The Role Of Pdcd4in LPS/D-GaIN-Induced Acute Liver Injury

ObjectiveProgrammed cell death4(PDCD4) has been identified as a tumor suppressor gene, which suppresses tumor growth, infiltration and metastasis by regulating the expression of various genes related to tumor promotion and progression at transcriptional and translational levels. Our previous studies also demonstrated that PDCD4played an important role in the development of ovarian cancer and glioma. Recent studies have shown that PDCD4is also involved in various inflammatory diseases. It has been reported that PDCD4-deficient mice are resistant to streptozotocin-induced type Ⅱ diabetes and experimental autoimmune encephalomyelitis, and are also protected from the lethality of LPS. These results suggest that PDCD4is a pro-inflammation protein. However, some other reports have shown that knockdown of PDCD4increases TNF-α protein production in LPS-stimulated RAW264.7macrophages and indicate that PDCD4is an anti-inflammatory factor. Therefore, the exact role of PDCD4in inflammatory diseases remains to be investigated.Intestinal endotoxemia-induced acute liver injury is a common inflammatory disease leading to acute liver failure and death in the clinic. It is induced by cell wall components of Gram-negative bacteria—lipopolysaccharide (LPS), which is a potent activator of kupffer cells and endothelial cells. LPS stimulates kupffer cells to produce and release inflammatory factors such as tumor necrosis factor alpha (TNF-α), Interleukin (IL)-1β, IL-6, etc. The inflammatory response mainly depends on the activation of mitogen-activated protein kinases (MAPK) and NF-κB pathways.14These inflammatory factors, especially TNF-α, can further induce hepatocyte apoptosis and necrosis. During this process, kupffer cells play a crucial role in determining the inflammatory response to LPS and hepatocytes are the major target cells. D-galactosamine (D-GalN) is a kind of amino sugar, which induces the depletion of uridine triphosphate (UTP) pool and increases the hepatotoxicity of LPS. LPS in combination with D-GalN can induce specific liver injury, but has no effect on other organs, such as the heart, spleen and lung. Therefore, LPS/D-GalN-induced hepatitis is a well-established model to mimic liver injury induced by intestinal endotoxemia in a clinical setting.The present study aims to examine the role and mechanism of PDCD4in LPS/D-GalN-induced liver injury. Results from the study will not only be helpful for clarifying the regulatory effect of PDCD4in inflammation, but also lay the solid foundation for exploring the pathogenesis of endotoxin-induced acute liver injury and looking for new targets.Methods1. The effect of PDCD4deficiency on LPS/D-GalN-induced acute liver injuryPDCD4-deficient mice at the age of6-8wk and body weight of16-18g were used in all the experiments, with age-and sex-matched wild type C57BL/6mice as controls. For induction of acute liver injury, mice were administered intraperitoneally LPS (50μg/kg) and D-GalN (800mg/kg). Mice were sacrificed at0,3,5h after LPS/D-GalN administration. Serum and Liver tissues were collected for detection.(1) The gross appearance of livers was obsearved and liver-to-body weight ratios were calculated in the experimental and control mice.(2) Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured by an automatic biochemical analyzer (Siemens, Dimension RLMax).(3) Liver paraffin sections were stained with hematoxylin and eosin (H&E) for pathological analysis. The percentage of apoptosis cell was detected by situ end labeling technique (TUNEL) of fragmental DNA.(4) The activation of caspase-3was detected by immunohistochemical staining and western blot analysis. 2. The effect of PDCD4deficiency on the levels of inflammatory cytokine in mice(1) The inflammatory cytokines, including TNF-α, MCP-1,IL-6, IL-10, IL-12p70, in serum of experimental and control mice were measured by flow cytometry staining with BD Cytometric Bead Array (CBA) MOUSE Inflammation Kit. IL-1β was messured by enzyme-linked immunosorbent assay(ELISA).(2) The mRNA expression of some inflammatory cytokines (Tnfa, I11b,116, Mcp1,1110,I112p40,I112p35) in liver tissues was messured by RT-PCR.3. The effect of PDCD4deficiency on the activation of inflammatory signaling pathways(1) The activation of MAPK pathway was detected by western blot in the two groups of mice.(2) The activation of NF-κB pathway was detected by western blot and immunohistochemical analysis in the two groups of mice.4. The effect of PDCD4deficiency on mouse peritoneal macrophage in vitro Mouse peritoneal macrophages of C57BL/6and PDCD4-/-were obtained, andwere treated with LPS. At Oh,3h and5h after LPS treatment, the culture supernatantand the macrophage proteins were collected for detection.(1) The levels of TNF-a and IL-6in the culture supernatant were messured by ELISA.(2) The two important signaling pathways, MAPK and NF-κB, were detected by western blot in mouse peritoneal macrophages from the two types of mice.Result1. PDCD4deficiency promotes hepatocytes apoptosis and necrosis in LPS/D-GalN-induced acute liver injury(1) Five hours after injected with LPS/GalN, significantly higher liver-to-body weight ratios accompanied with more obvious liver swelling and congestion were observed in PDCD4-deficient mice compared with wild type mice.(2) Five hours after LPS/D-GalN administration, serum ALT and AST levels in PDCD4-deficient mice were significantly higher than those in wild type mice.(3) Five hours after injection, the results of H&E staining showed that the liver tissues of PDCD4-deficient mice displayed the loss of structural integrity. PDCD4-deficient mice had more obvious degenerative, necrotic hepatocytes, the infiltration of some lymphocytes and few neutrophils and liver internal hemorrhage than wild type mice.(4) At5h after injection, the result of TUNEL assay showed the number of TUNEL-positive cells in liver tissues of PDCD4-deficient mice was significantly increased compared with wild type mice. In addition, the results from the immunohistochemical and western blot analysis showed that the expression of cleaved caspase-3was significantly higher in the liver tissues of PDCD4-deficient mice than those in wild type mice.2. PDCD4deficiency increased serum and hepatic inflammatory cytokines levels in LPS/D-GalN-induced acute liver injury(1) At three hours after injection of LPS/D-GalN, serum levels of TNF-a, IL-6and MCP-1in PDCD4-deficient mice were significantly higher than those in wild type mice.(2) Three hours after LPS/D-GalN administration, there was significantly increased expression of proinflammatory cytokines Tnfa, I1lb,116and Mcpl mRNA in PDCD4-deficient mice compared with wild type mice.3. PDCD4deficiency promotes the activation of MAPK and NF-κB in the liver(1) At3h after LPS/D-GalN injection, p-JNK levels were significantly higher in PDCD4-deficient mice than wild type mice. At five hours after administration, both p-ERK1/2and p-P38levels were significantly higher in PDCD4-deficient mice than wild type mice.(2) The level of p-P65expression was significantly higher in PDCD4-deficient mice than wild type mice at3h and5h after injection.4. PDCD4deficiency promotes LPS-induced activation of mouse peritoneal macrophages(1) TNF-a level in supernatant was significantly up-regulated in PDCD4-deficient mice at3h and5h after treatment, IL-6level was higher in PDCD4-deficient mice compared with wild type mice at5h after treatment.(2) The p-ERK1/2and p-P38expression levels were significantly increased in peritoneal macrophages from PDCD4-deficient mice at3h after treatment. The levels of p-JNK expression were significantly increased in peritoneal macrophages from PDCD4-deficient mice at5h after treatment.(3) Three and five hours after treatment, the levels of p-P65were higher in peritoneal macrophages from PDCD4-deficient mice than those from wild type mice.Conclusions&OriginalityIn conclusion, the current study indicates that PDCD4could inhibit the activation of the MAPK and NF-κB pathways, decrease the production and release of inflammatory cytokines and provide protective roles for liver in LPS/D-GalN-induced acute liver injury. It is first suggested that there was an inhibitory action of PDCD4in LPS/D-GalN-induced acute liver injury.These results not only provide new research ideas for the functions of PDCD4, but also have important theoretical and potential application values for exploring the pathogenesis of endotoxin-induced acute liver injury and looking for new preventionand treatment methods.