Gadolinium Chloride Restores the Function of the Gap Junctional Intercellular Communication between Hepatocytes in a Liver Injury

Abstract

Background: Gadolinium chloride (GdCl₃) has been reported to attenuate liver injury caused by a variety of toxicants. Gap junctional intercellular communication (GJIC) is thought to be essential in controlling liver homeostasis and pathology. Here we evaluate the effects of GdCl₃ on functional GJIC and connexin expression in mouse models and primary hepatocytes.

Methods: Mice were administered GdCl₃ intraperitoneally the day before a carbon tetrachloride (CCl₄) injection or bile duct ligation (BDL) operation. Primary hepatocytes were treated with CCl₄ or lipopolysaccharides (LPS), with or without GdCl₃. A scrape loading/dye transfer assay was performed to assess the GJIC function. The expression of connexins was examined by real-time reverse transcription polymerase chain reaction (RT-PCR), western blot and immunofluorescent staining.

Results: CCl₄ treatment or the BDL operation led to the dysfunction of GJIC and a down-regulation of Cx32 and Cx26 in injured liver. GdCl₃ administration restored GJIC function between hepatocytes by facilitating the transfer of fluorescent dye from one cell into adjacent cells via GJIC, and markedly prevented the decrease of Cx32 and Cx26 in injured liver. In primary hepatocytes, CCl₄ or LPS treatment induced an obvious decline of Cx32 and Cx26, whereas GdCl₃ pretreatment prevented the down-regulation of connexins. In vivo GdCl₃ protected hepatocytes and attenuated the liver inflammation and fibrosis in liver injury mouse models.

Conclusion: GdCl3 administration protects functional GJIC between hepatocytes, and prevents the decrease of connexin proteins at mRNA and protein levels during liver injury, leading to the alleviation of chronic liver injury.

Keywords: connexin; gadolinium chloride; gap junctional intercellular communication; hepatocyte; liver injury.

Figure 1

The expression pattern of multiple connexins in carbon tetrachloride (CCl₄)-injured mouse liver. The mRNA expression of different connexins was examined by a quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) in liver injury mice induced by CCl₄. Data are presented as the mean ± SEM, and were analyzed by Student’s t-test for analysis of variance. * p < 0.05 vs. OO-treated mice.

Figure 2

Cx32 and Cx26 expression in injured liver tissue with or without gadolinium chloride (GdCl₃) administration. Four weeks after GdCl₃ administration in CCl₄-treated mice or two weeks after GdCl₃ administration in bile duct ligation (BDL) mice, liver tissue was collected. Immuno-fluorescence staining, western blot and qRT-PCR analysis for the expression of Cx32 and Cx26 were performed. (A) Cx32 and Cx26 mRNA expression in CCl₄-injured liver. (B) Cx32 and Cx26 protein expression in CCl₄-injured liver. (C) Immunofluorescence staining for Cx32 and Cx26 in OO-treated, CCl₄-treated, GdCl₃ plus OO-treated and GdCl₃ plus CCl₄-treated liver. Scale bars, 20 μm. (D) Cx32 and Cx26 mRNA expression in BDL-injured liver. (E) Cx32 and Cx26 protein expression in BDL-injured liver. Typical autoradiograms are shown. Expression of tubulin was checked to correct for variations in protein loading and transfer. All results were confirmed in three independent experiments. Data are presented as the mean ± SEM, and were analyzed by ANOVA for analysis of variance. * p < 0.05 vs. OO-treated mice. # p < 0.05 vs. CCl₄-treated mice without GdCl₃ administration.

Figure 3

The gap junctional intercellular communication (GJIC) function in CCl₄-treated mice with or without GdCl₃ administration. (A) Digital images of Lucifer yellow dye transfer in liver treated with OO, CCl₄, GdCl₃ plus OO, GdCl₃ plus CCl₄. Scale bars, 100 μm. (B) GJIC function and integrity was determined by quantifying the distance of the Lucifer yellow dye transfer from the incision. Data are presented as the mean ± SEM and were analyzed by ANOVA for the analysis of variance. * p < 0.05 vs. OO-treated mice. # p < 0.05 vs. CCl₄-treated mice without GdCl₃ administration.

Figure 4

Cx32 and Cx26 expression in CCl₄- or lipopolysaccharide (LPS)-injured primary hepatocytes with or without GdCl₃ pretreatment. The expression of Cx32 and Cx26 mRNA (A) and protein (B) in primary hepatocytes which were treated with CCl₄ of different concentrations. Cx32 and Cx26 mRNA expression in response to CCl₄ (C) or LPS (D) with the pre-treatment of 200 μM GdCl₃, 1 mM MgCl₂ or 1 mM CaCl₂ for 1 h, following 2 h of CCl₄ or LPS treatment. All results were confirmed in three independent experiments. Data are presented as the mean ± SEM, and were analyzed by ANOVA for analysis of variance. * p < 0.05 vs. Ctrl. # p < 0.05 vs. CCl₄-treated hepatocytes without GdCl₃ administration.

Figure 5

The expression of Cx32 and Cx26 in CCl₄-treated liver tissue with or without clodronate administration. Clodronate liposome (10 L/g B.W.) injection was performed once 72 h before CCl₄ treatment to delete Kupffer cells. The qRT-PCR, western blot analysis and immunofluorescence staining for the expression of Cx32 and Cx26 were performed. (A) Cx32 and Cx26 mRNA expression in injured liver. (B) Cx32 and Cx26 protein expression in injured liver. Typical autoradiograms are shown. Expression of tubulin was checked to correct for variations in protein loading and transfer. (C) Immunofluorescence staining for Cx32 and Cx26 in injured liver. Scale bars, 100 μm. All results were confirmed in three independent experiments. Data are presented as the mean ± SEM, and were analyzed by ANOVA for analysis of variance. * p < 0.05 vs. Ctrl. # p < 0.05 vs. CCl₄-treated mice alone.

Figure 6

Immunofluorescence staining for Cx32 and Cx26 in CCl₄-injured primary hepatocytes with or without GdCl₃ pretreatment. Immunofluorescence staining for Cx32 (A) and Cx26 (B) in Ctrl-treated, CCl₄-treated, GdCl₃ plus CCl₄-treated and GdCl₃ plus CCl₄-treated hepatocytes. Scale bars, 20 μm. The cell membrane was labeled with DiOC18, a membrane dye. Scale bars, 10 μm. DAPI was used to visualize nuclei (blue). Data are presented as the mean ± SEM, and were analyzed by ANOVA for the analysis of variance. * p < 0.05 vs. Ctrl. # p < 0.05 vs. CCl₄-treated hepatocytes without GdCl₃ administration.

Figure 7

The effect of GdCl₃ on liver inflammation and fibrosis in BDL mouse models. (A) Serum ALT and AST activity in BDL mice with or without GdCl₃ administration. (B) Representative H&E-staining liver sections after GdCl₃ administration in BDL liver. Scale bars, 50 µm. Inset: H&E-staining for Sham livers. (C) Quantification of inflammatory areas. (D) Representative images of Sirius Red staining after GdCl₃ administration in BDL liver. Scale bars, 50 µm. Inset: Sirius Red staining for Sham livers. (E) Quantitative analysis of liver fibrosis. Ten randomly-selected fields were quantitated for each mouse using the Leica QWin V3 software. (F) Expression of Col α1(I), Col α1(III), and α-SMA mRNA levels in liver, measured by qRT-PCR. Data are presented as the mean ± SEM, and were analyzed by ANOVA for analysis of variance. ∗ p < 0.05 compared with the Sham group. # p < 0.05 compared with the BDL mice without GdCl₃ administration.