Huangqi decoction inhibits apoptosis and fibrosis, but promotes Kupffer cell activation in dimethylnitrosamine-induced rat liver fibrosis

Abstract

Background: Previously, Huangqi decoction (HQD) has been found to have a potential therapeutic effect on DMN-induced liver cirrhosis. Here, the mechanisms of HQD action against liver fibrosis were investigated in relation to hepatocyte apoptosis and hepatic inflammation regulation.

Methods: Liver fibrosis was induced by DMN administration for 2 or 4 weeks. Hepatocyte apoptosis and of Kupffer cells (KC) and hepatic stellate cells (HSC) interaction were investigated using confocal microscopy. The principle cytokines, fibrogenic proteins and apoptotic factors were investigated using western blot analysis.

Results: Compared with the DMN-water group, HQD showed decreased hepatocyte apoptosis and reduced expression of apoptotic effectors, cleaved-caspase-3, and fibrotic factors, such as smooth muscle α-actin (α-SMA), transforming growth factor beta-1 (TGF-β1). However, the KC marker CD68 increased significantly in DMN-HQD liver. Confocal microscopy demonstrated widespread adhesion of KCs to HSCs in DMN-water and DMN-HQD rats liver.

Conclusions: HQD exhibited positive protective effects against liver fibrosis; its mechanism of action was associated with protection from hepatocyte apoptosis and the promotion of CD68 expression in the devolopment of liver fibrosis to cirrhosis development.

Figure 1

Effects of Huangqi decoction (HQD) on histological changes (A, Sirius Red staining original magnification × 100; B, α-SMA staining original magnification × 100), C, hydroxyproline content, and D, the expression of pro-fibrotic factors of rat livers in DMN-induced liver fibrosis. ^▴ p < 0.05, ^▴▴p < 0.01, vs. DMN-water rats; ^△p < 0.05, ^△△p < 0.01 vs. 2 weeks DMN rats. Western blot analysis of total protein extracts was performed by antibodies recognizing α-SMA, TIMP-1, TGF-β1 and GAPDH respectively. Results are mean ± SD.

Figure 2

Effects of HQD on hepatocyte apoptosis (A), caspase-3 and HGF expression (B) in DMN-induced liver fibrosis in rats. Hepatic cryosections were stained with TUNEL (green) and hepatocytes paraffin antigen (Heppar, red). TUNEL (green) located in or near Heppar (red) means hepatocyte apoptosis. The arrows with blue color indicate apoptotic hepatocytes, the arrows with white color indicate non-hepatocyte apoptosis. (Original magnification × 400).

Figure 3

Effects of HQD on CD68 using immunofluorescence staining (A) and the expression of pro-inflammatory factors (B) using western blot analysis in DMN-induced liver fibrosis. Western blot analysis of total protein extracts was performed by antibodies recognizing CD68, TNF-α, MIP-1, IL-1, and GAPDH respectively. CD68, red; DAPI, blue; (original magnification × 200).

Figure 4

Effects of HQD on interaction of KCs and HSCs in DMN-induced liver fibrosis in rats. Hepatic cryosections were stained with α-SMA (blue) and CD68 (red). The pink color means the colocalization of (CD68) KCs and (α-SMA) HSCs. (Original magnification × 200).