Antifibrotic effect of N-acetyl-seryl-aspartyl-lysyl-proline on bile duct ligation induced liver fibrosis in rats

Abstract

AIM: To investigate the preventive effect of N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) on bile duct ligation (BDL)-induced liver fibrosis in rats.

METHODS: Liver fibrosis in rats was induced by BDL and AcSDKP was infused subcutaneously for 2 wk via a osmotic minipump (Alzet 2ML4) immediately after BDL operation. After scarifying, serum and liver specimens were collected. Hematoxylin and eosin staining, Sirius red staining, enzyme linked immunosorbent assay, Western blot or real-time polymerase chain reaction were used to determinate liver functions, histological alterations, collagen deposition, mRNA expression of markers for fibroblasts, transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-7 (BMP-7).

RESULTS: When compared to model rats, chronic exogenous AcSDKP infusion suppressed profibrogenic TGF-β1 signaling, α-smooth muscle actin positivity (α-SMA), fibroblast specific protein-1 (FSP-1) staining and collagen gene expression. Col I, Col III, matrix metalloproteinase-2, tissue inhibitors of metalloproteinase-1 and tissue inhibitors of metalloproteinase-2 mRNA expressions were all significantly downregulated by AcSDKP infusion (2.02 ± 1.10 vs 14.16 ± 6.50, 2.02 ± 0.45 vs 10.00 ± 3.35, 2.91 ± 0.30 vs 7.83 ± 1.10, 4.64 ± 1.25 vs 18.52 ± 7.61, 0.46 ± 0.16 vs 0.34 ± 0.12, respectively, P < 0.05). Chronic exogenous AcSDKP infusion attenuated BDL-induced liver injury, inflammation and fibrosis. BDL caused a remarkable increase in alanine transaminase, aspartate transaminase, total bilirubin, and prothrombin time, all of which were reduced by AcSDKP infusion. Mast cells, collagen accumulation, α-SMA, TGF-β1, FSP-1 and BMP-7 increased. The histological appearance of liver specimens was also improved.

CONCLUSION: Infusion of exogenous AcSDKP attenuated BDL-induced fibrosis in the rat liver. Preservation of AcSDKP may be a useful therapeutic approach in the management of liver fibrosis.

Key Words: N-acetyl-seryl-aspartyl-lysyl-proline; Liver fibrosis; Transformating growth factor-β1; α-smooth mucle actin; Bone morphological protein-7; Fibroblast specific protein-1; Epithelial-mesenchymal transition

INTRODUCTION

N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is an endogenous tetrapeptide normally present in the plasma and organs of humans and experimental animals[1-3]. It is released locally in tissues from its precursor thymosin-β4 (Tβ4) most likely by prolyl oligopeptidase (POP), a serine proteinase found in mammalian tissues[4,5]. AcSDKP is cleaved to an inactive form by the NH₂-terminal catalytic domain of angiotensin converting enzyme (ACE)[6].

Originally described as a natural inbibitor of hematopoietic stem cell proliferation, AcSDKP is now recognized as a critical negative regulator for extracellular matrix (ECM) accumulation in organs under both physiological and pathological conditions. Decreased basal levels of endogenous AcSDKP by ACE over expression or by POP inhibitors promote cardiac fibrosis and/or glomerulosclerosis[7,8]. Exogenous AcSDKP infusion reduces collagen deposition in rats heart and/or kidney under hypertensive and ischemic conditions[9]. AcSDKP also mediates the antifibrogenic effect of ACE inhibitors in the heart[10]. The mechanism of action of AcSDKP includes suppression of inflammation, ECM-producing cell proliferation, collagen production, and more importantly transforming growth factor-β1 (TGF-β1) signaling[7-9,11,12].

Indeed, these key cellular and molecular mechanisms are critical in regulating ECM accumulation in multiple organs, in particular the liver[13]. Moreover, ACE inhibition is beneficial in several liver fibrosis models where there is increased ACE activity and potentially excessive AcSDKP degradation, Tβ4 and significant POP activity are present in the liver, where AcSDKP is produced locally, and may play a role in the regulation of hepatic cell responses in vivo[14,15].

Our previous studies had revealed that AcSDKP ameliorated carbon tetrachloride (CCl₄)-induced liver fibrosis and liver functions in the rat liver. The current study was aimed to investigate the preventive effect of AcSDKP on bile duct ligation (BDL)-induced liver fibrosis in rats. The potential mechanisms involved were also examined.

We explored the effects of AcSDKP on liver fibrosis by infusion of exogenous AcSDKP into the BDL rat models. Our results demonstrate that exogenous AcSDKP preserves basal levels of AcSDKP in the liver and significantly reduces the development of liver fibrosis in this model. Based on these findings, we propose that AcSDKP plays an important role in attenuating liver fibrosis. The underlying mechanisms may involve decreased production of profibrotic cytokines and reduced collagen expression and accumulation.

MATERIALS AND METHODS

Materials

BDL-induced rat liver fibrosis models: all animal handling and experimental procedures were approved by the Animal Care and Use Committee of the Shanghai Jiaotong Uinversity School of Medicine. Male Sprague-Dawley rats (200-250 g) were obtained from the Shanghai Experimental Animal Center (Shanghai, China). The rat model of liver fibrosis was induced by BDL. Upon sacrifice, blood was collected and serum and/or plasma were obtained. Liver tissue was either fixed in 10% neutral buffered formalin, frozen in optimal cutting temperature, or snap frozen in liquid nitrogen and stored at -80 °C.

Experiment: AcSDKP-infused BDL-treated rats and the BDL model were established as above (n = 8-10). AcSDKP-infused BDL-treated rats were infused with AcSDKP at 800 μg/kg per day through a subcutaneous osmotic minipump (Alza Corp, Palo Alto, CA) beginning simultaneously with BDL. Rats were sacrificed at 2 wk. This dosage was used because it increased plasma AcSDKP to a concentration similar to that induced by captopril (100 μg/kg per day, 3- to 5-fold-change), without any adverse effect on the circulatory system[9].

Serum assays

Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, and albumin in serum and prothrombin time in plasma were measured using an automated analyzer.

Histological analysis

Formalin-fixed paraffin sections of the liver were stained with hematoxylin and esosin for pathological analysis or Sirius red for collagen. Collagen was quantified with Image Quant 5.1 software as previously described[16]. Positive cells were enumerated in 10 randomly selected fields at 400 × magnification.

Gene expression

Total RNA was extracted from livers using Trizol and was reverse-transcribed using an iscript cDNA synthesis kit. Real-time polymerase chain reaction (PCR) was performed on an iCycler system using the SYBR green Master Mix. Primer specificity was confirmed by sequencing PCR products. β-actin was the internal control. Data were presented according to the ΔΔC_t method.

Western blot

Frozen liver tissue was homogenized in ice-cold RIPA buffer containing protease and phosphatase inhibitors. A full list of antibodies is available in Supplemental data. Western blot was performed as previously described[17]. Bands were quantified by Scion Image 4.0.3. The loading control was tubulin.

Hydroxyproline content

Hydroxyproline content in liver tissue was determined as previously described[18].

Statistical analysis

Data are expressed as means ± SE. Comparisons were performed using analysis of variance. Least significant difference procedure analyses were performed when > 2 groups were present. P < 0.05 was considered statistically significant.

RESULTS

Chronic exogenous AcSDKP infusion suppressed profibrogenic TGF-β1 signaling,α-SMA, eibroblast specific protein-1 and bone morphogenetic protein-7 staining and collagen gene expression

When compared to model rats, TGF-β1 was significantly downregulated in AcSDKP-infused BDL-treated rats (Figure 1A). In contrast, bone morphogenetic protein-7 (BMP-7) staining in the liver of BDL-treated rats was increased by AcSDKP (Figure 1B). α-SMA, fibroblast specific protein-1 (FSP-1), collagen I, collagen III, tissue inhibitor of metalloproteinase-1 and 2 mRNA all were downregulated by AcSDKP infusion (Figure 1C and D). Collagen I, collagen III, matrix metalloproteinases-2, tissue inhibitors of metalloproteinase-1 and tissue inhibitors of metalloproteinase-2 mRNA expressions were all significantly downregulated by AcSDKP infusion (2.02 ± 1.10 vs 14.16 ± 6.50, 2.02 ± 0.45 vs 10.00 ± 3.35, 2.91 ± 0.30 vs 7.83 ± 1.10, 4.64 ± 1.25 vs 18.52 ± 7.61, 0.46 ± 0.16 vs 0.34 ± 0.12, respectively, P < 0.05). Matrix metalloproteinase-2 expression was increased in BDL-treated rats but suppressed by AcSDKP.

Figure 1 Western blotting and quantitive analysis. A: Transforming growth factor-β1 (TGF-β1); B: Bone morphogenetic protein-7 (BMP-7); C: α-smooth muscle actin positivity (α-SMA); D: Fibroblast specific protein 1 (FSP-1).

Chronic exogenous AcSDKP infusion attenuated BDL-induced liver injury, inflammation and fibrosis

BDL caused a remarkable increase in ALT, AST, total bilirubin, and prothrombin time, all of which were reduced by AcSDKP infusion (Table 1). The histological appearance of liver specimens was also improved (Figure 2A-C). Marked collagen accumulation was observed in AcSDKP-infused BDL-treated vs model rats, which was attenuated by AcSDKP infusion (Figure 2D-F). The reduction in total collagen was further confirmed by decreased hydroxyproline content. When compared to model rats, hyaluronic acid, ammonia terminal procollagen β peptide and hydroxyproline were all significantly decreased by AcSDKP infusion (127.4 ± 31.8 vs 267.2 ± 99.4, 6.9 ± 0.5 vs 35.2 ± 4.3, 162.3 ± 42.4 vs 398.2 ± 60.4, respectively, P < 0.05). Total mast cells decreased in AcSDKP vs model BDL-treated rats (Figure 2G-I).

Table 1 Comparision of liver functions.

Group	n	ALT (IU/L)	AST (IU/L)	TBIL (μmol/L)	AKP (IU/L)	PT (S)	ALB (g/L)
Normal	10	71.0 ± 32.8	146.0 ± 36.7	1.0 ± 0.8	221.8 ± 96.5	9.3 ± 1.6	38.1 ± 0.9
Model	8	66.9 ± 46.7	472.1 ± 236a	56.4 ± 53.9a	299.1 ± 37.4a	16.6 ± 4.6a	29.7 ± 9.5a
BDL + AcSDK	8	70.0 ± 34.9	245.7 ± 92.8c	6.3 ± 3.3c	265.4 ± 77.5	11.8 ± 1.0c	34.0 ± 4.9

^aP < 0.05 vs normal;

^cP < 0.05 vs model. BDL: Bile duct ligation; AcSDK: N-acetyl-seryl-aspartyl-lysyl-proline; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; TBIL: Total bilirubin; AKP: Alkaline phosphatase; PT: Prothrombin time; ALB: Albumin.

Figure 2 Hematoxylin and eosin, Sirius red, Giemsa staining for liver tissues in each group. A: Normal, hematoxylin and eosin (HE) (× 100); B: Model, HE (× 100); C: N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), HE (× 100); D: Normal, Sirius red (× 100); E: Model, Sirius red (×100); F: AcSDKP, Sirius red (× 100); G: Normal, Giemsa (× 200); H: Model, Giemsa (× 200); I: AcSDKP, Giemsa (× 200).

DISCUSSION

Here, we demonstrate that in the liver, chronic exogenous AcSDKP infusion preserves basal levels of AcSDKP and attenuates BDL-induced fibrosis. This is supported by other studies showing an important role of AcSDKP in preventing heart[19,20] and kidney[21,22] fibrosis at basal concentrations. Our previous studies had also revealed that AcSDKP ameliorated CCL₄-induced liver fibrosis and liver functions in rats.

Attenuation of liver fibrosis by AcSDKP is associated with suppressed inflammation and TGF-β signaling. Our results show that AcSDKP suppressed mast cells infiltration, TGF-β1 signaling and myofibroblasts in vivo.

Recent evidence suggests that epithelial-to-mesenchymal transition (EMT) may also contribute to liver fibrogenesis[23]. TGF-β1 is still generally considered to be the main positive regulator of EMT and ECM accumulation[23]. Indeed, our results show that AcSDKP suppressed TGF-β signaling and reduced the EMT markers α-SMA and FSP-1 in vivo[24,25]. In addition, AcSDKP increased BMP-7. BMP-7 counteracts the effects of TGF-β1 and is a prototypical negative regulator of EMT. Nevertheless, a more sophisticated study is required to fully elucidate the possible role of AcSDKP-induced inhibition of EMT in the attenuation of liver fibrosis.

There are some limitations in this study. We did not include a group of control rats infused with AcSDKP primarily because exogenous infusion of AcSDKP restored the peptide levels to control levels and this dose has been shown to have no adverse effects systemically. Secondly, the cellular mechanisms of AcSDKP action were not fully elucidated in our current study. The presence of an AcSDKP receptor on cells has been suggested[26]. We speculate that AcSDKP may directly affect liver cells by binding and activating its receptor on the cell surface, resulting in suppression of certain profibrogenic intracellular signaling pathways. Further studies to clone the receptor or develop specific receptor antagonists will enable full characterization of the cellular mechanisms involved in the antifibrotic effects of AcSDKP in vivo and in vitro.

In summary, this study shows that chronic exogenous AcSDKP infusion preserves basal levels of AcSDKP and attenuates liver fibrosis induced by BDL in rats. Our study strongly suggests a significant role for AcSDKP in the development of liver fibrosis and potentiates the usefulness of this tetrapeptide in the prevention of this disease. Additional studies are needed to gain further insight into the biological effect of AcSDKP in the liver and further studies are ultimately warranted in the human.

COMMENTS

Background

N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is an endogenous tetrapeptide in vivo which has antifibrogenic effects on the heart, the lung and the kidney. The authors’ previous studies had revealed that AcSDKP ameliorated carbon tetrachloride-induced liver fibrosis and liver functions in the rat liver.

Research frontiers

Originally described as a natural inhibitor of hematopoietic stem cell proliferation, AcSDKP is now recognized as a critical negative regulator for extracellular matrix accumulation in organs under both physiological and pathological conditions.

Innovations and breakthroughs

This is the first study to investigate the preventive effect of endogenous AcSDKP in bile duct ligation (BDL)-induced fibrosis in the rat liver and the potential mechanisms involved were also examined. The results strongly suggest a significant role for AcSDKP in the development of liver fibrosis and potentiates the usefulness of this tetrapeptide in the prevention of this disease.

Applications

Preservation of AcSDKP may be a useful therapeutic approach in the management of liver fibrosis.

Peer review

This is a good descriptive study in which authors analyze the preventive effect of AcSDKP on BDL-induced liver fibrosis in rats. The results are interesting and suggest that infusion of exogenous AcSDKP attenuated BDL-induced fibrosis in the rat liver. Preservation of AcSDKP may be a useful therapeutic approach in the management of liver fibrosis.