Introduction
Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide, which occurs in individuals who deny significant alcohol consumption (Abd El-Kader & El-Den Ashmawy, 2015). NAFLD encompasses a broad spectrum of liver pathologies ranging from simple steatosis (a benign enhanced fatty infiltration within the liver) to the more severe nonalcoholic steatohepatitis (NASH), characterized by inflammation, fibrosis and hepatocellular damage (ballooning), which may progress to fibrosis and end-stage liver disease, including cirrhosis and hepatocellular carcinoma (HCC) (Abd El-Kader & El-Den Ashmawy, 2015; Angulo, 2002; Starley, Calcagno, & Harrison, 2010). However, the multifactorial pathogenesis of NAFLD has not yet been fully understood, and highly effective therapeutics are still far from being available. The majority of hepatocellular lipids are stored as triglycerides (TGs), but other lipid metabolites, such as free fatty acids (FFAs) and cholesterol, may play a role in the development of the disease through the damaging effect of lipotoxicity (Abd El-Kader & El-Den Ashmawy, 2015; Feldstein et al., 2004). Intrahepatic lipid accumulation (i.e., excessive TGs content) and oxidative stress, in terms of generation of reactive oxygen species (ROS) and lipid peroxidation, are two of the main mechanisms involved in the etiology of liver diseases, including NAFLD, in which they play a crucial role in the onset and progression (Abd El-Kader & El-Den Ashmawy, 2015; Adachi & Ishii, 2002). Interestingly, some recent studies, which used in vitro models of hepatic steatosis, showed the protective effect of two natural polyphenols, namely Hydroxytyrosol and Quercetin, against the development of NAFLD and in the reduction of intrahepatic lipogenesis (Hur et al., 2012; Priore, Siculella, & Gnoni, 2014; Vidyashankar, Sandeep Varma, & Patki, 2013). Two of these reports observed a beneficial effect of Oleuropein and its metabolite HT (the main polyphenols present in green olives, olive leaves and extra virgin olive oil) on lipogenesis and hepatic lipid synthesis, using mouse and human hepatoblastoma cell lines as well as primary-cultured rat hepatocytes (Hur et al., 2012; Priore et al., 2014). Similarly, Quercetin (a polyphenolic flavonoid compound from dietary origin and with known antioxidant and hypolipidemic activities) has been shown to attenuate and contrast NAFLD symptoms by reducing intrahepatic TG accumulation, oxidative stress and inflammatory cytokine production caused by oleic acid-induced hepatic steatosis in HepG2 cells (Vidyashankar et al., 2013).
Despite these findings, the traditional in vitro models of hepatic steatosis are not suitable for recreating the entire liver morphology and thus investigating the pathogenesis of NAFLD, as they rely on 2D cell culture monolayers that do not accurately reproduce the 3D physiological microenvironment of hepatic tissue; as such, they cannot recapitulate the chronicity of a multifactorial disease as complex as NAFLD (Chavez-Tapia, Rosso, & Tiribelli, 2012; Gómez-Lechón et al., 2007a; Gori et al., 2014; Ricchi et al., 2009). In this regard, the emerging organ-on-chip technology can be the real game changer: these platforms represent dynamic cell culture systems that offer great promise for simulating and studying human diseases (Liu et al., 2019; Rothbauer et al., 2019), including NAFLD, at tissue and organ level (Bovard et al., 2018; Chang et al., 2017; Li, George, Vernetti, Gough, & Taylor, 2018; Lu et al., 2018; “Organ network in transparent chip to study how cancer cells spread,” n.d.). Recently, our group4 and others (Bulutoglu et al., 2019; Ehrlich et al., 2018; Lee & Sung, 2018) have proposed alternative and more complex models of hepatic steatosis in a microfluidic chip to overcome the bottlenecks and limitations of the canonical in vitro cell culture models.
Furthermore, organs-on-chips are being adapted to high-throughput screening for drug discovery, also in combination with high-content imaging systems (Lin, Ballinger, & Khetani, 2015; Peel et al., 2019; Tan et al., 2019; van den Berg, Mummery, Passier, & van der Meer, 2019).
Thus, leveraging this approach/technology, here we disclose an advanced three-dimensional (3D) culture model of NAFLD (using perfused HepG2 cells) within a liver-on-a-chip microfluidic device, under dynamic culture conditions. This platform, by mimicking the endothelial-parenchymal interface of a liver sinusoid in an optimized 3D cell culture microenvironment, allowed us to investigate the pathogenesis of NAFLD, through the high-content analysis (HCA) of different parameters of liver steatosis at the cell level. Finally, the NAFLD-on-a-chip model has been exploited to study the protective effects of the two aforementioned naturally occurring plant-derived polyphenols, namely Quercetin and Hydroxytyrosol, on the development of NAFLD as potential therapeutic treatment.
Materials and methods