| Objective: To investigate autophagy in intestinal epithelial cells injury induced by altitude hypoxia and regulation mechanism of it mediated by phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt) signaling pathway which may provide new theoretical basis for targeted prevention and treatment of intestinal disorders in plateau area in further research in this process.Methods: Experimental subject is rat intestinal crypt epithelial cells(IEC-6) in vitro. Establishing IEC-6 cells injured hypoxia model as the experimental groups(Hypoxia) using three gas incubator simulated high altitude hypoxia(5%O2) and IEC-6 cells containing normal oxygen incubator as the control groups(Control):(1) 3-MA toxic effects at different time points on IEC-6 cells containing normal oxygen with 6 h, 12 h, 24 h and 48 h as well as the cells viability rate of IEC-6 cells treated hypoxia joining concentrations screened of 3-MA(3-MA+Hypoxia) with 6 h, 12 h, 24 h and 48 h were detected by MTT assay.(2) Autophagy and autolysosomal punctate fluorescent particles, autophagic double membrane structure and expression of autophagy-related proteins LC3 and Beclin-1 in IEC-6 cells treated 6 h, 12 h, 24 h and 48 h with hypoxia were detected by monodansylcadaverine(MDC) staining, transmission electron microscopy and Western blot;(3) After selecting the most obvious autophagy traeted with hypoxic and adding autophagy inhibitor 3-MA, autophagy and autolysosomal punctate fluorescent particles of MDC positive cells expressing were analyzed under a fluorescence microscope. Autophagic double membrane structure in IEC-6 cells was observed by transmission electron microscopy. The expression of autophagy-related protein LC3 and Beclin-1 as well as PI3K/Akt signaling pathway-related protein Akt and p-Akt were analyzed by Western blot.Results:(1) Results of MTT for cytotoxicity of 3-MA on IEC-6 cells showed: with increasing the concentration of 3-MA and duration of action, the inhibition rate of IEC-6 cells proliferation was increasing gradually, showing time and concentration dependence. When the concentration of 3-MA was ≤5 mmol/L, the inhibition rate and drug toxicity of 3-MA on cells proliferation of IEC-6 decreased. Therefore, 5 mmol/L was chosen as the experimental concentration of 3-MA;(2) Results of MTT for IEC-6 cells proliferation in hypoxia showed: the cell viability of 6h and 12 h has declined, but the difference was not significantly than normoxic group(P>0.05); After treated for 24 h and 48 h, the cells viability decreased significantly(P<0.05). After hypoxia adding autophagy inhibitor 3-MA, compared with hypoxia group, the cell viability decreased significantly from 12 h, as well 24 h and 48 h decreased more significantly(P<0.05);(3) MDC staining showed: autophagy punctate fluorescent particles within normoxic cells were no significant. Punctate fluorescent particles mostly around the nucleus began to appear in IEC-6 cells treated hypoxia for 6 h. Autophagy punctate fluorescent particles peaked at 24 h. After hypoxia 48 h, the total number of ICE-6 cells and intracellular punctate fluorescent particles began to decline;(4) Results of TEM showed: autophagic double membrane structure began to appear in hypoxia 6 h group; With prolonged hypoxia time, the number of autophagic double membrane increased gradually; Increasing of autophagosomes was the most obvious among hypoxic 24 h group; within hypoxic 48 h cells, the autophagosomes were still seen, but the structure of cells were damaged, and cell membranes and nucleus were not the degree of fragmentation;(5) Western blot assay showed: expression of LC3 and Beclin-1 were significantly higher in hypoxia 6 h, 12 h, 24 h, 48 h groups than in the normxic group, but their expression significantly decreased after hypoxia 48 h(P<0.05);(6) After hypoxia adding autophagy inhibitor 3-MA, transmission electron microscopy and MDC staining results show that a significant reduction in IEC-6 nucleus around autophagy low oxygen group; western law blot test results showed expression of LC3 and Beclin-1 were significantly reduced compared with hypoxia group(P<0.05); expression of intracellular phospho-Akt(p-Akt) protein in hypoxia group was significantly higher than the normal group(P<0.05); expression of p-Akt was significantly decreased in hypoxia adding autophagy inhibitor 3-MA(P<0.05); after hypoxia IEC-6 cell injury, Akt was no significantly changes(P>0.05).Conclusions:(1) simulated high altitude hypoxia IEC-6 cell damage can induce autophagy;(2) PI3K/Akt signaling pathway may be simulated high altitude hypoxia IEC-6 cell damage induced autophagy regulation. |
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