Development of the microfluidic model of hepatic steatosis
In the present work, we first developed an in vitro model of hepatic
steatosis, under 3D dynamic culture conditions, into a microfluidic
sinusoid-like device (Liver-on-a-Chip device in Fig. 1) based on the
model previously published (Gori et al., 2016). Hepatic steatosis was
induced in HepG2 cells providing a fat overload with mixtures of two
FFAs (PA and OA), added to the medium alone or in combination at 1:2 and
2:1 molar ratios, and at 1mM final concentration for 48h. Depending on
the different proportions of saturated and unsaturated fatty acids used,
may represent hepatic cellular models of steatosis that mimic benign
chronic steatosis or a more severe and acute condition of steatosis
[6]. Then, through the use of confocal microscopy-based HCA we
evaluated in real-time the following parameters of steatosis:
intrahepatic FFA accumulation in the form of TGs (Fig. 2a, b) and their
cytotoxicity (Fig. 3a, b) as well as global levels of ROS/RNS and
Superoxide species (Fig. 4a-c) generated as a result of the different
FFA overloads.
[Figure 1]
Analysis of intrahepatic triglyceride accumulation, cytotoxicity and
oxidative stress in our microfluidic model of NAFLD
After the treatments with the different mixtures of FFAs for 48h (Fig.
2a, and b in the representative confocal micrographs), only an overload
of 1mM OA produced a statistically significant intracellular
accumulation of triglycerides [a four-fold increase vs. control
(ctrl), p<0.05]. The intracellular storage of triglycerides
observed in the other treatments, was proportional to the prevalence of
OA compared to PA. The analysis of FFA-induced cytotoxicity (Fig. 3a,
and b in the representative confocal micrographs) showed a remarkable
decrease in cell viability with 1mM PA (approximately 30% reductionvs. control, p<0.0001), followed by the treatments with
PA:OA 2:1 (p<0.01) and PA:OA 1:2 (p<0.01), implying
a clear dependence on the amount of PA in the FFA mixture. On the
contrary, the 1mM administration of OA alone did not cause any
significant reduction in cell viability compared to the ctrl. Lastly,
the evaluation of the oxidative stress derived from exogenous FFA
overload showed a statistically significant increase of both total
ROS/RNS levels only in the PA and PA:OA 2:1 treatments (Fig. 4a and c in
the representative confocal micrographs), and Superoxide levels only in
the PA group (Fig. 4b and c in the representative confocal micrographs)
compared to ctrl. Conversely, no significant rise was detected in any of
the other treatments, thereby underlining a direct effect of PA in the
generation of oxidative stress.
[Figure 2]
Overall, it turned out that while OA is more steatogenic than PA (Fig.
2a, b), the latter is more cytotoxic than OA (Fig. 3a, b), which is
consistent with the literature (Gómez-Lechón et al., 2007b; Ricchi et
al., 2009). Finally, in agreement with previous works (Gori et al.,
2016; Ricchi et al., 2009), the overload of FFAs produced moderate
levels of total ROS/RNS and Superoxide species (Fig. 4a-c) in every
treatment except those with PA and PA:OA 2:1, which showed higher
increases most likely responsible for the cytotoxic effects observed in
Fig. 3 and that may then lead to inflammation and apoptosis (Assaily et
al., 2011; Gori et al., 2014) correlated to disease progression and
severity (Angulo, 2002; Feldstein, Canbay, Angulo, et al., 2003;
Feldstein, Canbay, Guicciardi, et al., 2003).
[Figure 3]
Quercetin and Hydroxytyrosol individually reduce intrahepatic
triglyceride accumulation induced by the FFAs
Next, we investigated the role of dietary concentrations of the two
polyphenols in FFA-induced hepatic steatosis: i) Quercetin, a flavonoid
widely distributed in nature in many foods, especially in vegetables,
fruits and tea, at 10 μM final concentration, which is in the average
circulating human plasma concentrations, and in line with previous
reports (Radtke et al., 2002; Vidyashankar et al., 2013); ii)
Hydroxytyrosol, produced by the hydrolysis of oleuropein, which is a
polyphenol peculiar to olives and olive oil (Hur et al., 2012), at
physiological concentration of 10 μM (Hur et al., 2012). These two
natural compounds have been selected for their antioxidant,
anti-inflammatory and hypolipidemic properties (Hur et al., 2012;
Marcolin et al., 2012; Park, Choi, Um, Yoon, & Park, 2011; Peres et
al., 2000; Priore et al., 2014; Vidyashankar et al., 2013) and, in turn,
for the possibility to restrain or prevent the development of NAFLD in
our microfluidic model (i.e., reducing lipid accumulation, and the
related lipotoxicity and oxidative stress). Interestingly, when 10 μM
Quercetin (Fig. 2c, and 2d in the representative confocal micrographs)
and 10 μM Hydroxytyrosol (Fig. 2e, and 2f in the representative confocal
micrographs) were separately added for 48h to the cells, along with the
different mixtures of FFAs, intrahepatic triglyceride accumulation was
remarkably reduced compared to FFAs alone (Fig. 2a, b), with a
statistically significant difference in the OA treatment (Fig. 2c, e,
p<0.05 and p<0.01 for Que and HT, respectively) that
is the most steatogenic. Hence, such hypolipidemic effect of Que and HT
showed a very similar trend in all treatments and thus a relatively
comparable effect in the context of hepatic steatosis, which was further
confirmed by the log2-fold change (log2 F.C. in Fig. 1Sa and b) analysis
of the AdipoRed experiment. Indeed, the present analysis showed that the
more the prevalence of OA in the FFA mix and the stronger the
lipid-lowering effect of both Que and HT with the latter that,
regardless of the treatment, presents more statistically significant
fold changes compared to Que.
Quercetin and Hydroxytyrosol protect from the lipotoxicity of FFAs
Furthermore, a corresponding significant decrease in cytotoxicity was
also observed in almost all the treatments with Quercetin (Fig. 3c and
representative confocal micrographs in 3d) and Hydroxytyrosol (Fig. 3e
and representative confocal micrographs in 3f) compared to the FFAs
alone. Notably, regarding the strong lipotoxicity of PA, its effect was
partially reduced by both polyphenols compared to the treatments w/o
polyphenols, except for the PA:OA 1:2 condition in which the cell
viability increase, both after Que and HT administration, became
statistically significant (as shown in Fig. 3c, p<0.01, for
Que, and in Fig. 3e, p<0.01, for HT). Nevertheless, when the
combined treatments of PA with Que (Fig. 3c) and HT (Fig. 3e) were
compared to that with PA alone (white bars), we detected a slight
increase in cell viability that, however, was not statistically
significant in either case.
Quercetin and Hydroxytyrosol lower oxidative stress generated by the
FFAs
Finally, our data showed that also total ROS/RNS and Superoxide levels
were dramatically lowered with the addition of Quercetin (Fig. 4d, e and
f in the representative confocal micrographs) and Hydroxytyrosol (Fig.
4g, h and i in the representative confocal micrographs) compared to the
FFAs alone (Fig. 4a, b and c in the representative confocal
micrographs), as also proved by the log2-fold change analysis for both
polyphenols in the (log2 F.C. in Fig. 1Sc-f). In particular, the most
powerful effect of both polyphenolic compounds was observed against the
PA-induced ROS/RNS and Superoxide species in which the downregulation
was statistically significant (p<0.01 in Fig. 4d and e for
Que, and p<0.05 in Fig. 4g and h for HT). In addition, in the
PA:OA 2:1 treatment, the ROS/RNS and Superoxide production was
significantly lowered by both Que and HT (Fig. 4d and e with
p<0.001 for Que; Fig. 4g and h with p<0.01 for HT,
respectively), highlighting a slightly stronger effect, in terms of
statistical significance, of Quercetin against oxidative stress compared
to Hydroxytyrosol at the chosen concentrations.
[Figure 4]
Discussion
Drug discovery is currently hindered by the inability of conventional 2D
cell culture models as well as animal experiments to accurately predict
human responses. Liver-on-a-chip platforms may revolutionize this
scenario by reproducing the natural 3D microenvironment of the cells and
recapitulating some functionality of the hepatic tissue, allowing us to
imitate liver pathophysiology more closely to its in vivocounterpart. Hence, we leveraged on such disease-on-a-chip technology to
model the condition of hepatic steatosis, and to investigate at the cell
level the protective effects of dietary concentrations of two natural
polyphenols against some important features of the disease, in
particular the intrahepatic fat accumulation and its related
lipotoxicity and oxidative stress. Regarding Quercetin, its therapeutic
potential and hepatoprotective effect has been thus far attributed to
its antioxidant, anti-inflammatory and hypolipidemic activity (Marcolin
et al., 2012; Peres et al., 2000; Vidyashankar et al., 2013). Instead,
the beneficial effect in human health of extra virgin olive oil has been
long ascribed to its high content of oleic acid (Carluccio, Massaro,
Scoditti, & De Caterina, 2007; María-Isabel Covas, Konstantinidou, &
Fitó, 2009; Esposito & Giugliano, 2010). However, more recently, also
the important role played by the phenolic components (such as the
oleuropein-derivative Hydroxytyrosol) has been increasingly emerging,
not only for their known anti-oxidant and anti-inflammatory properties,
but also for their lipid-lowering ability (Bendini et al., 2007;
Carluccio et al., 2003; M.-I. Covas, 2008; Gordon, Paiva-Martins, &
Almeida, 2001; Hur et al., 2012; Jemai, Fki, et al., 2008; Jemai,
Bouaziz, Fki, El Feki, & Sayadi, 2008; Park et al., 2011;
Pérez-Jiménez, Ruano, Perez-Martinez, Lopez-Segura, & Lopez-Miranda,
2007; Priore et al., 2014). Indeed, in this work we observed a stronger
effect of HT, compared to Que, in mitigating the steatogenic effect of
OA as well as the different mixtures of FFAs (see Fig. 2 and Fig. 1S).
Actually, our in vitro results on the effects of Que and HT in
the framework of NAFLD confirmed in vitro as well as in
vivo data from the literature (Hur et al., 2012; LI et al., 2013;
Pirozzi et al., 2016; Porras et al., 2017; Priore et al., 2014;
Valenzuela et al., 2017; Vidyashankar et al., 2013) but in a more
realistic scenario that is closer to the in vivo situation. In
fact, our microfluidic model of NAFLD, providing us a more physiological
setting than conventional static 2D culture systems, may represent a
more suitable platform for simulating the chronicity of the disease and,
as such, getting closer to the animal model, despite its intrinsic
limitations that have yet to be completely overcome and improved.
These findings very importantly show a protective and comparable effect
of Quercetin and Hydroxytyrosol against FFA-induced hepatic steatosis
(confirming their lipid-lowering activity), lipotoxicity and oxidative
stress, in which they are able to scavenge free radicals. Therefore,
their role in counteracting excessive ROS/Superoxide generation and
boosting the antioxidant defenses of hepatic cells, along with the
reduction of excessive fat accumulation, seems to promote cell
viability, and may represent an appealing strategy for the treatment of
NAFLD. To date, no regulatory agency- approved cure for NAFLD has been
found yet. As a matter of fact, the results reported herein together
with the many beneficial pharmacological effects of Quercetin and
Hydroxytyrosol on liver damage may promote their future clinical
application as safe and effective therapeutic agents (Cao et al., 2014;
Echeverría et al., 2018; Marcolin et al., 2012; Peres et al., 2000;
Pirozzi et al., 2016; Tang et al., 2016; Valenzuela et al., 2017).
Future experiments will also include: i) the combined administration of
the two polyphenols to steatotic cells, in order to investigate a
possible synergistic effect of the two compounds; ii) the use of a more
complex liver microarchitecture that will involve different cell types,
including other parenchymal (i.e., primary human hepatocytes or
iPSC-derived hepatocytes) and non-parenchymal liver cells (e.g.,
endothelial cells, Kupffer cells and hepatic stellate cells) to enable
also the analysis of the expression levels of inflammatory and
fibrogenic cytokines (e.g., IL-6, IL-8, IL-1β, TNF-α, TGF-β1, CTGF),
involved in the development of NAFLD, from the supernatants of such a
multicellular hepatic microenvironment. In conclusion, our
NAFLD-on-a-chip approach may also pave the way to the technological
advancement of drug research, providing a promising tool to face the
challenges of drug screening with the final goal, in the next future, to
connect different tissues or even organs into a complex model system for
the study of human development and disease.