Graphical Abstract
Keywords: Liver fibrosis; picroside Ⅱ; M1 macrophage; hepatic stellate cell; natural killer cell; neutrophil; CXCL16.
Abbreviation: ALT, alanine aminotransferase; AST, aspartate aminotransferase; AKP, alkaline phosphatase; CL, clodronate liposome; Col1a1, collagen type I alpha I chain; Csf3r, colony-stimulating factor 3 receptor; CM, culture medium; CXCL16, C-X-C motif chemokine ligand 16; CXCR6, C-X-C motif chemokine receptor 6; ECM, extracellular matrix; GZMB, granzyme B; HSC, hepatic stellate cell; IFN-γ, interferon-gamma; IL-2, interleukin-2; JAK1, Janus kinase 1; Mdr2, multidrug resistance protein 2; MMP2, matrix metallopeptidase 2; MPO, myeloperoxidase; NETs, neutrophil extracellular traps; PSC, primary sclerosing cholangitis; PIC Ⅱ, picroside Ⅱ; STAT, signal transducer and activator of transcription; TBA, total bile acid; TGF-β, transforming growth factor-β; Timp1, tissue inhibitor of metalloproteinase 1; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; WGCNA, weighted gene co-expression network analysis; α-SMA, α-smooth muscle actin.
Introduction
Hepatic fibrosis is a complex pathological process of recurrent inflammation induced by various hepatic injuries and is characterized by fibrous scar formation, due to the dysregulated overproduction and gradual excessive accumulation of excessive extracellular matrix (ECM) (Parola & Pinzani, 2019). If get out of control, this process may continue to deteriorate severe liver diseases such as cirrhosis and hepatocellular carcinoma. Throughout the entire process, inflammatory reactions triggered by hepatocellular injury or necrosis of hepatic parenchymal or non-parenchymal cells are the major risk factors for the initiation and development of liver fibrosis, since the infiltration of inflammatory immune cells and their released chemotactic peptides may interact with each other and finally amplify the reaction of hepatic stellate cell (HSC) activation (Ma et al., 2017). To break through the bottleneck of anti-fibrosis drug development, ocaliva, an agonist of the nuclear receptor farnesoid X receptor (FXR) that emerged as a vital regulator in hepatic fibrosis, has been approved by the FDA for the treatment of biliary fibrosis. However, its side effects including but not limited to, pruritus, fatigue, abdominal pain, immunologic derangement, dizziness, and even in severe cases, acute liver failure, have restricted its popularization and application (Shah & Kowdley, 2020). Hence, there is an urgent need to explore the pathogenesis and specific targets of novel drugs for alleviating liver fibrosis.
The deficiency of multidrug resistance protein 2 (Mdr2) protein, a crucial transport protein closely associated with bile secretion, is reported to result in the accumulation of toxic bile acids and subsequent hepatocyte and cholangiocyte injuries, effectively mimicking the clinical pathological characteristics in primary sclerosing cholangitis (PSC) (Liu et al., 2020; Ma et al., 2017). Additionally, the Mdr2 knockout (Mdr2-/-) mouse is a well-known fibrotic mouse model and has been widely used for investigating the pathogenesis of liver fibrosis and evaluating drug efficacy (Liu et al., 2019). Targeting inflammatory pathways or influencing immune cells in anti-fibrotic therapies is promising in recent years. Especially, M1- and M2-like monocyte-derived macrophages have been demonstrated to be tightly associated with sclerosing cholangitis in Mdr2-/- mice by means of chemokines like CCL2-CCR2/CCR5 axis and FXR-related pathways, reminding us that abnormal changes of macrophages are not only features but also indispensable initial participants of hepatic fibrosis (Guicciardi et al., 2018; Shi et al., 2022). Gevitha et al. focused on how the absence of interferon-gamma (IFN-γ)-mediated signaling shaped the production of chemokines and immune cell recruitment, and ultimately influenced liver fibrotic in Mdr2-/- mice model. They also confirmed that the increase of IFN-γ promotes the expression of granzyme B (GZMB) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) from CD8+ T and natural killer (NK) cells, further resulting in the inhibition of activated hepatic stellate cells (aHSCs) and the remission of liver fibrosis (Ravichandran et al., 2019). Besides, low doses of interleukin-2 (IL-2) upregulated the expression of CD39 in T regulatory cells to inhibit the hepatic CD8+lymphocyte proliferation, further leading to diminished fibrosis in Mdr2-/- mice (Taylor et al., 2018). However, the potential mechanisms of pathological transformation from hepatic inflammation to fibrosis and complex crosstalk between multiple immune cells especially for macrophages and NK cells that lead to hepatic fibrosis in Mdr2−/− mice have not been fully elucidated.
Picroside Ⅱ (PIC Ⅱ), one of the representative active components of the Picrorhizae rhizoma , has been demonstrated to exert a therapeutic potential on ischemia/reperfusion injury, liver damage, and organic cancer metastasis through inhibiting inflammation, oxidative stress and angiogenesis (Li et al., 2020; Ma et al., 2020). In recent years, researchers have gradually turned their attention on the hepatic protective role and immunoregulative function of PIC Ⅱ. In the alpha-naphthyliso thiocyanate-induced cholestasis mice model, PIC Ⅱ significantly activated FXR to regulate the activities of bile acid efflux transporters and bile acid metabolizing enzymes in livers (Li et al., 2020). Meanwhile, bile acids were reported to have a potential effect in regulating the whole-body immune system via influencing the balance of gut microbiome or inducing signal transduction as messengers (Duan et al., 2022), which reminds us PIC Ⅱ might possess a direct influence on disparate immune cells in the liver. Meanwhile, although there is no direct regulatory effect on immune cells, PIC Ⅱ could alleviate apoptosis and inflammation in sepsis by decreasing the expression of toll-like receptor 4 (TLR4) and tumor necrosis factor-alpha (TNFα) in an ischemic injured rat model (Huang et al., 2016). Of note, PIC Ⅱ also inhibited NF-κB activation and achieved the alleviation of inflammatory response and apoptosis in endotheliocytes by regulating the sirtuin 1 (SIRT1)/lectin-like oxLDL receptor-1 (LOX-1) signaling pathway (Wang et al., 2019). However, whether PIC Ⅱ was able to protect the liver from Mdr2 deficiency-induced liver fibrosis and the potential interaction between the PIC Ⅱ and different immune cells in fibrotic livers still needs to be clarified.
In the current study, we aimed to systematically investigate the pathological role of multiple immune cells involved in Mdr2 deficiency-induced liver fibrosis and to explore whether PIC Ⅱ could achieve its hepatoprotective effects by influencing the function of and interaction between different immune cells in the fibrotic liver. Our findings not only evaluate how different immune cells regulate the immune microenvironment and HSCs activation during the development of liver fibrosis, but also offered a kind of natural ingredients as a novel therapeutic option for removing the bottleneck of antifibrosis.
Methods and materials