A role for intestinal alkaline phosphatase in preventing liver fibrosis

Abstract

Rationale: Liver fibrosis is frequently associated with gut barrier dysfunction, and the lipopolysaccharides (LPS) -TLR4 pathway is common to the development of both. Intestinal alkaline phosphatase (IAP) has the ability to detoxify LPS, as well as maintain intestinal tight junction proteins and gut barrier integrity. Therefore, we hypothesized that IAP may function as a novel therapy to prevent liver fibrosis. Methods: Stool IAP activity from cirrhotic patients were determined. Common bile duct ligation (CBDL) and Carbon Tetrachloride-4 (CCl4)-induced liver fibrosis models were used in WT, IAP knockout (KO), and TLR4 KO mice supplemented with or without exogenous IAP in their drinking water. The gut barrier function and liver fibrosis markers were tested. Results: Human stool IAP activity was decreased in the setting of liver cirrhosis. In mice, IAP activity and genes expression decreased after CBDL and CCl4 exposure. Intestinal tight junction related genes and gut barrier function were impaired in both models of liver fibrosis. Oral IAP supplementation attenuated the decrease in small intestine tight junction protein gene expression and gut barrier function. Liver fibrosis markers were significantly higher in IAP KO compared to WT mice in both models, while oral IAP rescued liver fibrosis in both WT and IAP KO mice. In contrast, IAP supplementation did not attenuate fibrosis in TLR4 KO mice in either model. Conclusions: Endogenous IAP is decreased during liver fibrosis, perhaps contributing to the gut barrier dysfunction and worsening fibrosis. Oral IAP protects the gut barrier and further prevents the development of liver fibrosis via a TLR4-mediated mechanism.

Keywords: Liver fibrosis; TLR4; gut barrier; intestinal alkaline phosphatase.

Figure 1

IAP expression is decreased in liver fibrosis in both mouse and human. (A) Stool IAP activity from liver cirrhosis patients VS. control measured by pNPP assay; (B) Stool IAP level of liver cirrhosis patients in different liver Child-Pugh classifications measured by pNpp assay; (C-D) Stool IAP activity in CBDL (C) and CCl4 (D) induced liver fibrosis models measured by pNpp assay at different time-points after the procedure. (E-F) Mouse duodenal AKP3 (duodenal IAP) and AKP6 (global IAP gene) gene expression detected by qPCR methods in both models. *: p<0.05.

Figure 2

IAP regulates intestinal tight junction protein expression in models of liver fibrosis in mice. (A) Terminal ileum tight junction protein genes (ZO-1, ZO-2, ZO-3, Occludin) expression in sham or common bile duct ligated mice detected by qPCR in WT and IAP KO animals. (B) Serum FITC-dextran level 4-hour after gavage at indicated groups. (C) Serum LPS concentration measured by LAL assay. (D) Terminal ileum level of tight junction proteins (ZO-1, ZO-2, ZO-3, Occludin) expression after 8 weeks of CCl4 induced liver fibrosis determined by qPCR. Serum FITC-dextran (E) level 4-hour after gavage and serum LPS concentration (F) at indicated groups measured by LAL assay. *: P<0.05.

Figure 3

Lack of IAP leads to a severe liver fibrosis after CBDL and CCL4 treatments. (A-C) WT and IAP KO mice underwent CBDL for 3 weeks, liver levels of ACTA-2, TIMP-2 and Collagen-1 genes were determined by qPCR, and compared with WT mice; (D,E) 3 weeks of CBDL mice were tested for fibrillar collagen by Sirius red staining (×100) and expression of a-SMA was determined by immunohistochemistry (×200). (F-H) WT and IAP KO mice underwent CCl4 treatment for 8 weeks, liver levels of ACTA-2, TIMP-2 and Collagen-1 genes were determined by qPCR; I, J: 8 weeks of CCl4 injected mice were tested for fibrillar collagen by Sirius red staining (×100) and expression of a-SMA was determined by immunohistochemistry (×200). *: P<0.05.

Figure 4

IAP supplementation rescues the gut barrier in murine models of liver fibrosis. (A) The level of ileal tight junction protein (ZO-1, ZO-2, ZO-3, Occludin) expression in mice treated with vehicle or IAP determined by qPCR, 3 weeks after CBDL. (B) Systemic serum FITC-dextran level 4-hour after oral gavage, and, (C) portal serum LPS concentration measured by LAL assay, 3 weeks after CBDL. (D) The level of tight junction protein (ZO-1, ZO-2, ZO-3, Occludin) expression was determined by qPCR, 8 weeks of CCl4 induced liver fibrosis. Systemic serum FITC dextran (E) and portal serum LPS concentration (F) after 8 weeks of CCl4 induced liver fibrosis. *: P <0.05.

Figure 5

Supplementation of IAP prevents the development of liver fibrosis in murine liver fibrosis models. (A-C) liver levels of ACTA-2, TIMP-2 and Collagen-1 genes in WT mice treated with oral vehicle or IAP at 3 weeks after CBDL, determined by qPCR; (D,E) Liver fibrosis tested for fibrillar collagen by Sirius red staining (×100) and expression of a-SMA determined by immunohistochemistry (×200) mice underwent CBDL. (F-H) WT mice with supplementation of IAP underwent CCl4 treatment for 8 weeks, and levels of ACTA-2, TIMP-2 and Collagen-1 gene expression in the liver were determined by qPCR; (I, J) Mice treated with 8 weeks of CCl4 were tested for fibrillar collagen by Sirius red staining (×100) and expression of a-SMA was determined by immunohistochemistry (×200). *: P<0.05.

Figure 6

Supplementation of IAP prevents the IAP KO mice development of liver fibrosis. (A-C) IAP KO mice with oral supplement IAP and 3 weeks after CBDL, liver levels of ACTA-2, TIMP-2 and Collagen-1 genes were determined by qPCR; (D, E) Mice 3 weeks after CBDL were tested for fibrillar collagen by Sirius red staining (×100) and expression of a-SMA was determined by immunohistochemistry (×200). (F-H) IAP KO mice with supplementation of IAP underwent CCl4 treatment for 8 weeks, and levels of ACTA-2, TIMP-2 and Collagen-1 gene expression in the liver were determined by qPCR; (I, J) Mice treated with 8 weeks of CCl4 were tested for fibrillar collagen by Sirius red staining (×100) and expression of a-SMA was determined by immunohistochemistry (×200). *: P<0.05.

Figure 7

IAP supplementation does not attenuate fibrosis in TLR4-KO mice. (A-C) 3 weeks after CBDL in TLR4 KO mice, ACTA-2, TIMP-2 and Collagen-1 gene expression in the liver was compared from mice with and without IAP treatment. Sirius red staining (×100) and ɑ-SMA expression (×200) was also compared between the groups (D, E). The CCl4 induced liver fibrosis model was also used in TLR4 mice with IAP in drinking water. After 10 weeks, liver ACTA-2, TIMP-2 and Collagen-1 gene expression were detected by qPCR (F-H) and liver collagen fiber was detected by Siruis red staining (×100) (I), while ɑ-SMA was detected by immunochemistry method (×200) (J). *: P <0.05.