Selective inhibition of protein kinase C β2 attenuates the adaptor P66 Shc-mediated intestinal ischemia-reperfusion injury.
Apoptosis is a major mode of cell death occurring during ischemia-reperfusion (I/R) induced injury. The p66(Shc) adaptor protein, which is mediated by PKCβ, has an essential role in apoptosis under oxidative stress. This study aimed to investigate the role of PKCβ2/p66(Shc) pathway in intestinal I/R injury. In vivo, ischemia was induced by superior mesenteric artery occlusion in mice. Ruboxistaurin (PKCβ inhibitor) or normal saline was administered before ischemia. Then blood and gut tissues were collected after reperfusion for various measurements. In vitro, Caco-2 cells were challenged with hypoxia-reoxygenation (H/R) to simulate intestinal I/R. Translocation and activation of PKCβ2 were markedly induced in the I/R intestine. Ruboxistaurin significantly attenuated gut damage and decreased the serum levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Pharmacological blockade of PKCβ2 suppressed p66(Shc) overexpression and phosphorylation in the I/R intestine. Gene knockdown of PKCβ2 via small interfering RNA (siRNA) inhibited H/R-induced p66(Shc) overexpression and phosphorylation in Caco-2 cells. Phorbol 12-myristate 13-acetate (PMA), which stimulates PKCs, induced p66(Shc) phosphorylation and this was inhibited by ruboxistaurin and PKCβ2 siRNA. Ruboxistaurin attenuated gut oxidative stress after I/R by suppressing the decreased expression of manganese superoxide dismutase (MnSOD), the exhaustion of the glutathione (GSH) system, and the overproduction of malondialdehyde (MDA). As a consequence, ruboxistaurin inhibited intestinal mucosa apoptosis after I/R. Therefore, PKCβ2 inhibition protects mice from gut I/R injury by suppressing the adaptor p66(Shc)-mediated oxidative stress and subsequent apoptosis. This may represent a novel therapeutic approach for the prevention of intestinal I/R injury.
Department of General Surgery, Second Affiliated Hospital, Dalian Medical University, 116023 Dalian, China.
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Z
Chen
Department of General Surgery, Second Affiliated Hospital, Dalian Medical University, 116023 Dalian, China.
G
G
Wang
Department of Pharmacology, Dalian Medical University, 116044 Dalian, China.
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X
Zhai
Department of Pharmacology, Dalian Medical University, 116044 Dalian, China.
Y
Y
Hu
Department of Pharmacology, Dalian Medical University, 116044 Dalian, China.
D
D
Gao
Department of General Surgery, Second Affiliated Hospital, Dalian Medical University, 116023 Dalian, China.
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L
Ma
Department of Pharmacology, Dalian Medical University, 116044 Dalian, China.
J
J
Yao
Department of General Surgery, Second Affiliated Hospital, Dalian Medical University, 116023 Dalian, China.
X
X
Tian
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D018938:Caco-2 Cells; D015687:Cell Hypoxia / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007249:Inflammation / Q000473:pathology; D007422:Intestines / Q000098:blood supply* / Q000201:enzymology / Q000473:pathology; D008297:Male; D008813:Mice, Inbred ICR; D018384:Oxidative Stress / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D064546:Protein Kinase C beta / Q000037:antagonists & inhibitors* / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology / Q000627:therapeutic use*; D021381:Protein Transport / Q000187:drug effects; D015427:Reperfusion Injury / Q000188:drug therapy* / Q000201:enzymology* / Q000473:pathology; D055765:Shc Signaling Adaptor Proteins / Q000378:metabolism*; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013755:Tetradecanoylphorbol Acetate
D047428:Protein Kinase Inhibitors; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; C528034:Shc1 protein, mouse; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D064546:Protein Kinase C beta; D013755:Tetradecanoylphorbol Acetate