Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease, encompassing a range of illnesses, from simple steatosis to nonalcoholic steatohepatitis. Its estimated global prevalence is about 25% [1
]. Oxidative stress and inflammation in the liver are believed to play an important role in the development and progression of this disease [2
]. Recent studies have demonstrated that accumulations of high cholesterol and cholesterol crystals in the livers increased cholesterol-induced NOD-like receptor protein 3 (NLRP3) inflammasome activation and thus resulting in an increase in caspase 1-mediated interleukin (IL)-1β protein release [3
Animal models of diet-induced steatohepatitis such as methionine and choline-deficient diet or high-fat and high-cholesterol (HFHC) diet can increase hepatic fat accumulation, inflammation, and liver injury, but these two models may also reduce body weight [4
]. Mice fed a HFHC diet develop hypercholesterolemia and accumulate cholesterol in the liver without the occurrence of obesity and insulin resistance [3
]. Although this animal model may still be controversial for patients with nonalcoholic steatohepatitis (NASH), it can be used as an experimental model for evaluating the testing compound on steatohepatitis and liver damage. In our previous study, mice fed a HFHC diet for six weeks showed increased hepatic cholesterol accumulation, NOD-like receptor protein 3 (NLRP3) inflammasome activation, and liver injury, as indicated by an increase in plasma alanine aminotransferase (ALT) activity and the presence of histological lesions in the liver [8
]. Long-term HFHC diet feeding (>6 weeks) has been shown to increase oxidative stress, inflammation, and fibrosis in the livers of mice [9
]. Mice fed a HFHC diet with active components of functional foods (e.g., green tea polyphenols or freshwater clam extract) have been demonstrated to prevent steatosis, inflammation, and liver injury [8
Phytonutrients are chemicals produced by plants with specific biological activities that can improve human health. Important bioactive phytonutrients include polyphenols, flavonoids, terpenoids, carotenoids, limonoids, glucosinolates, phytoestrogens, phytosterols, and anthocyanins, etc. [11
]. Recently, phytonutrients have received increased attention in studies on the prevention of many diseases, including NASH [10
]. Unlike clinical drugs, phytonutrients are natural products that possess relatively few or no side effects when they are used for therapy [11
]. Andrographis paniculata
(Burm. f.) Nees is a traditional medicine used in Chinese, Indian, and Thai remedies that are commonly used to treat infections, colds, and diarrhea [13
]. A. paniculata
and its related products are also used as functional foods in Taiwan. Terpenoids are the most attractive phytonutrients of A. paniculata
, and a number of diterpenoids have also been identified, including andrographolide (AND), 14-deoxy-11,12-didehydroandrographolide (deAND), neoandrographolide, 14-acetylandrographolide, and 14-deoxyandropholide [14
]. Of these, AND is the most abundant terpenoid in A. paniculata
and has been reported to have many biological functions, including liver-protective effects probably resulting from increases in antioxidant and anti-inflammatory activities [15
]. deAND, the second-most abundant diterpenoid in A. paniculata
, is present in a comparative concentration to AND in the leaves of the plant (~17.4 mg/g) [19
]. Although relatively less information is known about its biological functions compared with AND, deAND exerts no toxicity [20
] and has higher oral bioavailability than that of AND [15
]. In previous studies, the anti-cancer, anti-virus, anti-inflammation, and cardiovascular protective effects of deAND have been reported [20
Because AND has been demonstrated to have liver-protective effects [16
], it is possible that deAND may be an effective agent on treating liver diseases. This study was the first to investigate the hepatoprotective effects of deAND in a HFHC diet-induced steatohepatitis and liver injury in mice. The potential effects of deAND on antioxidant and anti-inflammatory activities in liver were determined.
In this study, deAND treatment reduced cholesterol accumulation, inflammation and lowered liver damage in mice fed a HFHC diet. In addition, deAND reduced NLRP3 inflammasome activation and oxidative stress in the liver. The Nrf-2-mediated downstream antioxidant enzyme activity and/or protein expressions in liver were upregulated by deAND. Although we could not distinguish whether the inflammation or oxidative stress in the liver induced by HFHC diet is derived from the hepatocytes or other immune cells, deAND treatment may ameliorate HFHC diet-induced steatohepatitis and liver damage possibly by increasing antioxidant and anti-inflammatory activities in the liver.
A previous study indicated that HFHC diet-induced histopathological changes in the livers of mice were accompanied by a significant accumulation of small cholesterol-containing droplets, which contained abundant cholesterol crystals [3
] and free cholesterol [36
]. The impact of dietary cholesterol was recently demonstrated to be a key factor in the transition from simple steatosis to NASH [37
]. The accumulation of cholesterol crystals and free cholesterol in the liver may lead to a dysregulated cholesterol synthesis pathway and cause liver damage [3
]. In this study, mice fed a HFHC diet for seven weeks showed morphological changes in the liver, alongside increased cholesterol accumulation and macrophage infiltration (Figure 2
), and increased plasma ALT and AST activities. In addition, a dramatically reduced hepatic GSH content (–90.7%), lowered antioxidant enzyme activities (GSH peroxidase and GSH reductase), and elevated hepatic levels of TNF-α (+77.6%) and IL-1β (+55.6%) was observed in mice fed a HFHC diet. A higher apoptosis index (caspase 3/pro-caspase 3 ratio) was found in liver after HFHC feeding. These results indicate that mice fed the HFHC diet increased oxidative stress, inflammation, and liver damage. Mice treated with 0.1% deAND showed mildly lowered oxidative stress, as indicated by increasing GSH content and GSH reductase activity, and a significantly reduced inflammation by lowering TNF-α level in liver. Because increased oxidative stress, lipotoxicity, and inflammation play key roles in the progression of many fatty liver diseases [2
], it is suggested that 0.1% deAND treatment may reduce HFHC diet-induced steatohepatitis and liver damage.
To further investigate the exact mechanisms of deAND involved in antioxidant and anti-inflammatory activities associated with HFHC diet-induced oxidative stress and inflammation in liver, mice were fed a HFHC diet for a longer feeding time (11 weeks). The results showed that NLRP3 inflammasome activation was induced by feeding of the HFHC diet. The increased NLRP3 inflammasome activation can be stimulated by the accumulation of cholesterol and cholesterol crystals in the livers [3
]. Shimada et al. also demonstrated that oxidized mitochondrial DNA can activate the NLRP3 inflammasome during apoptosis of cells [39
]. In this study, deAND treatment not only reduced hepatic cholesterol accumulation, but also lowered apoptosis in the liver (Figure 3
), factors that might lead to lower NLRP3 inflammasome activation as evidenced by attenuating caspase-1 mediated IL-1β release (Figure 5
c,d). In addition to inhibition of caspase-1 activation, down regulation of the expressions of NLRP3, caspase-1, and IL-1β mRNA by deAND treatment (Figure 5
e–f) further suggests that the anti-inflammatory property of this diterpenoid may act on transcriptional level by suppressing the activity of nuclear factor-kappa B (NFκB), which is responsible for expression of inflammatory cytokines [40
]. These observations were similar to a recent study showing that AND could inhibit NLRP3 inflammasome activation and reduce inflammation in choline-amino acid-deficient diet-induced NASH [41
Regarding oxidative stress and antioxidant activity in liver, mice fed the HFHC diet had higher hepatic lipid peroxide content and the activities of GSH peroxidase and GSH reductase. Notably, inductions of Nrf2 mRNA and HO-1 protein expressions were found in mice fed the HFHC diet, which may respond to cellular oxidative stress [42
]. Therefore, it is suggested that long-term feeding with a HFHC diet (11 weeks) may increase oxidative stress in liver and, thus, trigger the expression of Nrf2-mediated downstream antioxidant enzymes to overcome the imbalance in the redox status [42
]. In this study, deAND treatment lowered hepatic lipid peroxide and increased antioxidant enzyme activities could be explained by Nrf2 induction and, thereafter, lowered oxidative stress in the liver (Figure 6
). It is known that a constant increase in lipotoxicity, followed by increases in oxidative stress and inflammation may promote the progress from NAFLD to NASH and impair liver function [2
]. The present study is the first to demonstrate that deAND ameliorates steatohepatitis, liver fibrosis, and liver damage partially by enhancing hepatic Nrf2-mediated downstream antioxidant enzyme activities and suppressing NLRP3 inflammasome activation in HFHC diet-induced fatty liver disease.
It was noteworthy that supplementation of deAND in the HFHC diet also enhanced fecal excretions of cholesterol and total bile acids (Figure 4
). Increased bile acid excretion, the major route of cholesterol degradation in liver, after deAND treatment might accelerate the biosynthesis of bile acid using cholesterol as the substrate and, thus, lower cholesterol level in liver. In addition, a lower bile acid reabsorption from small intestine into liver may contribute to a lower bile acid level in liver, and thus reduce dysregulated cholesterol metabolism and cholestatic liver injury [45
]. Therefore, it is suggested that the enhancement of fecal excretions of cholesterol and total bile acids by deAND may lower bile acid and cholesterol contents in liver and, thus, reduce cholestatic liver injury in HFHC diet-induced steatohepatitis.
Metabolomics has been used as an effective diagnostic method to monitor specific metabolites produced by patients with hepatic steatosis and inflammation to allow early detection of liver disease [46
]. Glutamate is an abundant free amino acid in various tissues, particularly the muscle and liver, which can act as a substrate or intermediate for various biochemical reactions and maintain health. It is one of the most important biomarkers for monitoring the status of NAFLD/NASH [46
]. In this study, the plasma glutamate concentration increased in mice fed the HFHC diet, while the hepatic glutamate content decreased (Figure 7
). A lower hepatic glutamate level is found in NAFLD/NASH patients [46
]. Although the exact mechanism is unclear, the disturbance of glutamate homeostasis may play a role in the pathological changes in many diseases [48
]. Therefore, a higher plasma glutamate concentration may be attributed from an increase in glutamate release from muscle or other tissues (e.g., liver) due to chronic inflammation in HFHC diet-induced fatty liver and liver injury in mice. In the present study, deAND increased glutamate level in the liver. This result suggests that deAND may improve fatty liver disease, at least in part, by increasing the hepatic glutamate level. Further study is warranted to clarify this finding.
In summary, the present study demonstrated that deAND may reduce steatohepatitis, liver fibrosis and liver injury, upregulate Nrf-2 triggered increase in proteins and/or activities of antioxidant enzymes, and lower inflammation by attenuating NLRP3 inflammasome activation in mice fed a HFHC diet. Therefore, deAND is likely to lower steatohepatitis and liver injury by increasing antioxidant and anti-inflammatory activities.