Discussion
The TAA- and BDL-induced models of liver fibrosis in rats are good models for exploring the pathogenesis of liver fibrosis and evaluating the action of drugs for liver fibrosis and cirrhosis. Here, we found that BDL for one month significantly increased the ALT, AST, ALP, and Hyp levels, liver indexes and pathological score of liver fibrosis in female rats. TAA treatment for 23 weeks has similar effects on the male rats. However, the level of Hyp in the livers of BDL-treated rats was higher than that in those of TAA-treated rats, but the pathological score of liver fibrosis was much lower than that in TAA-treated rats, which suggested that the composition of liver fibrosis in BDL rats was different from that in TAA-treated rats, which may be due to the difference in sex.
Interleukins (IL) including a lot of cytokines which play an important role in liver injury or fibrosis.IL-1α and IL -6 correlates with severity of liver diseases (38, 39) while IL-4 levels can be used to predict advanced fibrosis in chronic hepatitis C (40). IL-1β, Il-6, IL-9, IL-10, IL-13, IL-18 and L-12 are higher in patients with cirrhosis (41-47), However, we found that with BDL or TAA treatment, IL-1α, IL- 4, IL-10, TNFα, MCP-1 and PDGF-BB decreased, but IL-18 and TGF-β 1 increased; BDL for a month upregulated IL-1β and MIP-1α in the livers of female rats, while TAA decreased IL-1β, IL-6 and IL-12p70 in the livers of male rats.
BDL significantly decreased p-NFκB, p-P38MAPK, p-Akt, and p-STAT5 and increased p-p70s6k in the livers of female rats. TAA significantly decreased the levels of p-CREB, p-JNK, p-NFκB, p-Akt, p-p70s6k, p-STAT3 and p-STAT 5 in male rats but had no significant effect on the levels of p-P38MAPK and p-ERK1/2.
IL-1 refers to two similar cytokines (IL-1α and IL-1β) that bind to the same receptor and play an important role in acute and chronic inflammation. IL-1β is produced by monocytes, macrophages and neutrophils; it also recruits and activates these cells and induces local inflammation. In addition, IL-1β can activate HSCs, which contributes to fibrosis. When IL-1β levels are elevated, depletion of IL-1R1 ameliorates this fibrotic phenotype in mouse models of liver fibrosis, and the absence of IL-1 signaling attenuates thioacetamide-induced liver fibrogenesis in rats. IL-1 may regulate fibrosis and tissue remodeling by modulating the expression of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases. IL-18 is constitutively expressed in myeloid cells and epithelial cells; it signals via a heteromeric receptor, increases the production of nitric oxide (NO) and chemokines, and increases cell adhesion molecules for leukocyte trafficking. The maturation and secretion of IL-1β and IL-18 are mediated by inflammatory caspases within inflammasomes containing the NOD-like receptor (NLR) protein (NLRP) 3. Large amounts of IL-1β are produced by Kupffer cells, which also express most NLRs. Inflammasome signaling often induces pyroptosis that allows the passive release of alarmins, including IL-1α. Inflammasomes may regulate liver fibrosis directly by mediating inflammasome expression in HSCs or indirectly by HSC activation via Kupffer cell-derived IL-1β and IL-18. In addition, depletion of Nlrp3 significantly reduced the expression of TGF-β1 and collagen-1α1 in carbon tetrachloride-induced or TAA-induced liver fibrosis mouse models.
p-p70s6k is the active form of p70s6k, which plays an important role in protein synthesis and cell cycle control. p70s6k is activated by growth factors and hormones through the phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathway. IL-1β increases the p-p70s6k protein level in a time-dependent manner(48) .
Overall, BDL for one month increased the degree of liver fibrosis by augmenting the production of TGFβ1, induced the activation of HSCs by increasing the production of IL-1β and IL-18 to enhance inflammasome function and accelerated the formation of the collagenous connective tissue via the p70s6k signaling pathway. However, a key function of IL-18 is that it cooperates with IL-12 to induce IFN-γ production from T helper cells and NK cells, leading to NK cell activation, but BDL slightly decreased the levels of IL-12p70 and IFN-γ.
However, the level of MIP-1α markedly increased in the livers of BDL rats. MIP-1α is also known as CCL3 and is strongly expressed on T cells, macrophages, neutrophils, endothelial cells and HSCs. It may recruit several cell types, such as T cells, neutrophils and eosinophils, to the site of inflammation by interacting with its receptors CCR1 and CCR5. HSCs also express CCR5, which is the target of CCL3 in the liver. CCR1 and CCR5 deficiency can alleviate liver fibrosis from chronic carbon tetrachloride treatment or BDL.
CREB, JNK, NFκB, P38MAPK, ERK1/2, Akt, STAT3 and STAT5 are related to liver injury or fibrosis. Unexpectedly, most of the ILs and phosphorylated signaling pathway proteins we assessed were decreased by BDL for one month or by TAA treatment for 23 weeks. Under a microscope, we found that liver tissue slices from TAA-treated male rats exhibited a high degree of hyperplasia of connective tissue and scar tissue formation, and this effect was more serious than that of BDL female rats. There was no inflammation or necrotic tissue, which may be the cause of the decrease in inflammation-related signaling pathways in the two liver fibrosis groups, especially in TAA-treated rats.