PKC? Phosphorylates TRAF2 To Protect Against Injury Induced By Intestinal Ischemia-reperfusion

Background:Intestine ischemia-reperfusion?I/R?injury is a common pathological damage process caused by a variety of disease states,leading to systemic inflammatory response syndrome.Injury of tissues and organs not only confined to the small intestine,can cause multiple organ dysfunction syndrome or even death.Excessive apoptosis play a indispensable role in intestinal I/R injury.Recent researchs indicated that TRAF2 could inhibit apoptosis induced by oxidative stress and TNF-?stimulation.In addition,the related research investigated that TRAF2 is phosphorylated by PKC?at Ser⁵⁵.Moreover,TRAF2 play a protective role in myocardial ischemia reperfusion injury.However,the role of PKC?/TRAF2 signaling pathway in intestinal I/R injury remains unclear.Caco-2 cells which derived from highly differentiated human colon adenocarcinoma are similar to intestinal epithelial cells in structure and function.Caco-2 cells are often used in intestinal drug absorption and metabolism studies in vitro.Objective:This study is aimed to examine the role of the phosphorylated TRAF2 at Ser⁵55 which is mediated by PKC?in the pathogenesis of intestinal I/R injury and to explore whether activation of PKC?by the activator phosphatidylcholine?PC?can alleviate the intestinal I/R injury via phosphorylating TRAF2 at Ser⁵⁵.Methods:1.Forty C57BL/6 male mice were randomly divided into the following 5groups?n=8 in each group?:?1?sham group,?2?sham+PC?0.3mM?group;?3?I/R group;?4?I/R+PC?0.3mM?;?5?I/R+PC?0.3mM?+ATM?60mg/kg/d?group.Miniature noninvasive mouse arterial clamp was used to trap the model of the mesenteric artery in the model group for 45 min followed by 1h,2h,4h reperfusion.All of the mouse mesenteric arteries in the sham operation group were treated with free but not clipping.The rats in the agonist-treated group were given the appropriate concentration?0.3 mM?of the mice in the agonist group at intervals of 1 hour at 4hours before the operation PC?0.2ml/h?;the corresponding dose of ATM?60mg/kg/d?was administered to all the mice in the control group by intraperitoneal injection for 7 days?once daily?.The same dose of saline was administered to the mice in the untreated group by the same route of administration?intragastric or intraperitoneal injection?.At the end of reperfusion time?1h,2h or 4h?,the peritoneal dissection was performed again and the appropriate length was cut.The small intestine tissue of each mouse was stored under appropriate conditions.The apoptosis of small intestinal epithelial cells was detected by TUNEL method.The pathological changes of small intestine were observed by hematoxylin-eosin?HE?staining and pathologic score.Intestinal related tissue proteins,including PKC?,PKC?,phosphorylated PKC??p-PKC??,TRAF2,phosphorylated TRAF2-Ser55/11?p-TRAF2-Ser55/11?,Bcl-2 and cleaved caspase-3 protein were detected by Western-blot.2.Caco-2 cells were randomly divided into two groups,including control group and H/R group.To simulate in vivo intestinal ischemia,unless otherwise noted,cellular hypoxic conditions were created.For the hypoxic conditions,cells were incubated in a microaerophilic system?Thermo Fisher Scientific 8000,Marietta,GA,USA?at 5%CO2 and 1%O2,and balanced with 94%N2 gas for 12 h.The cells were then cultured in normoxic conditions for 6 h of reoxygenation.After H/R,we conducted the immunofluorescence and co-immunoprecipitation expriment.Results:Intestinal I/R leads to significant increases in intestinal dysfunction and mucosal injury,resulting to the membrane translocation and phosphorylation of PKC?as well as enhanced the phosphorylation of TRAF2.After Caco-2 cells H/R,PKC?colocalizes with TRAF2 at membranen.In addition,the activation of PKC?by PC,a activator of PKC?,significantly attenuated I/R-induced damage via inhibitting apoptosis.Additionally,activation of PKC?increased the phosphorylation of TRAF2at Ser⁵55 but not at Ser¹¹,which resulted in the decrease of caspase-3 cleavage and the increase of Bcl-2 expression.Conclusion:Our research indicated that intestinal I/R injury or Caco-2 cells H/R could induce the translocation and phosphorylation of PKC?,which resulted in the phosphorylation of TRAF2 at Ser⁵55 but not at Ser¹¹.The TRAF2 Ser⁵55 phosphorylation can protect against I/R-induced injury via suppressing the apoptosis of intestinal epithelial cells.