김단백질에 의한 acetaminophen 간독성 억제작용기전

Abstract

Seaweeds have received increasing attention because these traditional food sources can both contribute to the maintenance of good health by providing nutritional benefits and be used to treat disease. Some seaweeds contain high amounts of proteins, vitamins, and minerals, and several polysaccharides found in seaweeds have diverse biological activities, including effects on the immune system and cancer. Some marine algae, for example, contain large amounts of polysaccharides,such as alginate, fucoidan, carrageenan, and agarose. Porphyran, a polysaccharide produced by the red alga Porphyra umbilicalis, is produced in large amounts in South Korea and has been shown to decrease cholesterol levels and to have antibiotic and anticancer effects, while fucoidan is known for its anticoagulant and antioxidant effects. In this study, we extracted a 14-kDa protein from the marine alga Porphyrayezoensis and examined its biological effects in vitro and in vivo. Acetaminophen (AAP) is a safe and effective analgesic when used at therapeutic levels. An overdose of AAP, however, can induce severe hepatotoxicity in experimental animals and in humans. Recent advances suggest that reactive metabolite formation, glutathione depletion, and alkylation of proteins, particularly mitochondrial proteins, are key initiating events in AAP toxicity. As in other types of liver injury, the roles of oncotic necrosis and apoptosis in AAPinduced liver damage have been debated. When AAP causes extensive ATP depletion, ATP depletion-dependent necrotic cell death results. However, when fructose and glycine are used to prevent ATP depletion, necrosis is blocked and caspasedependent apoptosis increases. MPT occurs under both fructose and glycinedeficient and sufficient conditions. The addition of cyclosporin A decreases necrosis and apoptosis. Thus, AAP toxicity is an example of "necrapoptosis," in which necrosis and apoptosis represent alternate outcomes of the same mitochondrial death pathway. We cultured Chang liver and HepG2 cells inEagle’s minimum essential medium (MEM) containing glycine (0.0075g/L) to block necroticcell death. AAP treatment and caspase-3 activation induced DNA fragmentation, indicating that the cells underwent apoptosis. However, DNA fragment results were not consistent with DNA laddering, a characteristic of apoptosis, although some DNA smearing, a feature of necrosis, was observed. Thus, we concluded that the mode of AAP-induced cell death was necrapoptosis because both necrosis and apoptosis apparently occur. Oxidative stress-induced cell death is a mitochondria-independent pathway of AAP cytotoxicity. Early investigations into the mechanism ofoxidative stress-induced cell death identified glutathione (GSH) as a critical factor in the detoxification of the reactive metabolite of AAP. While this initial breakthrough resulted in novel therapeutic strategies in the clinic, the mechanism of cell injury and liver failure is still not completely understood. In particular, the role of reactive oxygen species (ROS) in the pathophysiology of liver damage continues to be debated, despite three decades of research. Studies using a variety of experimental models have established that severe hepatocellular injury can lead to intracellular, mitochondriaderived oxidant stress. Elevated hepatic and mitochondrial GSSG levels are indicators of mitochondrial ROS formation, and AAPinduced cell death may be a result of oxidative cytotoxicity. Recent studies have shown that both endogenously produced and exogenously added ROS can regulate the activity of mitogen-activated protein kinase (MAPK) pathways that may be involved in cellular responses,such as proliferation, differentiation, and apoptosis. Three major MAPK kinase types exist:extracellular signal-regulated kinases (ERKs), p38 kinases, and c-Jun N-terminal kinases (JNKs). These kinases have several isoforms generated by alternative splicing of pre-mRNA. ERKs are generally activated by mitogenic and proliferative stimuli,such as growth factors involved in cellular proliferation and differentiation. JNKs and p38 kinases are primarily activated by extracellular stresses, such as UV irradiation, inflammatorycytokines, heat, and arsenic trioxide. Activation of these protein kinases causes a variety of cellular responses, depending on the cell type. In our experimental system, AAP induced ERK activation, but not JNK or p38 kinase activation. Thus, AAP-induced oxidative stress involves ERK activation. To further examine AAP-induced oxidative stress, we evaluated glutathione levels in a hepatocyte culture model of AAP-induced injury and in Sprague-Dawley rats. The results clearly demonstrated that AAP acted to decrease GSH levels, suggesting that AAP-induced injury involvesoxidative stress. We also examined whether a protein from P. yezoensis (PYP) could protect against AAP-induced injury. Ourresults indicate that co-treatment with PYP and AAPcausedcells to recover from AAP-induced injury in vivo.Specifically, AAP increased caspase-3 activity and DNA fragmentation, decreased GSH levelin liver tissue, and increased GOT/GPT levels in serum. However, when PYP was coadministered, caspase3 activity recovered, and DNA fragmentation, GSH levels, and GOT/GPT levels did not differ from those of the controls. Thus, PYP protected cells against AAP-induced liver injury. Furthermore, in the hepatocyte models, using Chang liver and HepG2 cells, AAP administration induced sub-G1 cell cycle arrest and decreased cell proliferation and GSH levels, while increasing caspase-3 activity, DNA fragmentation, and poly(ADP-ribose) polymerase (PARP) cleavage. This AAP-induced liver injury was reversed by the coadministration of AAP and PYP. That is, AAP plus PYP co-treatment induced cell proliferation, increased GSH levels, and inhibited PARP cleavage induced by AAP treatment. Insulin-like growth factors (IGFs) are synthesized in both fetal and adult hepatocytes and secreted into the extracellular fluid. Their interactions with the type-I IGF receptor (IGF-IR) play pivotal roles in the proliferation of a variety of cell types, control of the cell cycle progression in G1, regulation of the early phases of tumorigenicity, maintenance of the tumorigenic phenotype, andprevention of apoptosis. The variety of ways in which apoptosis can be induced suggests that wild-type IGF-IR and its ligands have widespread anti-apoptotic effects on several death signals. The binding of IGF-I to the IGF-IR activates multiple signal transduction cascades. One of these involves the MAPKs, a family of serine/threonine protein kinases activated by many stimuli, including IGF-IR. A key MAPK pathway contains ERK1/2. Upon IGF-IR autophosphorylation, the protein Shc is recruited to the receptor and becomes phosphorylated on tyrosine residues. Activated Shc then binds the adaptor protein Grb2 in an IRS-1-independent manner, leading to activation of the Ras-ERK pathway. This IGF-IR signaling pathway is most closely associated with cell differentiation and migration, but can also regulate the machinery of apoptosis in some cases, as in the detachment-induced death, or anoikis, of fibroblasts. Among the two dominant IGF-I signaling pathways, PI3-K and MAPK, members of the MAPK family, which include the ERK, JNK, and p38 kinase subgroups, play important roles in cell proliferation, apoptosis, and differentiation. Interestingly, these three family members also have roles in oxidative stress. Signaling through JNK is a proximal component of the cell death pathway and oxidative stress also activates JNK signaling, while the ERK pathway regulates cell growth and differentiation. Oxidative stress-induced ERK activation has been reported to function as an anti-apoptotic signal in homeostasis. Treatment with AAP induced ERK1/2 activation, and the addition of IGF-I to AAP-treated cells resulted in enhanced ERK1/2 activation. Nevertheless, IGF-I protected cells against AAP-induced cell death, as shown by the results of the MTS assay and PARP cleavage levels. We propose that ERK activation is pivotal in inducing the protective effect of IGF-I. Further study is required to clarify the mechanism. Both IGF-IR and PYP protected cells against AAP cytotoxicity.We hypothesized that PYP affected the IGF-IR signaling pathway and thus appeared to provide protective effects against AAP cytotoxicity. In Chang liver cells, AAP decreased IGF-IR phosphorylation, while PYP and AAP co-treatment reversed it. Furthermore, AAP decreased the electrophoretic mobility of IRS-1, IRS-1 protein expression, and binding of the p85/p110 complex to IRS-1. In contrast, these changes were reversed after AAP and PYP co-treatment.Furthermore, PYP heightened the decrease in Shc phosphorylation and binding of Grb2 to Shc by AAP. Thus, PYP inhibitedAAP cytotoxicity through two IGF-I signaling pathways: activation of the PI3K pathway and the MAPK pathway. In HepG2 cells, AAP treatment slightly inhibited IGF-I and IRS-1 phosphorylation. Furthermore, binding of IRS-1 and p85 markedly decreased after AAP treatment AAP blocked the PI3K pathway, leading to cell death. In contrast, PYP heightened the decrease in phosphorylation of IGF-I and IRS-I, and binding of p85 to IRS-1 by AAP. Furthermore, PYP enhanced the binding of Grb2 to Shc. Thus, PYP apparently inhibited AAP cytotoxicity via the PI3K and MAPK pathways in Chang liver and HepG2 cells. Finally, to mimic the metabolic process in the body, we hydrolyzed the 14-kDaPYP with pepsin. In amino acid sequencing, we detected a sequence of 21 amino acids that had high homology with the cytochromec sequence in P. umbilicalis and synthesized a peptide with this sequence (Pep21). We measured the effects of the pepsin hydrolysate and Pep21 on AAP cytotoxicity using the MTS assay in which both the hydrolysate and Pep21 protected against AAP cytotoxicity. Taken together, these results suggest that AAP induces liver injury via oxidative stress in vitro and in vivo. AAP and PYP co-treatment inhibited AAP cytotoxicity in vivo, as shown by the recovery in caspase-3 activity, and glutathione, GOT/GPT, and DNA fragmentation levels similar to those of the controls. In vitro, co-treatment with AAP and PYP resulted in increased glutathione levels, cell viability, and PARP cleavage. We also demonstrated that growth factors inhibitedAAP cytotoxicity. For example, IGF-I protected against AAP cytotoxicity, apparently via ERK activation. The protective effect of PYP also involved the ERK pathway. Our findings suggest that a protein extracted from the marine alga P. yezoensis (PYP) may be effective in protecting against AAP-induced liver injury.