1. Emerging functional role of bovine
lactoferrin and Naringenin in CCL4
and high fat diet induced NAFLD
2. • Non-alcoholic fatty liver disease (NAFLD) is closely associated
with metabolic syndrome. The incidence of non-alcoholic fatty
liver disease (NAFLD) is rising annually.
• NAFLD is now emerging as a common medical problem due to
its high incidence and the difficulty of treatment. The latest
epidemiology shows that NAFLD has become the second largest
liver disease after viral hepatitis, the incidence of which is 20–
30% (Sharma et al.,2015; Bellentani et al.,2000), and the
incidence of obesity in the population is up to 57.74% ( Starley
et al.,2010).
• Prevalence of metabolic risk factors including diabetes mellitus,
obesity, etc. is rapidly increasing in India putting this population
at risk for NAFLD.
• Patients with NAFLD are at increased risk for liver-related
morbidity and mortality and also cardiovascular disease risk and
increased incidence of diabetes mellitus on long-term follow-up
(Ajay Duseja et al.,2015).
• NAFLD ranges from simple steatosis in the absence of excessive
alcohol intake to non-alcoholic steatohepatitis (NASH) with or
without cirrhosis (Ekta Dhamija et al.,2019).
3. • It currently attracts more attention than in the past decades.
Although it is considered as a relatively benign form of chronic
liver injury, about 20% of NAFLD patients could progress to
cirrhosis and liver cancer, if not successfully retarded or
reversed.
• Unlike alcoholic fatty liver disease (AFLD), NAFLD is not induced by
the abuse or over-consumption of alcohol (30g alcohol/day for men
and 20 g alcohol/day for women).(Alkhouri et al.,2009)
• NAFLD is classified into four types according to the severity:
• Type 1 NAFLD is defined by steatosis with no inflammation or
fibrosis;
• Type 2 NAFLD is steatosis with non-specific lobular inflammation
but absence of fibrosis or hepatocyte ballooning;
• Type 3 NAFLD is steatosis with inflammation and fibrosis of variable
levels [non-alcoholic steatohepatitis (NASH)];
• Type 4 NAFLD is steatosis with inflammation, hepatocyte
ballooning, and fibrosis or Mallory-Denk bodies (NASH).
(Argo et al.,2009; Caldwell et al .,2002; Matteoni et al.,1999.).
4. • In addition, as a hepatic manifestation of metabolic syndrome
(Machado et al.,2006), NAFLD is associated with visceral
overweight or obesity, dyslipidemia, hyperinsulinemia or type
2 diabetes, and arterial hypertension, which is associated with
an increased risk of mortality (Haas et al.,2016).
• Considering all these , it is urgent to further explore the
mechanism and the progression of the disease in order to
develop methods for better targeting and therapeutic
strategies for individual patients.
• Furthermore, the occurrence and progression of NAFLD/NASH
can take a long period of time even several decades, which
limits the quality of data.
• Therefore, animal models are necessary to elucidate the
pathophysiological mechanisms and therapeutic targets of
NAFLD/NASH.
5. • High-Fat Diet &Carbon Tetrachloride:
• Liver damage induced by carbon tetrachloride (CCl4) is a well-
established general model for liver fibrosis.
• CCl4 induces an oxidative stress response in the liver, which
leads to the accumulation of toxic lipid and protein
peroxidation products and to a strong necrotic response.
• In mice, peritoneal injection of CCl4 brings about extensive
liver damage with degenerated and ballooned, necrotic
hepatocytes, as well as a mild mononuclear cell infiltration
and features of macro- and microsteatosis in the affected
areas.
• Transaminase and triglyceride levels are substantially higher
compared to those of control animals (injected with vehicle
only) ( Domitrovic et al., 2009; Zhan et al.,2016 ).
6. • Most importantly, CCl4 induces a dose-dependent
fibrosis that regresses after discontinuing CCl4
administration. ( Hansen et al., 2017).
• Combined model of HFD feeding and CCl4 administration
was developed and demonstrated that, in contrast to
mice exclusively fed an HFD, multiple administration of
CCl4 to HFD-fed mice induced not only steatosis, but also
recruitment of inflammatory cells, hepatocellular
ballooning, centrolobular fibrosis, both pericellular and
perisinusoidal, hypertriglyceridemia and significantly
increased transaminase levels, total cholesterol and
glucose levels were lower in the HFD + CCl4 group
compared to both the control group and the HFD group
(Kubota et al.,2013; ).
7. • Lactoferrin :
• Raghuveer et al. (2002) reported that including LF in the diet of
preterm infants also attenuated iron-induced oxidation products.
• Britigan et al. (1986) and Satue-Gracia et al. (2000) also showed
that LF had an antioxidant activity because the iron bound to the
protein was unable to act as a catalyst for the generation of the
hydroxyl radical which acts as an oxidative stress detoxificant.
• The protective effects of LF and the damaging effects of the
trauma may be due to the mediation of relative amounts of both
anti-inflammatory and pro-inflammatory components (Kruzel et al.
2002).
• When LF was administered for protection against CCl4 it caused
generalized deactivation of the monocytes and macrophages as
manifested by a significant reduction of both pro- and anti-
inflammatory mediators of inflammation
8. • Lactoferrin has been demonstrated to have protective effects
against cutaneous inflammation, colitis, and rheumatoid
arthritis by inhibiting the levels of the proinflammatory
cytokines tumor necrosis factor-α and IL-1b and stimulating
the expression of the antiinflammatory cytokine IL- 10
(Cumberbatch et al., 2000; Togawa et al., 2002).
• Lactoferrin has also been demonstrated to possess
antiinflammatory activity, as it can directly modulate cytokine
production by immune cells, such as macrophages and
lymphocytes, through receptor-mediated signaling pathways
(Van Snick and Masson, 1976; Mazurier et al., 1989).
• Lactoferrin can also down regulate inflammatory responses by
preventing iron-catalyzed free radical damage at inflammation
sites (Chodaczek et al., 2007).
9. • Naringenin :
• Flavonoids are a large group of phenolic compounds with
broader therapeutic effects( Cook et al.,1996)
• More than 4,000 different types of these compounds are
present in fruits, medical herbs, vegetables, nuts, and
beverages ( Nijveldt et al .,2001)
• Naringenin is the bitter constituent of grapefruit and is found
in the juice, flower, and rind of the fruit (Orhan et al.,2015
Wilcox et al., 1999).
• Naringenin supplementation also lowered elevated plasma
lipid concentrations in high-fat-diet–fed rats (Pu et al.,2012)
and decreased plasma lipids and cholesterol in high-
cholesterol-diet–fed rats (Shin et al .,1999).
10. • The cholesterol-lowering effect of naringenin was observed
in LDL receptor (LDLR) knockoutmice (Li et al.,2004).
• Naringenin (1% or 3% wt: wt of diet) prevented
hyperinsulinemia, leading to a reduction in hepatic sterol
regulatory element binding protein (SREBP) 1c and hepatic
lipogenesis in the fasted state .
• The reduction in hepatic TG availability due to naringenin
supplementation also contributed to VLDL-TG production
and VLDL-apoB secretion and attenuated dyslipidemia
(Mulvihill et al.,2009 ).
• Recent evidence suggests that naringenin decreases
expression of proinflammatory mediators such as TNF-a,
cyclooxygenase-2, and inducible NO synthase in 3-
nitropropionic acid–induced rats (Gopinath et al.,2012).
11. • Statins:
• Statins are (3-hydroxy-3-methyglutaryl coenzyme A reductase
inhibitor) are used worldwide for the treatment of lipid
disorders; in particular, they reduce the high levels of low
density lipoprotein cholesterol (LDL-C), decreasing thus
cardiovascular events and mortality (C. Stancu and A. Sima,
2001).
• However, there are accumulating data in the literature
suggesting that statins, such as simvastatin (SIM), may also
exert antiinflammatory effects, such as inhibition of cytokine
formation, adhesion molecule expression, and reduction of
nitric oxide production [Zhang et al.,2018, Athyros et al.,2018,
Park et al.,2016, Ajamieh et al.,2015), all of which could be of
value in protecting against pathological inflammation and
tissue damage.
12. • Statins for the treatment of non-alcoholic fatty liverdisease
and non-alcoholic steatohepatitis: Statins have anti-
inflammatory, anti-oxidant and anti-thrombotic effects that
are independent of their lipid-lowering activity (Pignatelli et
al., 2012, Violi et al.,2013). Therefore, they have been
proposed for the treatment of NAFLD and NASH, since in
these conditions both inflammation and oxidative stress play
an important pathogenetic role(Del Ben et al.,2014)
• In a recent study (Wang et al.,2013) simvastatin treatment
improved the prognosis of NASH-related fibrosis in rat models
of steatosis, by increasing the expression of endothelial nitric
oxidesynthase (eNOS), decreasing the expression of inducible
nitric oxidesynthase (iNOS), and inhibiting the activation of
hepatic stellatecells (HSC).
