抑制PKCβ/p66shc信号通路对肠缺血再灌注多器官损伤的保护作用
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摘要
小肠缺血再灌注（ischemia reperfusion，I/R）是一种临床上常见的伴随着危重病症的病理生理过程，可以引起小肠屏障功能受损、肠内细菌移位和全身炎症介质激活等一系列级联反应，最终导致全身炎症反应综合征和多器官功能衰竭综合征，具有较高的发病率和死亡率。在发生损伤的多种器官中，肝脏由于直接收集肠道回流的血液，在其他器官损伤之前即可发生急性肝损伤；肺脏由于急性肺损伤（acute lung injury，ALI）常进展为凶险的急性呼吸窘迫综合征（acute respiratorydistress syndrome，ARDS），死亡率较高。小肠I/R引发多器官损伤的发病机制复杂，新近研究发现，小肠I/R引起的活性氧自由基（reactive oxygen species，ROS）释放以及细胞凋亡的诱导是引起多器官损伤的重要原因，此过程受体内复杂而精密的信号传导网络调控，具体机制不明。又有研究指出，蛋白激酶C（protein kinaseC，PKC）β依赖的衔接蛋白p66shc（p66shc）的磷酸化参与ROS的生成和细胞凋亡的诱导过程。
PKC家族包括一组多功能的蛋白激酶，它们通过磷酸化修饰，在细胞应激反应的信号转导中发挥重要作用。研究表明，在机体受到I/R损伤后，PKC蛋白可以由胞浆转位至胞膜，并发生磷酸化激活，进而调控体内的氧化应激及细胞凋亡信号。P66shc是ShcA蛋白家族的成员，其他成员包括p46shc和p52shc。内源或外源性的应激反应可以导致p66shc在丝氨酸36的位点发生磷酸化，进而导致细胞氧化应激和细胞凋亡。
我们作出如下假设：抑制PKCβ可以进一步抑制p66shc介导的氧化应激和细胞凋亡，进而保护由小肠I/R引起的多器官损伤。首先，为证明PKCβ在小肠I/R引起的远隔肝肺损伤中的作用，我们构建了小鼠小肠I/R模型，并使用PKCβ特异性抑制剂LY333531抑制小鼠体内PKCβ的激活。再灌注结束后，收集血清和组织进行后续实验分析。实验结果发现，小肠I/R对肝肺造成严重损伤，包括组织病理学改变，炎性细胞浸润，氧化应激损伤和细胞凋亡等。应用PKCβ抑制剂LY333531后，以上损伤均得到不同程度的减轻，表现为减轻组织学损伤，减少炎性介质释放，减轻肝肺组织的氧化应激和细胞凋亡。
其次，为证明LY333531可以抑制PKCβ的激活，进而抑制p66shc的磷酸化来减轻小肠I/R后ALI和急性肝损伤，我们进一步在体内外检测了PKCβ和p66shc之间的关系、p66shc在细胞凋亡中的作用及LY333531能否通过减少p66shc的激活在小肠I/R后的远隔肝肺损伤中发挥保护作用。数据表明，小肠I/R引起p66shc在肝肺组织中显著激活，若体内抑制PKCβII或体外PKCβIIRNA干扰均可抑制p66shc的诱导激活，并进一步减少p66shc的线粒体转位及其与细胞色素C的结合，减少细胞色素C和H2O2的释放，增加了GSH和GSH-PX的活性。以上数据表明PKCβ抑制剂对肠缺血再灌注引起的远隔脏器损伤的保护作用可能和PKCβII–p66shc–细胞色素C轴相关，而应用PKCβ抑制剂来预防小鼠肠缺血再灌注引起的远隔肝肺损伤是可靠而有效的。
最后，为证明天然植物中存在的抗氧化活性成分在小肠I/R引起的多器官损伤中是否发挥保护作用，以及保护作用与p66shc通路的关系。我们使用原儿茶酸（protocatechuic acid，PCA）进行预处理，检测小鼠小肠I/R后肠道及肝脏损伤及p66shc通路相关蛋白的表达。研究发现，PCA预处理可显著减轻小肠I/R引起的小肠原位器官损伤和急性肝损伤，表现为改善组织学损伤、肝功能和全身炎症，减轻p66shc相关的氧化应激和细胞凋亡。
综上所述，PKCβ依赖的p66shc激活在小肠I/R引起的多器官损伤中发挥重要作用，特异性抑制PKCβ可减轻p66shc介导的多器官损伤。另外多酚化合物PCA可抑制p66shc的激活，在小肠I/R致多器官损伤中发挥保护作用。本研究为小肠I/R损伤所致的多器官损伤提供了新的作用靶点，同时也为开发多酚化合物PCA以预防小肠I/R致多器官损伤的提供了有利的依据。
第一部分LY333531抑制PKCβ磷酸化对肠缺血再灌注远隔肝肺损伤的保护作用
目的：探讨PKC家族在肠缺血再灌注肝肺损伤中的变化及特异性抑制PKCβ磷酸化在此病理生理过程中所发挥的保护作用。
方法：健康成年雄性ICR小鼠40只，随机分为4组：假手术组（Sham组）、模型组（I/R组）、假手术给药组（Sham+LY333531）、模型给药组（I/R+LY333531）。各I/R组行肠系膜上动脉夹闭45分钟，再灌注45分钟、90分钟或180分钟以构建小肠I/R模型；各Sham组行假手术处理。假手术给药组及模型给药组于术前3天连续灌胃给10mg/kg的LY333531，每天1次。观察小肠、肝脏、肺脏组织病理学变化，检测血清中丙氨酸氨基转氨酶（alanine aminotransferase, ALT）、天冬氨酸氨基转氨酶（aspartate aminotransferase, AST）、肿瘤坏死因子α（tumor necrosisfantor-α，TNF-α）及白细胞介素-6（interleukin-6，IL-6）水平。检测肝、肺组织匀浆H2O2、谷胱甘肽（glutathione，GSH）、谷胱甘肽过氧化物酶（glutathione peroxidase，GSH-PX）、丙二醛（malondialdehyde，MDA）、髓过氧化物酶（myeloperoxidase，MPO）水平。采用脱氧核糖核酸转移酶原位末端标记技术（TdT-mediated dUTPNick-End Labeling，TUNEL）检测肝、肺组织细胞凋亡情况。采用Western Blot法检测肝、肺组织PKCβI、PKCβII、PKCγ、PKCδ、PKCε、phospho-PKCβII、锰超氧化物歧化酶（manganese superoxide dismutase，MnSOD）、cleaved-caspase-3蛋白表达水平。
结果：
1.与Sham组相比，I/R组：肝和肺组织PKCβII膜蛋白及磷酸化PKCβII蛋白表达水平明显升高，而其他PKC家族膜蛋白表达无明显变化；小肠组织病理示小肠绒毛排列明显紊乱，固有层崩解并形成明显溃疡，肝组织病理示肝索排列紊乱肝细胞核固缩，伴有胞浆嗜伊红细胞增多，肺组织病理示大量炎性细胞浸润，肺泡壁增厚并伴有肺泡出血；血清TNF-α、IL-6、ALT、AST水平显著升高；肝、肺组织H2O2、MDA和MPO水平显著增高，GSH和GSH-PX水平降低；肝和肺组织TUNEL凋亡检测显示凋亡细胞显著增多；肝和肺组织MnSOD蛋白表达水平明显降低，cleaved-caspase-3表达水平显著升高；Sham+LY333531组未见显著变化。
2、与I/R组相比，I/R+LY333531组：肝和肺组织PKCβII膜蛋白及磷酸化PKCβII蛋白表达水平明显降低；小肠、肝脏和肺脏组织病理示损伤均有所减轻；血清TNF-α、IL-6、ALT、AST水平明显下降；肝、肺组织H2O2、MDA和MPO水平显著降低，GSH和GSH-PX水平升高，肝肺组织TUNEL凋亡检测显示凋亡细胞显著减少；肝和肺组织MnSOD蛋白水平表达明显升高，cleaved-caspase-3蛋白表达水平明显降低。
结论：
1.小肠I/R可引起肝、肺组织中PKCβII的激活。
2.利用LY333531特异性抑制PKCβII的激活可对小肠I/R致远隔肝肺损伤有保护作用，可以有效减少肠肝肺组织学损伤、肝脏和肺脏的氧化应激和细胞凋亡。
第二部分抑制PKCβ依赖的p66shc磷酸化保护肠缺血再灌注远隔器官损伤
目的：探讨PKCβ依赖的p66shc磷酸化在小肠I/R远隔器官损伤中的作用，阐明LY333531预处理能否通过调控PKCβ/p66shc通路在肠I/R致肝肺损伤中发挥保护作用。
方法：体内实验分组及模型构建如第一部分所述，采用免疫共沉淀及WesternBlot法检测肝、肺组织中p66shc、磷酸化p66shc、PKCβ、磷酸化PKCβ、细胞色素C的蛋白表达。
体外实验分为两部分：1.人肝L02细胞和肺A549细胞，分为以下4组：对照组（Control组）、模型组（PMA组）、模型+干扰PKCβ组（PMA+si-PKCβ组）、模型+LY333531组（PMA+LY333531组）。采用Western Blot法检测p66shc、磷酸化p66shc、PKCβ、磷酸化PKCβ蛋白表达水平变化。
2.人肝L02细胞和肺A549细胞，分为以下4组：对照组（Control组）、干扰p66shc组（si-p66shc组）、模型组（H2O2组）、模型+干扰p66shc组（H2O2+si-p66shc组）。采用caspase-3活性试剂盒检测caspase-3活性、流式细胞术检测细胞凋亡。
结果：
1.体内实验：与sham组相比，I/R组肝肺组织中p66shc磷酸化显著增高，且呈再灌注时间依赖性；肝肺组织线粒体蛋白中p66shc含量显著增高；肝肺组织中p66shc和细胞色素C结合增多；肝肺组织中线粒体释放细胞色素C增多。
2.体内试验：与I/R组相比，I/R+LY333531组肝肺组织中p66shc磷酸化显著降低；肝肺组织线粒体蛋白中p66shc含量显著降低；肝肺组织中p66shc和细胞色素C结合减少；肝肺组织中线粒体释放细胞色素C减少。
3.体外实验第一部分：与Control组相比，PMA组p66shc磷酸化显著增高，而PMA+si-PKCβ组和PMA+LY333531组磷酸化p66shc较PMA组显著降低。
4.体外实验第二部分：与Control组相比，H2O2组caspase-3活性显著增强，凋亡细胞比例增高，而H2O2+si-p66shc组caspase-3活性和细胞凋亡比例较H2O2组显著降低。
结论：
1.小肠I/R组引起肝肺组织p66shc发生磷酸化，使p66shc发生线粒体转位，增强其与细胞色素C的结合，并促进细胞色素C的释放。
2. LY333531可以抑制p66shc蛋白的磷酸化，进而抑制其线粒体转位、与细胞色素C的结合及诱导细胞色素C释放，对肠I/R后的肝肺损伤发挥保护作用。
3. P66shc的磷酸化依赖于PKCβ的激活。
第三部分原儿茶酸调控p66shc磷酸化对小肠缺血再灌注多器官损伤的保护作用
目的：探讨多酚化合物PCA调控p66shc磷酸化信号通路在小肠I/R损伤和继性肝损伤中的作用。
方法：健康成年雄性ICR小鼠50只随机分为以下5组，每组10只：（1）假术组（Sham组）；（2）模型组（I/R组）；（3）假手术+PCA组（Sham+PCA0mg/kg group）；（4）模型+PCA低剂量组（I/R+PCA40mg/kg group）；（5）型+PCA高剂量组（I/R+PCA80mg/kg group）。各模型组采用Megison法构建肠I/R模型，行肠系膜上动脉夹闭45min，再灌注90min；各PCA处理组于模建立前3天给予PCA相应剂量腹腔注射，每天一次。再灌注结束后，收集血清本以检测TNF-α、IL-6、ALT和AST水平，收集小肠和肝脏组织标本进行病理织学评分和GSH、GSH-PX水平检测；利用RT-PCR法检测组织中p66shc mRNA达水平；利用Western-blot方法检测组织中p66shc、磷酸化p66shc、核因子κB p65NFκB p65）、Foxo3a、磷酸化Foxo3a、MnSOD、cleaved-caspase-3和Bcl-xL的白表达水平。
结果：
1.与Sham组相比，I/R组：小肠组织病理示肠绒毛排列紊乱，部分区域肠绒脱落，上皮下间隙增宽，毛细血管淤血；肝脏组织病理示肝索排列紊乱，可见量炎性细胞浸润，汇管区和肝血窦淤血现象严重，出现大量核固缩，核溶解；清TNF-α、IL-6、ALT、AST水平显著升高；NF-κB p65蛋白表达水平显著增加；SH含量和GSH-PX酶活力显著下降；小肠和肝脏组织p66shc mRNA水平和磷酸水平显著增加；Foxo3a磷酸化表达水平明显增加；MnSOD水平显著下降；leaved-caspase-3表达水平上调，Bcl-xL的表达下调。Sham+PCA组未见显著变化。
2.与I/R组相比，I/R+PCA（40mg/kg,80mg/kg）组：小肠组织病理示肠绒毛列整齐，上皮与固有层轻度分离，损伤程度明显减轻；肝脏组织病理示肝细胞列相对规则，炎性细胞浸润程度减轻，肝细胞分叶核形态正常，核固缩及核溶现象减轻。血清TNF-α、IL-6、ALT、AST水平显著降低；NF-κB p65蛋白表达平显著下降；GSH含量和GSH-PX酶活力显著增加；小肠和肝脏组织p66shcRNA水平和磷酸化显著降低；Foxo3a磷酸化表达水平明显降低；MnSOD水平著增加；cleaved-caspase-3表达降低，Bcl-xL的表达升高。
结论：
1. PCA对小肠I/R损伤和继发性肝脏损伤有保护作用。
2. PCA的保护作用与抑制p66shc的磷酸化及调控下游抗氧化和抗凋亡因子的表达相关。
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 reperfusion
Objective: 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 reperfusion
Objective: 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 damage
Objective: 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.
PKC家族包括一组多功能的蛋白激酶，它们通过磷酸化修饰，在细胞应激反应的信号转导中发挥重要作用。研究表明，在机体受到I/R损伤后，PKC蛋白可以由胞浆转位至胞膜，并发生磷酸化激活，进而调控体内的氧化应激及细胞凋亡信号。P66shc是ShcA蛋白家族的成员，其他成员包括p46shc和p52shc。内源或外源性的应激反应可以导致p66shc在丝氨酸36的位点发生磷酸化，进而导致细胞氧化应激和细胞凋亡。
我们作出如下假设：抑制PKCβ可以进一步抑制p66shc介导的氧化应激和细胞凋亡，进而保护由小肠I/R引起的多器官损伤。首先，为证明PKCβ在小肠I/R引起的远隔肝肺损伤中的作用，我们构建了小鼠小肠I/R模型，并使用PKCβ特异性抑制剂LY333531抑制小鼠体内PKCβ的激活。再灌注结束后，收集血清和组织进行后续实验分析。实验结果发现，小肠I/R对肝肺造成严重损伤，包括组织病理学改变，炎性细胞浸润，氧化应激损伤和细胞凋亡等。应用PKCβ抑制剂LY333531后，以上损伤均得到不同程度的减轻，表现为减轻组织学损伤，减少炎性介质释放，减轻肝肺组织的氧化应激和细胞凋亡。
其次，为证明LY333531可以抑制PKCβ的激活，进而抑制p66shc的磷酸化来减轻小肠I/R后ALI和急性肝损伤，我们进一步在体内外检测了PKCβ和p66shc之间的关系、p66shc在细胞凋亡中的作用及LY333531能否通过减少p66shc的激活在小肠I/R后的远隔肝肺损伤中发挥保护作用。数据表明，小肠I/R引起p66shc在肝肺组织中显著激活，若体内抑制PKCβII或体外PKCβIIRNA干扰均可抑制p66shc的诱导激活，并进一步减少p66shc的线粒体转位及其与细胞色素C的结合，减少细胞色素C和H2O2的释放，增加了GSH和GSH-PX的活性。以上数据表明PKCβ抑制剂对肠缺血再灌注引起的远隔脏器损伤的保护作用可能和PKCβII–p66shc–细胞色素C轴相关，而应用PKCβ抑制剂来预防小鼠肠缺血再灌注引起的远隔肝肺损伤是可靠而有效的。
最后，为证明天然植物中存在的抗氧化活性成分在小肠I/R引起的多器官损伤中是否发挥保护作用，以及保护作用与p66shc通路的关系。我们使用原儿茶酸（protocatechuic acid，PCA）进行预处理，检测小鼠小肠I/R后肠道及肝脏损伤及p66shc通路相关蛋白的表达。研究发现，PCA预处理可显著减轻小肠I/R引起的小肠原位器官损伤和急性肝损伤，表现为改善组织学损伤、肝功能和全身炎症，减轻p66shc相关的氧化应激和细胞凋亡。
综上所述，PKCβ依赖的p66shc激活在小肠I/R引起的多器官损伤中发挥重要作用，特异性抑制PKCβ可减轻p66shc介导的多器官损伤。另外多酚化合物PCA可抑制p66shc的激活，在小肠I/R致多器官损伤中发挥保护作用。本研究为小肠I/R损伤所致的多器官损伤提供了新的作用靶点，同时也为开发多酚化合物PCA以预防小肠I/R致多器官损伤的提供了有利的依据。
第一部分LY333531抑制PKCβ磷酸化对肠缺血再灌注远隔肝肺损伤的保护作用
目的：探讨PKC家族在肠缺血再灌注肝肺损伤中的变化及特异性抑制PKCβ磷酸化在此病理生理过程中所发挥的保护作用。
方法：健康成年雄性ICR小鼠40只，随机分为4组：假手术组（Sham组）、模型组（I/R组）、假手术给药组（Sham+LY333531）、模型给药组（I/R+LY333531）。各I/R组行肠系膜上动脉夹闭45分钟，再灌注45分钟、90分钟或180分钟以构建小肠I/R模型；各Sham组行假手术处理。假手术给药组及模型给药组于术前3天连续灌胃给10mg/kg的LY333531，每天1次。观察小肠、肝脏、肺脏组织病理学变化，检测血清中丙氨酸氨基转氨酶（alanine aminotransferase, ALT）、天冬氨酸氨基转氨酶（aspartate aminotransferase, AST）、肿瘤坏死因子α（tumor necrosisfantor-α，TNF-α）及白细胞介素-6（interleukin-6，IL-6）水平。检测肝、肺组织匀浆H2O2、谷胱甘肽（glutathione，GSH）、谷胱甘肽过氧化物酶（glutathione peroxidase，GSH-PX）、丙二醛（malondialdehyde，MDA）、髓过氧化物酶（myeloperoxidase，MPO）水平。采用脱氧核糖核酸转移酶原位末端标记技术（TdT-mediated dUTPNick-End Labeling，TUNEL）检测肝、肺组织细胞凋亡情况。采用Western Blot法检测肝、肺组织PKCβI、PKCβII、PKCγ、PKCδ、PKCε、phospho-PKCβII、锰超氧化物歧化酶（manganese superoxide dismutase，MnSOD）、cleaved-caspase-3蛋白表达水平。
结果：
1.与Sham组相比，I/R组：肝和肺组织PKCβII膜蛋白及磷酸化PKCβII蛋白表达水平明显升高，而其他PKC家族膜蛋白表达无明显变化；小肠组织病理示小肠绒毛排列明显紊乱，固有层崩解并形成明显溃疡，肝组织病理示肝索排列紊乱肝细胞核固缩，伴有胞浆嗜伊红细胞增多，肺组织病理示大量炎性细胞浸润，肺泡壁增厚并伴有肺泡出血；血清TNF-α、IL-6、ALT、AST水平显著升高；肝、肺组织H2O2、MDA和MPO水平显著增高，GSH和GSH-PX水平降低；肝和肺组织TUNEL凋亡检测显示凋亡细胞显著增多；肝和肺组织MnSOD蛋白表达水平明显降低，cleaved-caspase-3表达水平显著升高；Sham+LY333531组未见显著变化。
2、与I/R组相比，I/R+LY333531组：肝和肺组织PKCβII膜蛋白及磷酸化PKCβII蛋白表达水平明显降低；小肠、肝脏和肺脏组织病理示损伤均有所减轻；血清TNF-α、IL-6、ALT、AST水平明显下降；肝、肺组织H2O2、MDA和MPO水平显著降低，GSH和GSH-PX水平升高，肝肺组织TUNEL凋亡检测显示凋亡细胞显著减少；肝和肺组织MnSOD蛋白水平表达明显升高，cleaved-caspase-3蛋白表达水平明显降低。
结论：
1.小肠I/R可引起肝、肺组织中PKCβII的激活。
2.利用LY333531特异性抑制PKCβII的激活可对小肠I/R致远隔肝肺损伤有保护作用，可以有效减少肠肝肺组织学损伤、肝脏和肺脏的氧化应激和细胞凋亡。
第二部分抑制PKCβ依赖的p66shc磷酸化保护肠缺血再灌注远隔器官损伤
目的：探讨PKCβ依赖的p66shc磷酸化在小肠I/R远隔器官损伤中的作用，阐明LY333531预处理能否通过调控PKCβ/p66shc通路在肠I/R致肝肺损伤中发挥保护作用。
方法：体内实验分组及模型构建如第一部分所述，采用免疫共沉淀及WesternBlot法检测肝、肺组织中p66shc、磷酸化p66shc、PKCβ、磷酸化PKCβ、细胞色素C的蛋白表达。
体外实验分为两部分：1.人肝L02细胞和肺A549细胞，分为以下4组：对照组（Control组）、模型组（PMA组）、模型+干扰PKCβ组（PMA+si-PKCβ组）、模型+LY333531组（PMA+LY333531组）。采用Western Blot法检测p66shc、磷酸化p66shc、PKCβ、磷酸化PKCβ蛋白表达水平变化。
2.人肝L02细胞和肺A549细胞，分为以下4组：对照组（Control组）、干扰p66shc组（si-p66shc组）、模型组（H2O2组）、模型+干扰p66shc组（H2O2+si-p66shc组）。采用caspase-3活性试剂盒检测caspase-3活性、流式细胞术检测细胞凋亡。
结果：
1.体内实验：与sham组相比，I/R组肝肺组织中p66shc磷酸化显著增高，且呈再灌注时间依赖性；肝肺组织线粒体蛋白中p66shc含量显著增高；肝肺组织中p66shc和细胞色素C结合增多；肝肺组织中线粒体释放细胞色素C增多。
2.体内试验：与I/R组相比，I/R+LY333531组肝肺组织中p66shc磷酸化显著降低；肝肺组织线粒体蛋白中p66shc含量显著降低；肝肺组织中p66shc和细胞色素C结合减少；肝肺组织中线粒体释放细胞色素C减少。
3.体外实验第一部分：与Control组相比，PMA组p66shc磷酸化显著增高，而PMA+si-PKCβ组和PMA+LY333531组磷酸化p66shc较PMA组显著降低。
4.体外实验第二部分：与Control组相比，H2O2组caspase-3活性显著增强，凋亡细胞比例增高，而H2O2+si-p66shc组caspase-3活性和细胞凋亡比例较H2O2组显著降低。
结论：
1.小肠I/R组引起肝肺组织p66shc发生磷酸化，使p66shc发生线粒体转位，增强其与细胞色素C的结合，并促进细胞色素C的释放。
2. LY333531可以抑制p66shc蛋白的磷酸化，进而抑制其线粒体转位、与细胞色素C的结合及诱导细胞色素C释放，对肠I/R后的肝肺损伤发挥保护作用。
3. P66shc的磷酸化依赖于PKCβ的激活。
第三部分原儿茶酸调控p66shc磷酸化对小肠缺血再灌注多器官损伤的保护作用
目的：探讨多酚化合物PCA调控p66shc磷酸化信号通路在小肠I/R损伤和继性肝损伤中的作用。
方法：健康成年雄性ICR小鼠50只随机分为以下5组，每组10只：（1）假术组（Sham组）；（2）模型组（I/R组）；（3）假手术+PCA组（Sham+PCA0mg/kg group）；（4）模型+PCA低剂量组（I/R+PCA40mg/kg group）；（5）型+PCA高剂量组（I/R+PCA80mg/kg group）。各模型组采用Megison法构建肠I/R模型，行肠系膜上动脉夹闭45min，再灌注90min；各PCA处理组于模建立前3天给予PCA相应剂量腹腔注射，每天一次。再灌注结束后，收集血清本以检测TNF-α、IL-6、ALT和AST水平，收集小肠和肝脏组织标本进行病理织学评分和GSH、GSH-PX水平检测；利用RT-PCR法检测组织中p66shc mRNA达水平；利用Western-blot方法检测组织中p66shc、磷酸化p66shc、核因子κB p65NFκB p65）、Foxo3a、磷酸化Foxo3a、MnSOD、cleaved-caspase-3和Bcl-xL的白表达水平。
结果：
1.与Sham组相比，I/R组：小肠组织病理示肠绒毛排列紊乱，部分区域肠绒脱落，上皮下间隙增宽，毛细血管淤血；肝脏组织病理示肝索排列紊乱，可见量炎性细胞浸润，汇管区和肝血窦淤血现象严重，出现大量核固缩，核溶解；清TNF-α、IL-6、ALT、AST水平显著升高；NF-κB p65蛋白表达水平显著增加；SH含量和GSH-PX酶活力显著下降；小肠和肝脏组织p66shc mRNA水平和磷酸水平显著增加；Foxo3a磷酸化表达水平明显增加；MnSOD水平显著下降；leaved-caspase-3表达水平上调，Bcl-xL的表达下调。Sham+PCA组未见显著变化。
2.与I/R组相比，I/R+PCA（40mg/kg,80mg/kg）组：小肠组织病理示肠绒毛列整齐，上皮与固有层轻度分离，损伤程度明显减轻；肝脏组织病理示肝细胞列相对规则，炎性细胞浸润程度减轻，肝细胞分叶核形态正常，核固缩及核溶现象减轻。血清TNF-α、IL-6、ALT、AST水平显著降低；NF-κB p65蛋白表达平显著下降；GSH含量和GSH-PX酶活力显著增加；小肠和肝脏组织p66shcRNA水平和磷酸化显著降低；Foxo3a磷酸化表达水平明显降低；MnSOD水平著增加；cleaved-caspase-3表达降低，Bcl-xL的表达升高。
结论：
1. PCA对小肠I/R损伤和继发性肝脏损伤有保护作用。
2. PCA的保护作用与抑制p66shc的磷酸化及调控下游抗氧化和抗凋亡因子的表达相关。
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 reperfusion
Objective: 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 reperfusion
Objective: 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 damage
Objective: 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.
引文
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