Suppression Of PKCβ/p66shc Signaling Pathway Attenuates Multiple Organ Injury Induced By Intestinal Ischemia Reperfusion

Intestinal ischemia-reperfusion (I/R) is a serious clinical dilemma withhighmorbidity and mortality. Intestinal I/R often induced the impaired intestinal barrierfunction, intestinal bacterial translocation and activation of systemic inflammatorymediators, and such a cascade of reactions will ultimately lead to systemicinflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome(MODS). Remote organ damage, especially acute lung injury (ALI) and liver injury arecommon complications that contribute to the high mortality rate. Toxic reactive oxygenspecies (ROS) induced by intestinal I/R and subsequent apoptosis are known to promoteremote organ damage. Protein kinase C β (PKCβ)dependent adaptor protein p66shc(p66shc) phosphorylation is involved in the generation of ROS and induction ofapoptosis.The protein kinase C (PKC) family comprises a group of multi-functional proteinkinases thatplayimportant roles as signal transducers of cellular stress byphosphorylation of the serine and threonine residues. Activation of PKC, indicated bytranslocation of the protein from the cytoplasm to the membrane with subsequentphosphorylation, occurs in response to many conditions, such as I/R injury andhemorrhagic shock, to regulate the oxidative stress and apoptosis signal pathway. Adaptor protein p66shc (p66shc) is a member of the ShcA protein family, and iscomprised of two other proteins, p46shc and p52shc. Phosphorylation of the tyrosineresidues of p46shc and p52shc plays an important role in their interaction with theepidermal growth factor receptor. In contrast to p46shc and p52shc, p66shc has a uniqueN-terminal proline-rich domain with a serine phosphorylation site (serine36).Endogenous or exogenous stress, such as free radicals attack, results in serine36phosphorylation of the p66shc, contributing to cell oxidative stress and apoptosis.Thus, we hypothesized that the PKCβ inhibitor LY333531would suppressp66shc-mediated oxidative stress and apoptosis and protect against ALI and liver injuryinduced by intestinal I/R. First, to prove the role of PKCβ in remote organs injuryinduced by intestinal I/R, we constructed a murine intestinal I/R model and used PKCβinhibitor LY333531to suppress the activation of PKCβin mice. PKCβ inhibitorLY333531was administrated3days prior to I/R surgery. At the end of reperfusion,blood and tissues were collected for analysis. Intestinal I/R caused severe injury to thelung and liver, including histopathological changes, inflammatory cell infiltration,oxidative stress and apoptosis. PKCβ suppression by LY333531significantly attenuatedI/R-induced histologic damage, inflammatory cell infiltration, oxidative stress, andapoptosis in the lung and liver, and also alleviated systemic inflammation.Secondly, in order to prove that LY333531can inhibit PKCβ activation, as well asinhibiting the phosphorylation of p66shc to alleviate the intestinal I/R induced ALI andacute liver injury, we further examined the relationship between PKCβ and p66shcinvitro and in vivo, the role of p66shc in apoptosis, and whether LY333531could reducep66shc activation to play a protective rolein the liver and lung damage induced byintestinal I/R. Data showed that intestinal I/R induced p66shc activation in the liver andlung tissue, inhibiting PKCβII in vivo or in vitro with PKCβII RNA interferenceinhibited the activation induced p66shc,These resulted in the decrease of cytochrome-crelease and caspase-3cleavage, and an increase in glutathione (GSH) and glutathioneperoxidase (GSH-PX). These data indicated that PKCβ suppression protects againstremote organ injury, which may be partially attributed to the p66shc-cytochrome-c axis. The development of a PKCβ inhibitor for prophylaxis against intestinal I/R is promising,to prevent secondary ALI or liver injury.Finally, to demonstrate whether antioxidant ingredients in natural plants play aprotective role in multiple organ damage induced by intestinal I/R, as well as therelationship between the protective effect and p66shc pathway, we use PCApretreatment, to detect intestinal and liver damage induced by intestinal I/R and theexpression of p66shc pathway related protein. Our study found that PCA pretreatmentcan significantly reduce intestinal damage and acute liver injury induced by intestinalI/R, showing improvement in histological damage, liver function and systemicinflammation, reducing p66shc associated oxidative stress and apoptosis.In summary, PKCβ dependent p66shc activation plays an important rolein multipleorgan damage induced by intestinal I/R, specific inhibition of PKCβ reduce p66shcmediatedmultiple organ damage. What’s more, polyphenolic compoundsPCA inhibitsthe activation of p66shc to play a protective role in intestinal I/R-induced multiple organinjury.In this study, we apply mouse model ofintestinal I/R and H2O2oxidation modelof liver and lung cells to explore the mechanism of PKCβ/p66shc signaling pathway inintestinal I/R injury and protective role of PKCβ inhibitors or PCA pretreatment, toprovide not only an effective and reliable pretreatment drugs and targets as protection ofremote organ injury induced by intestinal I/R, but also a more favorable basis for thedevelopment of the PCA.Part IBlockade of PKCβ phosphorylation by LY333531protectsagainst remote liver and lung injury induced by intestinalischemia reperfusionObjective: To explore the role of PKCβ phosphorylation signaling pathway inintestinal I/R-induced multiple organs dysfunction. To determine the effects of LY333531on regulation of PKCβ membrane translocation, phosphorylation as well asantioxidative and proapoptotic genes related to PKCβ activation.Method: Forty adult male ICR mice were randomly divided into four groups,including Sham group, I/R group, Sham+LY333531group and I/R+LY333531group.Sham group is only given appropriate vascular separation without occlusion. The I/Rand I/R+LY333531groups were subjected to mesenteric arterial ischemia for45minutes and reperfusion for45,90,180minutes to establish ischemia-reperfusionmodel. In all LY333531-pretreated groups, the mice received intragastric LY333531administration for3consecutive days. Intestine, liver and lung tissues were harvestedfor histopathologic assessment, while serum specimens were collected for measuringthe level of ALT, AST, TNF-α and IL-6. Besides, liver and lung homogenates were alsoused to detect the level of H2O2,GSH, GSH-PX, MDA and MPO. TUNEL detectionwas used to observe the apoptosis of liver and lung cells. Liver and lung expressions ofPKCβI、PKCβII、PKCγ、PKCδ、PKCε、phospho-PKCβII、MnSOD、cleaved-caspase-3,were determined by Western blotting for protein level.Results: LY333531pretreatment markedly reduced I/R-induced multiple organsinjury such as intestine, liver and lung and they were indicated by histologicalalterations; improved the decreases in ALT, AST, TNF-α and IL-6expression levels;the decreases in H2O2, MDA and MPO expression levels and the increase in GSH andGSH-PX levels, the decreases of apoptosis of liver and lung cells; and the increases inmanganese superoxide dismutase, and the decreases in cleaved-caspase-3expressionlevels in the liver and lung. Moreover, LY333531treatment down-regulatedphospho-PKCβII expression.Conclusion: LY333531has a significant protective effect on multiple organsdysfunction induced by intestinal I/R injury. The protective effect of LY333531may beattributed to the suppression of PKCβII phosphorylation and the modulation ofdownstream antioxidative/proapoptotic factors. Part IISuppression of PKCβ-dependent p66shc phosphorylation protectsagainst remote liver and lung injury induced by intestinalischemia reperfusionObjective: To explore the effect and mechanism of PKCβ/p66shc signalingpathway in intestinal I/R-induced remote organs injury and H2O2-induced injury inA549and L02cells. To determine the mechanisms of p66shc phosphorylation inducingapoptosis. To demonstrate whether LY333531protects against remote organ injuryinduced by intestinal ischemia reperfusion through PKCβ/p66shc signaling pathway.Method: The groups and model buildings of animal experiments are as previouslydescribed. Human liver L02and lung A549cells were randomly divided into fourgroups: Control group, PMA group, PMA+si-PKCβ group, PMA+LY333531group.Another experiment groups: Control group, H2O2group, si-p66shc group, H2O2+si-p66shc. When the cell fusion to80%, replaced by serum-free medium, the cellsreceived LY333531, si-PKCβ or si-p66shc administration and cultured for6hours, thenthey were given PMA or H2O2to bulid model. Liver and lung expressions ofphospho-PKCβII, PKCβII, phospho-p66shc, p66shc and cytochrome c were determinedby Western blotting for protein level. Immunoprecipitation was used to detect therelationship between cytochrome c and p66shc. Caspase-3activity assay kit was usedfor the detection of caspase-3activity.Results: LY333531pretreatment reduced the level of phospho-PKCβII,phospho-p66shc and cytochrome c through the inhibition of mitochondrial translocationof p66shc, combination between p66shc and cytochrome c as well as the release ofcytochrome c. Suppression of PKCβII reduced phospho-p66shc, which caused apoptosisthrough the activation of caspase-3.Conclusion: The protective effect of LY333531may be attributed to thesuppression of p66shc phosphorylation, mitochondrial translocation, combination with cytochrome c and the activation of caspase-3.Part IIIModulating the p66shc phosphorylationwith protocatechuic acidprotects the intestine from ischemia-reperfusion injury andalleviates secondary liver damageObjective: To investigated the effect of Protocatechuic acid (PCA) pretreatmentfor protecting intestinal I/R-induced local intestine and remote liver injury in mice. Toexplore its molecular mechanism from a perspective of p66shc pathway.Method: Fifty ICR mice were randomly divided into five groups:1) control group,2) I/R group,3) control+PCA group,4) I/R+PCA low-dose group,5) I/R+PCAhigh-dose group. The I/R and I/R+PCA groups were subjected to mesenteric arterialischemia for45minutes and reperfusion for90minutes. In all PCA-pretreated groups,the mice received intraperitoneal PCA administration for three consecutive days. Serumspecimens were collected for measuring Alanine Aminotransferase (ALT), AspartateAminotransferase (AST), tumor necrosis factor-α (TNF-α) and interleukin6(IL-6),while intestine and liver tissues were harvested for histopathologic assessment includingGlutathione (GSH) and Glutathione Peroxidase (GSH-PX). Intestine and liverexpression of p66shc, phosphorylated p66shc, Foxo3a, phosphorylated Foxo3a,manganese superoxide dismutase (MnSOD), cleaved caspase-3, and Bcl-xL weredetermined by Western blotting for protein level and semiquantitative reversetranscription-polymerase chain reaction analysis for mRNAlevel.Results: PCA pretreatment markedly alleviated intestine and liver injury inducedby intestinal I/R as indicated by histological alterations, decreases in serological damageparameters and nuclear factor-kappa B and phospho-foxo3a protein expression levels,and increases in glutathione, glutathione peroxidase, manganese superoxide dismutaseprotein expression, and Bcl-xL protein expression in the intestine and liver. These parameters were accompanied by PCA-induced adaptor protein p66shc suppression.Conclusion: PCA has a significant protective effect in the intestine and liverfollowing injury induced by intestinal I/R. The protective effect of PCA may beattributed to the suppression of p66shc and the regulation of p66shc-relatedanti-oxidative and anti-apoptotic factors.