions:1. In vitro, using Caco-2cells, after establishing the ischemia-reperfusion injury model, the activity of cells declined obviously, and the relative expression of mRNA decreased significantly, but the expression of occludin protein had no significant change.2. After establishing the ischemia-reperfusion injury mode by Caco-2, glutamine promotes the proliferation of intestinal epithelium, and upgrades expression of occludin at the levels of gene and protein.