α2-Adrenergic Receptor in Liver Fibrosis: Implications for the Adrenoblocker Mesedin

Abstract

The noradrenergic system is proposed to play a prominent role in the pathogenesis of liver fibrosis. While α1- and β-adrenergic receptors (ARs) are suggested to be involved in a multitude of profibrogenic actions, little is known about α2-AR-mediated effects and their expression pattern during liver fibrosis and cirrhosis. We explored the expression of α2-AR in two models of experimental liver fibrosis. We further evaluated the capacity of the α2-AR blocker mesedin to deactivate hepatic stellate cells (HSCs) and to increase the permeability of human liver sinusoidal endothelial cells (hLSECs). The mRNA of α2a-, α2b-, and α2c-AR subtypes was uniformly upregulated in carbon tetrachloride-treated mice vs the controls, while in bile duct-ligated mice, only α2b-AR increased in response to liver injury. In murine HSCs, mesedin led to a decrease in α-smooth muscle actin, transforming growth factor-β and α2a-AR expression, which was indicated by RT-qPCR, immunocytochemistry, and Western blot analyses. In a hLSEC line, an increased expression of endothelial nitric oxide synthase was detected along with downregulated transforming growth factor-β. In conclusion, we suggest that the α2-AR blockade alleviates the activation of HSCs and may increase the permeability of liver sinusoids during liver injury.

Keywords: hepatic stellate cells; liver fibrosis; mesedin; norepinephrine; sinusoidal endothelial cells; sinusoidal permeability; α2-adrenoceptors.

Figure 1

Expression of α2 adrenergic receptor (α2-AR) subtypes in carbon tetrachloride (CCl₄)-treated and bile duct-ligated (BDL) mouse models of liver fibrosis/cirrhosis. (A–C) Expression of α2a-AR, α2b-AR, and α2c-AR mRNA in liver tissues from mice treated with CCl₄ and controls, measured after 4 weeks using RT-qPCR. (D–F) Hepatic expression of α2a-AR, α2b-AR, and α2c-AR in mice after 4 weeks of BDL vs a sham operation (SO), measured using RT-qPCR. mRNA levels are shown as the mean ± standard error of the mean (SEM) (n = 4). Differences between both groups were analyzed using Student’s t-test. Statistical significance is indicated as follows: p < 0.05 (*); p < 0.01 (**); p < 0.001 (***).

Figure 2

Influence of mesedin on the expression of α-adrenergic receptors in murine hepatic stellate cells (M1-4HSCs). (A–D) Immunofluorescence analysis of α1/α-ARs (red) and α-SMA (green) in M1-4HSCs treated with 10 µM mesedin vs untreated controls (ctrl.). The scale bar corresponds to 100 µm, and the cell nuclei were stained with DAPI. (E,F) Quantification of α1-AR⁺/α-SMA⁺ and α2-AR/α-SMA⁺ M1-4HSCs upon incubation with mesedin vs the control (ctrl). (G) Western blot and densitometric analysis of α2-AR and β2-AR in M1-4HSCs with and without mesedin. Student’s t-test, n = 4, p < 0.05 (*), p < 0.01 (**).

Figure 3

Altered expression of α2-AR in M1-4HSCs due to mesedin-treatment. (A–C) Quantification of three major subtypes of α2-AR (α2a-AR, α2b-AR, and α2c-AR) under mesedin treatment in comparison to control cultures (ctrl.) using RT-qPCR. Student’s t-test, n = 6, p < 0.01 (**).

Figure 4

Influence of mesedin on α-smooth muscle actin (α-SMA) expression in M1-4HSCs. (A) Quantification of α-SMA mRNA in mesedin-treated M1-4HSCs vs controls (ctrl.), n = 6. (B) Quantification of α-SMA⁺ M1-4HSCs with and without mesedin, n = 5. (C) Western blot and densitometric analysis of α-SMA in M1-4HSCs treated with mesedin, n = 4. Student’s t-test; data are shown as the mean ± SEM, p < 0.05 (*), p < 0.01 (**), p < 0.0001 (****).

Figure 5

Transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF) expression in mesedin-treated M1-4HSCs. (A) TGF-β (red) and α-SMA (green) staining of M1-4HSCs under control conditions (ctrl.) and incubation with mesedin. Cell nuclei were stained with DAPI; scale bar 100 µm. Quantification of α-SMA/TGF-β-positive M1-4HSCs (n = 9, p < 0.001 (***), Student’s t-test; data shown as the mean ± SEM). (B) TGF-β expression as analyzed by Western blot and a corresponding densitometric analysis of M1-4HSCs treated with mesedin vs the control (ctrl.) (n = 4, p < 0.05 (*), Student’s t-test, data shown as the mean ± SEM). (C) PDGF Western blot of M1-4HSCs with and without mesedin (ctrl.), n = 4.

Figure 6

Impact of mesedin on the permeability of human liver sinusoidal endothelial cells (hLSECs). (A) Western blot and densitometric analysis of eNOS in hLSECs with and without (ctrl.) mesedin, n = 4. (B) Immunofluorescence analysis of α1-AR vs α2-AR (red) and eNOS (green) in hLSECs. Cell nuclei were stained with DAPI. Scale bar corresponds to 100 µm. Quantification of α2-AR-positive cells in hLSECs (n = 6, p < 0.01 (**), Student’s t-test, data shown as the mean ± SEM). (C) Western blot and densitometric analysis of TGF-β in hLSECs 48 h after incubation with and without (ctrl.) mesedin. Student’s t-test, p < 0.01 (**).