ABSTRACT
Our previous work indicated exposure of Human liver cell 7702 (HL7702) cells to Microcystin-leucine-arginine (MC-LR) for 24 hours can disrupt insulin (INS) signaling by the hyperphosphorylation of specific proteins. For further exploring the time-dependent effect posed by MC-LR on this pathway, in the current study, HL7702 cells together with mice were exposed to the MC-LR with different concentrations under short-term treatment, and then, protein phosphatase 2A (PP2A) activity and expression of proteins related to INS signaling, as well as the characteristics of their action in the liver, were investigated. The results indicated, in HL7702 cells with 0.5, 1, and 6 hours of treatment by MC-LR, PP2A activity showed an obvious decrease in a time and concentration-dependent manner. While the total protein level of Akt, glycogen synthase kinase 3 (GSK-3), and glycogen synthase remained unchanged, GSK-3 and Akt phosphorylation increased significantly. In livers of mice with 1 hour of intraperitoneal injection with MC-LR, a similar change in these proteins was observed. In addition, the levels of total IRS1 and p-IRS1 at serine sites showed decreasing and increasing trends,respectively, and the hematoxylin and eosin staining showed that liver tissues of mice in the maximum-dose group exhibited obvious hepatocyte degeneration and hemorrhage. Our results further proved that short-term treatment with MC-LR can inhibit PP2A activity and disrupt INS signaling proteins' phosphorylation level, thereby interfering with the INS pathway. Our findings provide a helpful understanding of the toxic effects posed by MC-LR on the glucose metabolism of liver via interference with the INS signaling pathway.
Subject(s)
Insulin/metabolism , Liver/drug effects , Microcystins/toxicity , Water Pollutants, Chemical/toxicity , Animals , Cell Line , Dose-Response Relationship, Drug , Glycogen Synthase/metabolism , Glycogen Synthase Kinase 3/metabolism , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , Liver/metabolism , Liver/pathology , Male , Marine Toxins , Mice , Phosphorylation/drug effects , Protein Phosphatase 2/metabolism , Signal Transduction/drug effectsABSTRACT
Our previous studies indicated that α4 was involved in the toxicity of MC-LR on the cytoskeleton via the change of PP2A activity in HEK 293. To explore the role of α4 in MC-LR toxicity via PP2A regulation in different cell lines, the HL7702 cell overexpressing α4 protein was exposed to MC-LR, and the change of PP2A, cytoskeletal structure, and cytoskeleton-related proteins were investigated. The results showed that PP2A activity was decreased, PP2A/C subunit expression and phosphorylation (Tyr307) increased significantly, but methylation (Leu 309)clearly decreased. The structure of the actin filaments and microtubules (MTs) remained unchanged, and the expression and phosphorylation of the cytoskeleton-related proteins showed different changes. In addition, the main components of the MAPK pathway, JNK, P38, and ERK1/2, were activated together. Our results indicated that elevated α4 expression did confer some resistance to MC-LR-induced cytoskeletal changes, but the responses of different cell lines to MC-LR, under the α4-overexpression condition, are not exactly the same.
Subject(s)
Cytoskeleton/drug effects , Intracellular Signaling Peptides and Proteins/metabolism , Microcystins/toxicity , Protein Phosphatase 2/metabolism , Actin Cytoskeleton/drug effects , Actin Cytoskeleton/ultrastructure , Adaptor Proteins, Signal Transducing , Cell Line , Cytoskeleton/ultrastructure , Humans , Marine Toxins , Microtubules/drug effects , Microtubules/ultrastructure , Mitogen-Activated Protein Kinases/metabolism , Molecular Chaperones , PhosphorylationABSTRACT
Astragalus polysaccharide (APS) was obtained by hot water extraction, alcohol precipitation, gel-permeation chromatography and ultrafiltration. Fluorescence material 2-aminoacridone (2-AMAC) labeled APS bind to macrophage in a time- dependent manner and the binding can be remarkably inhibited by APS. Furthermore, the effect of APS on RAW264.7 macrophage demonstrated APS increase the level of cytokines including TNF-α, GM-CSF and the production of NO. NF-κB protein levels are increased in response to APS. Blocking NF-κB with specific inhibitor resulted in decreased levels of NO and TNF-α. The results suggested that APS possess potent immunomodulatory activity by stimulating macrophage and could be used as an immunotherapeutic adjuvant.
