Cytoprotection And Mechanism Of HSP90Involved In Post-burn Hypoxia Induced Intestinal Epithelial Cell Injury

Stress caused by burn, trauma or shock may lead to intestinal mucosalbarrier dysfunction. Bacteria and endotoxin may invade into systemic circulation via portalvein and lymphatic system, which causes bacteria translocation, endotoxemia and a cascadeof cytokines and other inflammatory mediators, and eventually results in multipleorgan dysfunction Syndrome (MODS). Therefore, the intestinal tract is regarded as the organwith early damage which initiates the following processes of MODS.The integrity of the intestinal mucosal barrier plays anvital role in maitaining intestinaltract function. The balance between regeneration and apoptosis of intestinal epithelial cells(IECs) is important in the maintenance of intestinal mucosal barrier function. Excessiveapoptosis of IEC damages the intestinal mucosal barrier function. Previous studies haveshown that severe burn and trauma can cause hypoxia. Heat shock protein90(HSP90),one of the molecular chaperone, is involved in regulating a variety of cellular signaltransduction and cell function. Kinase Akt the key element of phosphoinositide dependentkinase/protein serine threonine kinase (PI3K/Akt) signaling pathway, plays a significantpart in promoting cell survival and inhibiting cell apoptosis. Thus, it is very likely thatHSP90acts as a regulatory molecule of kinase Akt, and participates in cell protection.However, it has not been explored whether hypoxia can cause IEC apoptosis and whether thesignaling pathway of HSP90and PI3K/Akt is involved in protection of hypoxia-induced IECapoptosis.We have successfully constructed hypoxia model of the IECs and established HSP90overexpression and knockdown systems. Cell viability was assayed after overexpression orknockdown of HSP90in intestinal epithelia by CCK-8, Annexinv-APC/PI cellstreamingand TUNEL staining. The results showed that: hypoxia led to IEC apoptosis, which was themost prominent12hours after hypoxia. Cell apoptosis rate increased and cell viabilitygradually declined time dependently. Compared to the control group, the apoptosis rate of the HSP90over-expression group was significantly lower. In contrast， apoptosis rate wassignificantly higher in HSP90knockdown group. The results suggested that HSP90protectedintestinal cells from hypoxia-induced apoptosis. Then, we used several kinase inhibitors totreat IECs and used Annexinv-APC/PI flow to detect apoptosis rate, to explore the role ofthese signaling pathways in the hypoxia-induced apoptosis. The results showed that afterhypoxia treatment for12hours, the apoptosis rate of intestinal epithelia treated with aAkt-specific blocking agent, LY294002, increased significantly compared with other groups.Compared to the LY294002treated group, the apoptosis rate of cells overexpressed withHSP90was significantly higher, suggesting that the Akt signaling pathwaywas involved in the mechanism of HSP90protection of hypoxia-induced apoptosis of IECs.Our prior study confirmed that HSP90and Akt exert a protective role in apoptosis ofIECs induced by hypoxia. However, little is known about their roles in IEC survival underhypoxic condition and the underlying molecular mechanism(s).Western blot analysis was applied to detect expression of Akt, PI3K, PDK1, BAD,cytochrome C,caspase3and PARP under normal and hypoxic conditions in IECs. Resultsshowed that In the Caco2cells, hypoxia slightly increased the phosphorylation level of BAD,indicating that the cell is trying to counteract the detrimental effects of hypoxia. HSP90overexpression further increased pBAD abundance. In contrast, HSP90shRNA decreasedpBAD abundance. Accordingly, cytochrome C release from the mitochondria into the cytosolwas decreased by HSP90overexpression but reduced by HSP90knockdown. Overexpressionof HSP90had no effect on the amount of cleaved caspase3and cleaved PARP in the Caco2cells under normoxic condition. Hypoxia significantly increased the abundance of cleavedcaspase3and PARP. When HSP90expression was reduced by shRNA, the amount of cleavedcaspase3and PARP significantly increased2-fold and3-fold, respectively. OverexpressingHSP90suppressed the cleavage of caspase3and PARP. Our results suggest that HSP90inhibits the hypoxia-induced mitochondria-dependent apoptotic signaling pathway in theCaco2cells.Immunofluorescence and co-immunoprecipitation was applied to detect interactionbetween HSP90and pAkt. Results showed that HSP90was co-immunoprecipitated with pAktand conversely, and pAkt was co-immunoprecipitated with HSP90under normoxic andhypoxic conditions. Hypoxia tended to increase the amount of HSP90-pAkt complex. HSP90-overexpression increased the amount of HSP90and pAkt complex but HSP90shRNAgroup reduced the amount of this complex in the hypoxic group.What’s more, burn-modle of mice Animals also showed that AD-HSP90treatmentsignificantly decreased the injury index, alleviated pathological damage comparised with theBurn group.In conclusion, our data collectively indicate that Hypoxia induces apoptosis of the Caco2cells in a time-dependent manner, during which HSP90plays an important role in protectingthe Caco2cells from the hypoxia-induced injury. This is achieved by stabilizing pAkt thatforms a complex with HSP90. Activated Akt (pAkt) then phosphorylates BAD to prevent itfrom activating the apoptotic signaling pathway.These results show that HSP90may provide a potential and effective gene therapeutictarget of intestinal epithelial barrier dysfunction during intestinal hypoxia following severeburn or trauma.