Dachaihu decoction ameliorates pancreatic fibrosis by inhibiting macrophage infiltration in chronic pancreatitis

Abstract

Aim: To explore the role of macrophages in chronic pancreatitis (CP) and the effect of Dachaihu decoction (DCHD) on pancreatic fibrosis in mice.

Methods: KunMing mice were randomly divided into a control group, CP group, and DCHD group. In the CP and DCHD groups, mice were intraperitoneally injected with 20% L-arginine (3 g/kg twice 1 d/wk for 6 wk). Mice in the DCHD group were administered DCHD intragastrically at a dose of 14 g/kg/d 1 wk after CP induction. At 2 wk, 4 wk and 6 wk post-modeling, the morphology of the pancreas was observed using hematoxylin and eosin, and Masson staining. Interleukin-6 (IL-6) serum levels were assayed using an enzyme-linked immunosorbent assay. Double immunofluorescence staining was performed to observe the co-expression of F4/80 and IL-6 in the pancreas. Inflammatory factors including monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α) and IL-6 were determined using real time-polymerase chain reaction. Western blot analysis was used to detect fibronectin levels in the pancreas.

Results: Compared with the control group, mice with 20% L-arginine-induced CP had obvious macrophage infiltration and a higher level of fibrosis. IL-6 serum concentrations were significantly increased. Double immunofluorescence staining showed that IL-6 and F4/80 were co-expressed in the pancreas. With the administration of DCHD, the infiltration of macrophages and degree of fibrosis in the pancreas were significantly attenuated; IL-6, MCP-1 and MIP-1α mRNA, and fibronectin levels were reduced.

Conclusion: The dominant role of macrophages in the development of CP was mainly related to IL-6 production. DCHD was effective in ameliorating pancreatic fibrosis by inhibiting macrophage infiltration and inflammatory factor secretion in the pancreas.

Keywords: Dachaihu decoction; Interleukin-6; Macrophages; Pancreatic fibrosis.

Figure 1

Morphological changes and macrophage infiltration in pancreatic sections obtained from mice with chronic pancreatitis induced by L-arginine. A: Pancreatic morphology at different time points of L-arginine induction using hematoxylin and eosin staining (original magnification, × 100); B: Macrophage infiltration in pancreas was detected using immunohistochemistry. Macrophages were stained using an anti-mouse F4/80 antibody (original magnification, × 200).

Figure 2

Interleukin-6 production is related to macrophage infiltration. A: Double immunofluorescent signals from F4/80 and interleukin-6 in L-arginine-induced chronic pancreatitis tissue at different time points (original magnification, × 200); B: Interleukin-6 levels in the sera of mice with chronic pancreatitis. Values are shown as mean ± SD (n = 36). ^aP < 0.05, ^bP < 0.01 vs control.

Figure 3

Dachaihu decoction protects against pancreatic injury and pancreatic fibrosis in mice with chronic pancreatitis. A: Changes in pancreatic morphology at different time points were evaluated by hematoxylin and eosin staining (original magnification, × 100); B: Collagen deposition was detected by Masson staining (original magnification, × 100); C: Ratio of pancreas weight/body weight changes in the different groups; D: The expression of fibronectin in the pancreas following the development of chronic pancreatitis. Values are shown as mean ± SD (n = 9). ^aP < 0.05, ^bP < 0.01 vs control; ^cP < 0.05, ^dP < 0.01 vs CP group.

Figure 4

Dachaihu decoction attenuated macrophage infiltration and interleukin-6 expression in the pancreas. A: Immunohistochemistry of macrophages associated with chronic pancreatitis (original magnification, × 200); B: Interleukin-6 mRNA levels in the pancreas determined by RT-PCR. Values are shown as mean ± SD (n = 9). ^aP < 0.05, ^bP < 0.01 vs control; ^cP < 0.05, ^dP < 0.01 vs CP group.

Figure 5

Effects of Dachaihu decoction on MCP-1 and MIP-1α mRNA levels in the pancreas of mice with chronic pancreatitis. A: MCP-1 mRNA levels in the pancreas as determined by real time-polymerase chain reaction; B: MIP-1α levels in the pancreas as determined by real time-polymerase chain reaction. Values are shown as mean ± SD (n = 9). ^aP < 0.05, ^bP < 0.01 vs control; ^cP < 0.05, ^dP < 0.01 vs CP group.