The liver is innervated by sympathetic, parasympathetic, and peptidergic nerves, which contain afferent and efferent fibers. Several neuropeptides have been identified in the liver such as, neuropeptide Y (NPY), substance P (SP), and calcitonin gene-related peptide (CGRP). In particular, SP is synthesized by the liver, where it can regulate different activities including inflammation and proliferation. NAFLD is characterized by hepatic steatosis, liver inflammation, cirrhosis and ultimately liver failure. During this disease, ductular reaction (DR), represented by the activation of hepatic stem/progenitor cells (HpSCs), has been correlated with the severety of hepatic damage. It has been shown that SP plasma levels are elevated in patients with nonalcoholic cirrhosis. Knockout of SP receptor (NK-1R) reduces cholangiocyte hyperplasia and expression of fibrogenic markers in bile duct¿ligated (BDL) mice. However, no studies exist regarding the role of the SP/NK-1R axis in the development of non-alcoholic fatty liver disease. For that reason, the general aim of the present project will be to define the role of this axis in the activation of HpSC niche during NAFLD. Using experimental models and human biopsies, specific objectives will be: i) to evaluate liver morphology and hepatic steatosis in the different samples; ii) to investigate the localization of SP and NK1R in the different hepatic cells: hepatocytes, cholangiocytes, hepatic stellate cells (HSCs) and macrophages; iii) to study the expression of membrane metallopeptidase (MME); iv) to individuate the presence of inflammation markers, such as TNF-¿ and IL-6. Data obtained from this project will lead i) to understand cellular mechanisms at the basis of NAFLD progression; ii) to clarify the pathogenesis of cholangiopathies and biliary fibrosis; iii) to individuate novel molecular tools and therapeutic approaches targeting NAFLD.
The present study will clarify important aspects regarding: i) the role of SP/NK-1R axis in the activation of hepatic stem/progenitor cells (HpSCs) during NAFLD; and ii) the main relevant pathways implicated in this activation of HpSC niches in course of NAFLD both in vivo and in vitro.
The objective #1 of the present project will analyze and quantify the presence of SP and NK-1R at the level of hepatic cells in the development of NAFLD and the progression to fibrosis and cirrhosis. In particular, the results coming from this specific objective will assess the contribution of SP expression in the evolution of the disease. There is increasing information regarding the role of sensory innervation (¿-CGRP and the SP/NK1R axis) in the regulation of cholestatic liver diseases in animal models as well as diseased human samples. In fact, different studies have shown that there is an increase in ¿-CGRP and its receptor components; and enhanced expression of the SP/NK1R axis in cholestatic animal models and human primary sclerosing cholangitis (PSC) samples and patients with cirrhosis. For these reasons, this part of the study will have important implications regarding the clinical association between SP/NK-1R axis and NAFLD and will individuate new possible therapeutic target. In general, the correlation of histological aspects with data obtained from this part of the project will be useful to understand the relations between SP/NK-1R axis and NAFLD progression.
The objectives #2 and #3 of the present project will further clarify the possible correlations between HpSC activation and SP/NK-1R axis through different mechanisms. Firstly, we aim to analyze the HpSC niche activation. Then, we want to correlate the previous results with histological features and SP/NK-1R expressions. In the end, we aim to investigate the activation of signaling pathways such as, WNT, Notch1, Jag1, and Beta-catenin, together with the study of the membrane metallopeptidase and the inflammation, trying to find the main pathways involved in the pathogenesis of NAFLD through the activation of SP/NK-1R axis. Thus, the results of this part of the project would clarify whether the pathogenesis of NAFLD is associated with SP/NK-1R axis and HpSC activation and whether a specific mechamism is activated and participate in this process. Hepatic stem/progenitor cells (HpSCs) are bipotent progenitor cells, capable to differentiate into mature hepatocytes and cholangiocytes. They are are quiescent during physiological turnover of the organ but are activated in acute and chronic liver injuries. HpSCs respond to various stimuli and, once activated, they generate a peculiar morphological tissue response characterized by the ductular reaction. We will study the presence of DR in the several samples to correlate this presence with the activation of the SP/NK-1R axis. Then, since the main signaling pathways involved in HpSC niche are represented by Notch and WNT systems, we will evaluate the activation of these different mechanisms. Other studies have found that SP increases biliary mass and liver fibrosis through increased biliary senescence; and knockout of NK-1R decreases biliary proliferation/mass in BDL mice by down-regulation of cAMP/protein kinase A signaling. SP has also been shown to activate HSCs by TGF-ß1/Smad-3¿dependent signaling pathway. In addition, NK1R antagonists have been shown to prevent liver injury. In the end, overexpression of MME decreases SP-induced cholangiocarcinoma growth both in vitro and in vivo. A lot of mechanisms at the base of the SP/NK-1R axis activation, where it is very important to clarify the ffectiv epresence in NAFLD. Thus, the results coming from objectives #2 and #3 would be crucial to understand the relations between HpSC niche, NAFLD pathogenesis and SP/NK-1R axis, and their involvement in biliary damages. In general, data obtained from this part of the project will be important to better understand the pathogenesis of cholangiopathies and biliary fibrosis and to identify possible cellular, prognostic, and clinical target. Furthermore, the eventual disarrangement of these pathways during NAFLD will be assessed to test the hypothesis that such alterations could represent a main mechanism at the basis of biliary fibrosis progression.