Fig 1.
Bile duct ligation caused liver injury such as necrosis, fibrosis and bile duct proliferation and AGT3 partially improves them.
Histological findings of liver at 2 w after operation. SO, sham operated, control rats; BDL, rats with cholestatic liver disease treated by bile duct ligation; and AGT, BDL rats treated with intraperitoneal injection of GGT antibody (AGT3). Asterisk; necrotic area, arrow; fibrosis area and arrow head; bile duct proliferation area. H&E. Scare bars are 100μm.
Table 1.
PCR primers and probe used in this study.
Table 2.
Body weight and biochemical parameters.
Fig 2.
Upregulation of serum γ-glutamyl transpeptidase (GGT) in bile duct ligation animals induces reduction of bone mass.
(A) Bone mineral density of rat lower body (white square indicate analyzed area) at 2 w after operation measured by dual-energy X-ray absorptiometry (DEXA). SO, sham operated, control rats; BDL, rats with cholestatic liver disease treated by bile duct ligation; and AGT, BDL rats treated with intraperitoneal injection of GGT antibody (AGT3). (B) Representative μ-CT images of the distal femora of the rats in the SO, BDL, and AGT groups. White squares indicate the area analyzed. (C) Total bone density, (D) Mean cortical bone section area. Significant differences (*P < 0.05, ** P < 0.01) were observed between two experimental groups.
Fig 3.
Bile duct ligation animals exhibit upregulated bone resorption with reduced bone formation and AGT3 partially improves them.
(A) Histological findings: hematoxylin-eosin (H&E) staining: (a,d) SO, sham operated control rats, (b,e) BDL, rat with cholestatic liver disease treated by bile duct ligation, (c,f) AGT, BDL rats treated with intraperitoneal injection of GGT antibody (AGT3). Tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts: (g) SO rats (h) BDL rats and (i) AGT rats. (B) Histomorphometric analysis of cancerous bone: (a) Trabecular bone number (Tb.N), (b) Trabecular thickness (Tb.Th), (c) osteoblast surface (Ob.S), and (d) osteoid volume per total tissue volume (OV/TV). (C) Histomorphometric analysis of cortical bone: (a) medial cortical area (Med.Ct.Ar), (b) medial cortical area per medial endocortical (Med.Ct.Ar./Med.Ec), (c) lateral cortical area (Lat.Ct.Ar), and (d) lateral cortical area per lateral endocortical (Lat.Ct.Ar./Lat.Ec). (D) Number of TRAP-positive osteoclasts. Significant differences (*P < 0.05, **P <0.01) were observed between the two experimental groups.
Table 3.
Cytokine expression in bone marrow at 2 weeks after operation.
Fig 4.
γ-glutamyl transpeptidase (GGT) stimulates osteoclastogenesis-related gene expression in ST2 cells.
(A) (a) ST2 cells were treated with 100 ng/ml of recombinant human GGT (rhGGT) for 2, 4, 12 and 24 h. VEGF-A, MCP-1, LIX, IFN-γ, TNF-α, IL-1β, RANKL and OPG mRNA expression was analyzed by RT-PCR. ST2 cells were treated with 100 ng/ml of recombinant human GGT (rhGGT) for 1 and 2d. Quantificaation of TNF-α (b) and MCP-1 (c) was performed by realtime PCR. (B) 100 ng/ml of AGT3 was added 2 h before 100 ng/ml of rhGGT treatment. After 2 h of incubation with rhGGT, TNF-α mRNA expression was examined by RT-PCR. (C) Bone marrow cells were cultured with conditioned medium of GGT-stimulated ST2 with or without TNF-α neutralizing antibody (TNF-α Ab). The cell culture stained with TRAP solution.
Fig 5.
γ-glutamyl transpeptidase (rhGGT) inhibits osteoblastic differentiation of osteoblasts in vitro.
(A) Alkaline phosphatase (ALP) activity in MC3T3-E1 cells after 6d of incubation with rhGGT (10, 100 and 500 ng/ml). (B) Mineralization-related gene expression in MC3T3-E1 cells at 12 and 24 h after incubation with rhGGT (100 ng/ml). (C) Primary osteoblasts (OBs) were cultured in OB differentiation media for 2 w with rhGGT (10, 100, and 500 ng/ml); Alizarin red S staining is shown for each condition; (D) Quantification of stained Alizarin red.