[Effects of sodium arsenite exposure on activation and extracellular matrix secretion of human hepatic stellate cells]

Abstract

Objective: To explore the effects of sodium arsenite (NaAsO(2)) exposure on the activation and extracellular matrix secretion of human hepatic stellate cells, and to provide a theoretical basis for the mechanism study of arsenic induced hepatic fibrosis. Methods: Different doses of NaAsO(2) (0.0, 0.1, 1.0, 10.0, 50.0, 100.0 μmol/L) were exposed to human hepatic stellate cell line (Lx-2) for 24, 48 and 72 huors. CCK-8 assay was used to measure cell viability and IC(50) of NaAsO(2) on Lx-2 was then calculated; According to IC(50) results, 0.000, 1.875, 3.750, 7.500, and 15.000 μmol/L of NaAsO(2) were exposed to Lx-2 cells for 24 hours, besides, 7.500 μmol/L of NaAsO(2) was exposed to Lx-2 cells for 0, 12, 24, 48, and 72 hours, then collected cells and culture supernatant; HSC activation-related protein, including α-smooth muscle actin (α-SMA), and transforming growth factor-β1 (TGF-β1) expression levels were detected by Western blot analysis, the main extracellular matrix including laminin (LN) , hyaluronic acid (HA), collagen Ⅳ (COL-Ⅳ) and procollagen Ⅲ(P Ⅲ NP) secretion level was detected by Elisa assay. Results: CCK-8 assay showed that the cell viability of Lx-2 cells were increased obviously at low doses (≤1.0 μmol/L) of arsenic exposure, especially at 48 and 72 h. In contrast, with the increasing doses of arsenic exposure, the survival rate of Lx-2 cell was decreased gradually, and the survival rate of the high-dose (50, 100 μmol/L) arsenic exposure group at 24, 48 and 72 h were significantly lower than 0.0 μmol/L group, P<0.05. The IC(50) of NaAsO(2) on Lx-2 cells at 24, 48, 72 h were calculated as 72.75, 48.19 and 29.95 μmol/L, respectively; The expression levels of HSC activation-related protein showed that, after treated with 1.875, 3.750, 7.500, 15.000 μmol/L NaAsO(2) for 24 h, α-SMA and TGF-β1 protein level were higher than 0.000 μmol/L group. The increased expression of α-SMA and TGF-β1 protein were most significant in 7.500 μmol/L NaAsO(2) group (P<0.05). In addition, the expression levels of α-SMA and TGF-β1 also showed a time-dependent increasing in Lx-2 cells after treated with 7.500 μmol/L NaAsO(2) for 0, 12, 24, 48 and 72 h; Elisa assay showed that after treated with 1.875, 3.750, 7.500, 15.000 μmol/L NaAsO(2) for 24 h, the secretion levels of HA, LN, COL-Ⅳ and PⅢNP were obvious higher than 0.000 μmol/L group (P<0.05). Moreover, the secretion levels of HA, LN, COL-Ⅳ and P Ⅲ NP also showed a time-dependent increased manner in Lx-2 cells after exposed to 7.500 μmol/L NaAsO(2) for 0, 12, 24, 48 and 72 h (P<0.05). Conclusion: NaAsO(2) exposure to Lx-2 cells can upregulate the expression level of HSC activation-related proteins, induce its further activation, then increase ECM secretion level.

Keywords: Extracellular matrix; Hepatic stellate cell; Liver; Liver fibrosis; Sodium arsenite.