PF improved survival rates in DMN-indcued liver fibrosis in rats
During drug intervention, 2 rats died in the DMN-water group, and 1 in the DMN-GdCl3 group. Animal body, liver, lung, spleen, kidney, heart and brain weights were monitored during the formation of liver fibrosis (Table 1). Liver, spleen and lung weights increased significantly in the 2-week DMN rats compared with those in the 2-week normal group (p<0.05). Compared with 4-week normal rats, body, liver, heart and kidney weights decreased significantly in the 4-week DMN-water group (p<0.05), whereas the spleen weight in the 4-week DMN-water rats increased significantly (p<0.05). These results indicated that other organs such as spleen, lungs, kidneys and heart were damaged severely during cirrhosis. Compared with 4-week DMN-water rats, body, spleen and heart weights decreased notably in the DMN-GdCl3 rats (p<0.05), and spleen weight decreased significantly in the DMN-PF rats (p<0.05).
Liver/body weight ratio of the DMN-water group was significantly lower than that of the normal group (p<0.05). Spleen/body, lung/body, kidney/body, heart/body and brain/weight ratios were markedly increased (p<0.05) in the 4-week DMN-water rats compared with those in the 4-week normal rats (Additional file1: Table S2). The liver/body weight ratio of the PF rats was higher than that of the DMN-water group but there were no significant difference (p<0.05).
PF ameliorated liver function in DMN-induced liver fibrosis
Liver function parameters deteriorated over time in rats subjected to DMN (Table 2). DMN-treated rats developed hepatic injury as shown by significantly higher plasma concentrations of AST, ALT, ALP and TBil, and a lower concentration of TP and Alb compared with normal rats. PF treatment ameliorated significantly the increase of ALT, AST, ALP and TBil, and the decrease of Alb compared with 4-week DMN-water rats (p<0.05), while GdCl3 decreased ALP levels significantly (p<0.05).
PF improved histology in DMN rats
Pathological changes occurred in the liver during development of fibrosis, which was confirmed by H&E staining, and in the spleen, lungs and kidneys (Figure 1). In the liver, there was normal lobular architecture, with the central vein and radiating hepatic cords in the livers of normal rats (Figure 1). After DMN intoxication for 2 weeks, we observed massive hepatocyte necrosis, intense neutrophil infiltration, and initiation of fibrosis (Figure 1). In the DMN-water group, the liver sections revealed collagen fiber deposition, marked cirrhosis, and severe centrilobular necrosis. We observed marked reduction in the thickening of the collagen bundles in the DMN-PF group.
In normal spleen, the white pulp was circular or oval and consisted mainly of lymphocytes and the red pulp contained mainly red blood cells, and macrophages surrounded the white pulp (Figure 1). The spleen showed clear structural damage in the 2-week DMN rats; the white pulp was irregular in shape and the circular region had almost disappeared. The white pulp structure was restored in the 4-week DMN rats. GdCl3 had a positive protective effect on spleen structural restoration.
In normal lungs, there was no inflammatory cell infiltration and alveolar structure was normal. Alveolar structural damaged gradually developed after DMN treatment (Figure 1). There was a large amount of inflammatory cell infiltration in the alveolar walls and spaces. PF could reduce alveolar inflammatory cell invasion significantly in the lungs.
Normal kidney was characterized by clear organizational structure, and there was almost no macrophage infiltration (Figure 1). A lot of renal tubular epithelial cell swelling, necrosis and inflammatory cell infiltration appeared in 2-week DMN rats. Macrophage infiltration clearly decreased in kidneys of 4-week DMN rats. However, there was no obvious effect on the brain and heart compared with other organs in DMN-induced liver fibrosis (data not shown).
PF inhibited liver fibrosis in DMN rats
We evaluated whether PF inhibited hepatic fibrosis. Sirius Red staining clearly revealed accumulation of ECM, especially collagen. As shown in Figure 2A, collagen staining was scarcely observed in the normal liver samples, except in the area around the small central venous walls. In DMN-treated liver samples at 2 weeks, collagen stretched from the portal to lobular areas (Figure 2A). Additional cirrhotic nodule formation was seen in 4-week DMN-treated rats. Liver fibrosis was attenuated extensively by PF, which had better effects than GdCl3. Ridit analysis showed that there was a significant difference between the DMN-PF and DMN-water rats (p<0.05), but the DMN-GdCl3 intervention group did not differ significantly (Additional file1: Table S3).
Using antibody against desmin, a marker of stellate cell activation, we assayed the expression of this protein immunohistochemically (Figure 2B). In the normal rats, only vascular smooth muscle cells were strongly positive for desmin. After 2 weeks DMN treatment, desmin-positive HSCs were detected in areas of centrilobular and near periportal fibrotic bands. The number of desmin-positive HSCs was even higher in 4-week DMN-treated cirrhotic livers. After treatment with PF, the increased expression of desmin was markedly decreased. These results revealed the antifibrogenic effects of PF. Hepatic hydroxyproline content gradually increased in DMN-treated rats gradually (Figure 2C). PF treatment ameliorated significantly the increase of hydroxyproline content, compared with 4-week DMN-water rats (p<0.05).
Sustained deposition of ECM is mainly resulted from activation of HSCs; therefore, a correlation between accumulated collagen and activated HSCs was studied by detecting a marker of activated HSCs, α-SMA in liver sections described above. We detected the α-SMA and Collagen 1 α1 (Col1α1) by real-time PCR (Figure 2D). There was a gradual increase in their expressions and PF significantly attenuated the upregulation of α-SMA and Col1 (α1) significantly. Changes in expression of desmin and Col1 (α1) were also demonstrated by western blotting (Figure 2E).
Taken together, these results confirm that DMN administrations cause HSC activation and accumulation of ECM that might facilitate liver fibrosis and PF could inhibit DMN-induced liver fibrosis.
Macrophage activation disturbed in main organs in DMN-induced liver fibrosis
Liver macrophages/Kupffer cells were important contributors to fibrosis progression. However, macrophages in other organs have rarely been detected in previous studies. To elucidate the function of macrophages in liver fibrosis, a specific macrophage marker, CD68, has been used to monitor macrophage activation[4]. As shown in Figure 3, CD68-positive macrophages were present in hepatic sinusoids and were at very low levels in normal liver. After DMN treatment, CD68-positive macrophages with strong staining appeared not only in hepatic sinusoids, but also in portal areas and adjacent to fibrotic septa. PF and GdCl3 decreased CD68 expression compared with that in DMN-water rats.
The CD68-positive macrophages were at a relative high level and mainly located in the red pulp in normal spleen (Figure 3). CD68-positive macrophages increased significantly around the disrupted white pulp in 2-week DMN rats and decreased markedly in DMN-water rats. PF and GdCl3 inhibited CD68-positive macrophages markedly.
CD68-positive pulmonary macrophages with low expression in the lung in normal rat distributed widely in the alveolar, bronchial, pulmonary interstitial. Macrophage distribution increased gradually following DMN treatment and reached a peak in 4-week DMN-water rats. PF decreased the number of CD68-positive macrophages significantly; however, GdCl3 had no such effect.
Immunostaining with CD68 antibody showed marked accumulation of CD68-positive macrophages in the kidneys after 2 weeks DMN treatment. However, CD68-positive macrophages in the kidneys were decreased markedly in DMN-water rats. Administration of PF and GdCl3 had no obvious effects on macrophages in kidneys compared with that in DMN-water rats.
Real-time PCR showed that CD68 transcripts were upregulated about 6.7-fold after 2 weeks DMN treatment (Figure 4A). Thereafter, mRNA expression declined slightly in the 4-week DMN-treated liver. Similar changes at the protein level of CD68 were observed by western blotting (Figure 4B). Expression of CD68 in the spleen and kidneys was similar. CD68 expression in the spleen and kidneys in DMN-water rats decreased to a level below that in normal rats. PF and GdCl3 did not inhibit CD68 expression significantly in the spleen and kidneys. Unlike CD68 expression in the liver, kidneys and spleen, it increased gradually following DMN administration in the lungs. PF reduced CD68 expression, whereas GdCl3 even increased CD68 expression. These results were also confirmed by western blotting.
PF but not GdCl3 inhibited serum levels of TNF-α and IL-1β
One of important function of macrophages is to secrete proinflammatory factors, so we decided to detect proinflammatory cytokines in the serum. The level of TNF-α and IL-1β was low in normal serum and increased to 25.3- and 35.7-fold following 2 weeks DMN-treatment, respectively (Table 3), compared with that of the normal rats, and decreased markedly in DMN-water rats. PF but not GdCl3 decreased TNF-α and IL-1β significantly. The cytokines content in the serum represents the overall level in the body, and these proinflammatory cytokines are mainly derived from macrophages.
Inflammatory cytokines could derive from other organs besides the liver
We detected TNF-α and IL-1β in the liver, spleen, lungs and kidneys using real-time PCR. IL-1β transcripts were up-regulated 4.15-fold, 3.43-fold, 7.89-fold and 3.28-fold in the liver, spleen, lungs, and kidneys, respectively, in 2-week DMN rats (Table 4). Afterwards, IL-1β decreased in the liver, spleen and kidneys in DMN-water rats while it increased in the lungs in DMN-water rats (p>0.05) compared with that in the 2-week DMN rats. PF could inhibit the IL-1β transcription significantly in the lungs compared with DMN-water rats (Table 4).
GdCl3 treatment decreased liver TNF-α transcripts markedly compared with that of DMN-water rats. However, GdCl3 almost did not decrease TNF-α transcripts in the lungs. PF significantly suppressed the TNF-α expression in the spleen and lungs significantly (p <0.01 or 0.05) compared with that of DMN-water rats (Table 4).