Knockout of α-calcitonin gene-related peptide attenuates cholestatic liver injury by differentially regulating cellular senescence of hepatic stellate cells and cholangiocytes

Abstract

α-Calcitonin gene-related peptide (α-CGRP) is a 37-amino acid neuropeptide involved in several pathophysiological processes. α-CGRP is involved in the regulation of cholangiocyte proliferation during cholestasis. In this study, we aimed to evaluate if α-CGRP regulates bile duct ligation (BDL)-induced liver fibrosis by using a α-CGRP knockout (α-CGRP^-/-) mouse model. α-CGRP^-/- and wild-type (WT) mice were subjected to sham surgery or BDL for 7 days. Then, liver fibrosis and cellular senescence as well as the expression of kinase such as p38 and C-Jun N-terminal protein kinase (JNK) in mitogen-activated protein kinases (MAPK) signaling pathway were evaluated in total liver, together with measurement of cellular senescence in cholangiocytes or hepatic stellate cells (HSCs). There was enhanced hepatic expression of Calca (coding α-CGRP) and the CGRP receptor components (CRLR, RAMP-1 and RCP) in BDL and in both WT α-CGRP^-/- and BDL α-CGRP^-/- mice, respectively. Moreover, there was increased CGRP serum levels and hepatic mRNA expression of CALCA and CGRP receptor components in late-stage PSC samples compared to healthy control samples. Depletion of α-CGRP reduced liver injury and fibrosis in BDL mice that was associated with enhanced cellular senescence of hepatic stellate cells and reduced senescence of cholangiocytes as well as decreased activation of p38 and JNK MAPK signaling pathway. Cholangiocyte supernatant from BDL α-CGRP^-/- mice inhibited the activation and increased cellular senescence of cultured human HSCs (HHSCs) compared to HHSCs stimulated with BDL cholangiocyte supernatant. Taken together, endogenous α-CGRP promoted BDL-induced cholestatic liver fibrosis through differential changes in senescence of HSCs and cholangiocytes and activation of p38 and JNK signaling. Modulation of α-CGRP/CGRP receptor signaling may be key for the management of biliary senescence and liver fibrosis in cholangiopathies.

Figure 1. Increased expression of α-CGRP contributes to liver injury during cholestasis.

[A] Lack of α-CGRP induced CGRP receptor components expression (CRLR, RAMP-1 and RCP) in α-CGRP^−/− and BDL α-CGRP^−/− mice compared to WT and BDL WT mice, respectively, in isolated cholangiocytes (n=4). [B] Hepatic mRNA expression of CALCA (n=1) and CRLR (n=1), RAMP-1(n=5) and RCP (n=5) were increased in total liver from PSC patients compared to healthy controls (n=4). [C] Knockout of α-CGRP decreased SGPT and SGOT serum levels in BDL mice (n=3). [D]CGRP serum levels of PSC patient. CGRP serum levels were higher in PSC (n=5) patients than their control groups (n=4). *p<0.05 vs. WT mice or health control samples; ^#p<0.05 vs. BDL WT mice.

Figure 2. Lack of α-CGRP decreases cholestatic liver fibrosis induced by BDL.

[A] There was enhanced liver fibrosis in BDL WT compared to WT mice, but reduced liver fibrosis in BDL α-CGRP^−/− mice compared to BDL WT mice (n=3, Orig., magnification, 20×; scale bar=50μm). [B] Hydroxyproline levels in total liver samples from BDL CGRP^−/− mice was decreased compared to BDL WT mice (n=3). [C-D] Knockout of α-CGRP reduced mRNA expression of [C] α-SMA and [D] Fn1 in total liver samples from BDL α-CGRP^−/− mice compared to BDL WT mice. *p<0.05 vs. WT mice; ^#p<0.05 vs. BDL WT mice.

Figure 3. Lack of α-CGRP reduces BDL-induced fibrosis marker expression.

[A] There was decreased protein expression of α-SMA in total liver from BDL α-CGRP^−/− mice compared to BDL WT mice (n=3). [B] There was increased mRNA expression of TGF-β1 and Smad2 in total liver from BDL WT compared to WT mice, whereas the expression of these two genes was reduced in BDL α-CGRP^−/− mice compared to BDL WT mice (n=3). [C] TGF-β1 levels were higher in cholangiocyte supernatant from BDL WT mice than those in normal cholangiocyte supernatant from WT mice but decreased in cholangiocyte supernatant from BDL α-CGRP^−/− mice compared to BDL WT mice. [D-E] The protein expression of [D] p-JNK and [E] p-p38 in total liver was increased in BDL WT mice compared to WT mice, whereas decreased in BDL α-CGRP^−/− mice compared to BDL WT mice (n=3). *p<0.05 vs. WT mice; ^#p<0.05 vs. BDL WT mice.

Figure 4. Lack of α-CGRP reduces BDL-induced cellular senescence.

[A] Cellular senescence was enhanced in BDL WT mice compared to WT mice but reduced in BDL α-CGRP^−/− mouse liver compared to BDL WT mice, which was verified by SA-β-gal staining in liver sections (n=4, Orig., magnification, 40×; scale bar=100μm). [B-C] The mRNA expression of [B] p16, p21 and CCL2 and PAI-1 as well as [C] p16 protein levels were increased in total liver from BDL WT mice compared to WT mice but decreased in total liver from BDL α-CGRP^−/− mice compared to BDL WT mice (n=3). *p<0.05 vs. WT mice; ^#p<0.05 BDL WT mice.

Figure 5. Lack of α-CGRP decreases fibrosis and senescence marker expression in cholangiocytes from BDL mice.

[A] The mRNA expression of α-SMA, Col1α1 and Fn1 was decreased in cholangiocytes from BDL α-CGRP^−/−mice compared to BDL WT mice (n=4). [B] The mRNA expression of p16, p21 and PAI-1 was decreased in cholangiocytes from BDL α-CGRP^−/− mice compared to BDL WT mice (n=4). [C] The mRNA expression of SASP markers such as IL-6, IL-8, IL1-β and MMP2 was decreased in cholangiocytes from BDL α-CGRP^−/− mice compared to BDL WT mice (n=4). [D] Immunofluorescence demonstrated that p16 protein expression was decreased in cholangiocytes from BDL α-CGRP^−/− mice compared to BDL WT mice (n=3, Orig. magnification, 20×, scale bar= 50 μm). * p 0.05 vs. WT mice; ^#p<0.05 vs. BDL WT mice.

Figure 6. Lack of α-CGRP decreases fibrosis gene expression and increases senescence marker expression in HSCs from BDL mice.

[A] The mRNA expression of Col1α1 was decreased in HSCs isolated from BDL α-CGRP^−/− mice compared to BDL WT mice (n=3). [B] The p16 and p21 mRNA expression was increased in BDL α-CGRP^−/− mice compared to BDL WT mice (n=3). [C] Immunofluorescent staining showed that p16 protein expression was decreased in HSCs from BDL WT mice compared to BDL α-CGRP^−/− mice (n=3, Orig. magnification, 40×, scale bar= 25 μm). ^#p<0.05 vs. BDL WT mice.

Figure 7. Effect of α-CGRP on the expression of fibrosis and senescence genes in cultured HHSCs and IMCLs.

[A] Representative immunofluorescence picture of receptor activity-modifying protein 1 (RAMP1) in HHSCs and IMCLs were shown (n=4, Orig., magnification. 40×; scale bar=50μm). [B-C] α-CGRP stimulated the expression of α-SMA and Col1 α 1 in both [B] IMCLs and [C] HHSCs, which was prevented by CGRP_8–37 (n=4). [D] The expression of p16 and p18 was decreased in HHSCs while increased in ICMLs simulated by α-CGRP; these effects were partly reversed by incubation with CGRP_8–37 (n=4). *p<0.05 vs. Basal; ^#p<0.05 vs. α-CGRP-treated group.

Figure 8. Effect of stimulation with cholangiocyte supernatant on fibrosis and senescence gene expression in HHSCs.

[A-C] The mRNA expression of [A] TGF-β1, [B] TIMP1 and [C] Fn1 increased in HHSCs stimulated with cholangiocyte supernatant from BDL WT mice compared to HHSCs stimulated with cholangiocyte supernatant from WT mice but decreased in HHSCs treated with cholangiocyte from BDL α-CGRP^−/− mice compared to HHSCs stimulated with cholangiocyte supernatant from BDL WT mice (n=4). [D-F] The mRNA expression of [D] p16, [E] p18 and [F] p21 was increased in HHSCs treated with cholangiocyte supernatant from BDL α-CGRP^−/− mice compared to HHSCs stimulated with BDL cholangiocyte supernatant (n=4). *p<0.05 vs. HHSCs treated with cholangiocyte supernatant from WT mice; ^#p<0.05 vs. HHSCs treated with cholangiocyte supernatant from BDL WT mice.