Acute lung injury (ALI) is the leading cause of death in patients with respiratory failure. It may occur through direct injury, such as pneumonia and the inhalation of harmful substances, or through indirect damage, such as ischemia-reperfusion, the aspiration of gastric contents, sepsis and multiple injuries or acute pancreatitis, which may cause an unstable redox state that can cause DNA damage or protein and lipid oxidation [1
]. ALI can induce damaging hypoxia and ischemic stress or the release of bacterial endotoxins, such as lipopolysaccharide (LPS) [2
]. LPS can induce a serious release of inflammatory signals and cause lung damage characterized by inflammatory leukocyte infiltration. The severity of the pneumonia is caused by the great accumulation of neutrophils, increased reactive oxygen species (ROS) production, and enhanced proinflammatory cytokines production, such as tumor necrosis factor (TNF-α), interleukin-1β (IL-1β) and IL-6, which exacerbate the inflammation, leading to secondary diffuse lung parenchymal damage in the alveoli [3
]. Therefore, it is important to find an agent with the ability to inhibit the inflammatory response for the treatment of ALI.
The activation of the Toll-like receptor 4 (TLR4) signaling pathway leads to the production of inflammatory mediators, which results in the translocation of nuclear factor-κB (NF-κB) and the activation of mitogen-activated protein kinase (MAPK), which plays a key role in triggering inflammation after the LPS challenge [4
]. Phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling has been proposed as a significant functional regulator of TLR4/NF-κB activity [5
]. It can promote cell division and proliferation and plays key roles in suppressing apoptosis, immune regulation [6
], regulating anti-inflammatory cytokines and promoting the degradation of the extracellular matrix [7
Oxidative stress-mediated injury plays an essential role in lung failure. The protective mechanism is currently believed to be derived from antioxidant enzymes (catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx)) and nuclear factor erythroid 2-related factor (Nrf2)/heme oxygenase (HO-1) signaling against oxidative stress [8
]. Nrf2 is an inducible protein with cytoprotective and antioxidant activities. Under normal circumstances, Nrf2 binds to Kelch-like ECH-related protein 1 (Keap1) in the cytosol. When there is a redox imbalance in the cell, free Nrf2 binds antioxidant response elements in the nucleus, thereby activating detoxification gene expression [9
]. HO-1 catalyzes the breakdown of heme, thereby inhibiting neutrophil-, macrophage- and lymphocyte-mediated inflammatory responses [10
]. Oxidative stress and certain pro-inflammatory mediators can induce Nrf2 activation and HO-1 expression. HO-1 is a cytoprotective protein produced through the Keap1/Nrf2/HO-1 axis and can curtail the cytotoxicity of various sources of oxidative stress and inflammatory reactions [11
The endoplasmic reticulum (ER) serves many roles in the cell, including in protein folding, transportation, cellular calcium storage, lipid and steroid synthesis, and the metabolism of carbohydrates [12
]. Under cellular stress and inflammatory conditions, ER stress activates the unfolded protein response (UPR) to relieve this stress and restore ER homeostasis [14
]. Under pathological conditions (such as ALI, sepsis and infection), ER stress may lead to the accumulation of a large number of incorrect protein structures [15
]. Three ER transmembrane proteins are affected by ER stress: protein kinase R–like ER kinase (PERK), inositol-requiring kinase 1 (IRE1), and activating transcription factor 6 (ATF6). Glucose-regulated protein 78 (GRP78, also known as BiP) binds to IRE1, PERK and ATF6 in stress-free cells and dissociates from these UPR sensors during ER stress [17
]. C/EBP homologous protein (CHOP) and caspase-12 are also key molecules in ER-induced apoptosis. Severe and prolonged ER stress strongly induces CHOP [14
]. Additionally, caspase-12 induces the downstream death molecule caspase-3 when activated by ER stress [18
]. Thus, ER stress potentially affects survival in ALI [16
Autophagy maintains energy homeostasis, allowing cells to survive under nutrient restriction [19
]. Two signaling molecules tightly control the autophagy-mediated activation of AMP-activated protein kinase (AMPK), which induces autophagy and the mammalian target of rapamycin (mTOR) via the autophagy axis [20
]. Autophagy is achieved through the formation of autophagosomes, which are involved in the conversion of light chain 3-I (LC3-I) to LC3-II in the cytosol. Therefore, the ratio of LC3-I to LC3-II can be used as autophagy markers [21
]. Beclin 1 plays a key role in the localization of other autophagic proteins to the autophagosome prestructure. Thus, LC3-II and beclin 1 are often measured as autophagic markers [22
]. However, little is known about how autophagy is regulated during ALI development.
AMPK plays a central role in the control of energy balance and can sense the energy state of cells by detecting the amount of AMP. Its activation enhances energy production through liver kinase B1 (LKB1) and calcium/calmodulin-dependent protein kinase 2 (CaMKK2) [23
]. ER calcium release contributes to an increase in intracellular calcium, which causes the production of ROS and the activation of AMPK. Reports suggest that LKB1/CaMKK/AMPK signaling may promote lung tissue injury [24
Hispolon is a natural bioactive polyphenol from S. sanghuang
or Phellinus linteus
used as a herbal medicine in Taiwan, Korea, China and Japan [25
]. Our previous publication indicates that hispolon protects the livers against CCl4-induced acute liver injury [26
]. Other studies have shown antitumor actions for hispolon against human leukemia [27
] and liver cancer [28
] and human hepatoma cell metastasis [29
]. Hispolon can induce the protein expression of HO-1 and inhibit iNOS (inducible nitric oxide synthase) and NO (nitric oxide) productions in LPS or LTA-induced BV-2 cells [30
]. Although many studies have demonstrated the anti-inflammatory potential of hispolon, the mechanism of hispolon protecting against LPS-induced ALI has not been evaluated. Therefore, we hypothesize that the TLR4/PI3K/Akt/mTOR and Keap1/Nrf2/HO-1 signalings, the modulation of oxidative stress and the ER stress-induced apoptosis axis involve the anti-inflammation of hispolon in LPS challenge ALI. Thus, the purpose of this research was to investigate the antioxidative, anti-inflammatory, and anti-apoptotic functions of hispolon.
ALI is a severe lung disease and there is no good medicine to treat ALI clinically, so the development of ALI treatment strategies is urgently needed [2
]. LPS is considered to be one of the factors leading to ALI, which causes fluid to enter the lung tissue and increases microvascular permeability, causing the symptoms of acute inflammation in the tissue [2
]. LPS-induced ALI mouse models have been shown to be useful models for studying potential treatments against human ALI. The current study utilized an in vivo mouse model to study the protective effects of hispolon in LPS challenge ALI. The present study found that LPS induces neutrophil infiltration and pulmonary edema, and that treatment with hispolon significantly reduces these effects. Oxidative stress induces the activation of macrophages and lipid oxidation, leading to pro-inflammatory cytokine expression. The study also demonstrates that, in the first line of defense against infection, these stress markers ultimately release various pro-inflammatory mediators and recruit neutrophils. We measured the oxidative stress marker antioxidant enzymes and MPO. Surprisingly, hispolon significantly improved the oxidative stress conditions and decreased the pro-inflammatory cytokine expression caused by LPS. Thus, we discovered that hispolon treatment decreased LPS-mediated lung edema, neutrophil infiltration and inflammatory cytokine secretion in the BALF, while controlling the TLR4/PI3K/Akt/mTOR, Keap1/Nrf2/HO-1 pathway and suppressing oxidative stress and the ER stress signaling pathways. In short, the in vivo data indicate that hispolon has anti-inflammatory and antioxidant functions that prevent the lung damage caused by LPS.
Hispolon is an active polyphenolic compound. There is increasing evidence in the literature that hispolon has a wide range of medicinal properties, such as antioxidant, anti-inflammatory, immunomodulatory, antiviral, hepatoprotective and anticancer activities. Among them, the antitumor activity of hispolon has been detailed in different studies, and it has been observed to inhibit the growth of cancer cells by inducing cell cycle arrest, apoptosis and metastasis inhibition. [25
]. However, the toxicity of hispolon that causes these effects is not fully understood. A recent study has revealed that hispolon showed no significant toxicity for phorbol ester (TPA)-treated and untreated MDA-MB-231 cells between 0 and 40 µM (8.8 µg/mL) for 24 h, thus avoiding anti-proliferation due to hispolon interference caused by activity [38
]. Hispolon, with a concentration of 0 to 20 µM (0–4.4 µg/mL), induces the expression of HO-1 protein in BV-2 cells for 24 h under the stimulation of LPS or lipoteichoic acid (LTA), thereby effectively inhibiting the production of iNOS/NO and having no cytotoxicity [29
]. Hispolon was also shown to be less cytotoxic to normal cells [39
]. Furthermore, hispolon induces apoptosis in acute myeloid leukemia cells and inhibits AML xenograft tumor growth in vivo, where the high dose (10 mg/kg) used is the same as in this study [40
TLR4, a member of the Toll-like receptors, localizes to the cell membrane and cytoplasm and is a pattern recognition receptor for LPS. LPS-activated macrophages trigger a signaling cascade by releasing inflammatory mediators, resulting in multiple organ damage including ALI [30
]. Pro-inflammatory cytokines trigger macrophage expression by activating the NF-κB pathway in inflammatory diseases [37
]. In ALI patients, an increase in pro-inflammatory cytokines has been observed and are associated with major inflammatory disorders [4
]. In the current study, the administration of hispolon significantly ameliorated pulmonary inflammation, hemorrhage and the thickening of the alveolar septa. Furthermore, hispolon treatment diminished the levels of pro-inflammatory cytokines after LPS challenge. These preliminary results indicate that hispolon plays a favorable inhibitory role in LPS-induced ALI.
NF-κB serves as a signal regulator in inflammation, cell proliferation and differentiation [4
]. In clinical trials, the activation of NF-κB was increased in patients in response to the overexpression of NF-κBp65 in alveolar macrophages, under severe bacterial infection [37
], compared with in the control group. From the above experiments, we speculate that hispolon could increase IκBα during LPS challenge to suppress NF-κB, with the suppression of IKK phosphorylation in the cytosol, preventing NF-κB p65 nuclear translocation.
The MAPK (ERK1/2, p38MAPK, and JNK) cascade has been shown to play an extracellular signal transduction, such as LPS-induced inflammatory cytokine production [8
]. The defective activation or pharmacological inhibition of ERK or p38 reduces the production of LPS-induced pro-inflammatory cytokines [37
]. Thus, targeting TLR4 or its downstream MAPK signaling may prevent the abnormal immune response associated with ALI. In addition, hispolon activated ERK1/2, p38MAPK, and JNK phosphorylation in cancer cells, such as nasopharyngeal carcinomas cells [42
], cervical cancer cells [43
] and hepatocellular carcinoma cells [27
]. MAPK signaling plays a critical role in the chemotherapy drugs and metastasis [44
]. Furthermore, LPS is presented to the TLR4/MD2 complex via LPS binding protein and CD14. The formation of the TLR4/MD2/LPS complex causes the phosphorylation of MAPK and activates downstream NF-κB [46
]. Both TLR4/MD2 and MAPK/NF-κB are required for LPS-induced inflammation. Therefore, many natural compounds inhibit MAPK/NF-κB signaling in a variety of ways, showing the treatment or relief of lung inflammation. This indicates that it may be the target of acute inflammatory drug treatment. In addition, the inhibition of the MAPK pathway can reduce the transcription and oxidative stress of proinflammatory mediators [47
]. In this study, hispolon suppressed MAPK phosphorylation and decreased the pro-inflammatory cytokines expressions via NF-κB activation in the LPS-induced model. Thus, hispolon significantly prevented the degradation NF-κB and IκBα and the phosphorylation of MAPK in LPS-induced ALI mice.
Recent reports suggest that the PI3K/Akt/mTOR axis is a key point for TLRs/NF-κB to coordinate inflammatory responses [5
]. It is clear that the activation of TLR leads to the recruitment of PI3K, by allowing adaptor molecules to enter the receptor complex. Thus, PI3K/AKT/mTOR signaling regulates cell growth, proliferation, migration, invasion, survival, apoptosis and autophagy, and is tightly regulated in the TLR signaling pathway [30
]. This shows that the LPS-induced inflammatory reaction was mediated through the TLR4 receptor, which improved the PI3K, p-Akt, and p-mTOR protein levels. Pre-treatment with hispolon significantly decreased PI3K, p-Akt and p-mTOR levels after the LPS challenge. Thus, the PI3K/AKT/mTOR axis is a potential predictor for hispolon treatment in the LPS-challenged mice. It is proven by the above experiments that the PI3K/Akt/mTOR and TLR/NF-ĸB pathways are coordinated in regulating the inflammatory response triggered by LPS.
LPS significantly enhances ROS production, promotes inflammatory responses, decreases antioxidant enzyme (catalase, SOD and GPx) activity, and increases the MPO activity associated with neutrophil infiltration in the development of ALI [30
]. However, appropriate ROS levels help to protect humans from external stimuli or pathological damage. By contrast, the excessive production of ROS is thought to induce cell damage and oxidative stress. The data of this study indicate that hispolon treatment diminished the production of ROS, increased Nrf-2 expression in the nucleus and increased the phosphorylation of AMPK after LPS challenge. In addition, cells contain a variety of antioxidant enzymes to prevent cell damage by reducing oxidative stress [30
]. Our preliminary data showed that hispolon increases the expression of the antioxidant proteins in the ALI model.
In the presence of oxidative stress, the release of Nrf2 from Keap1 was activated. Nrf2 is involved in the induction of HO-1 and GP, which can eliminate ROS through oxidative damage [7
]. HO-1 may suppress oxidative stress through the activation of the PI3K/Akt or Nrf2 axis in sepsis-induced ALI [4
]. Of interest, HO-1 can prevent liver and endothelial cell apoptosis mediated by ER stress in diabetic animals [48
]. These results have suggested that the special protective effects of hispolon were mediated by inhibiting oxidative stress via the Keap1/Nrf2/HO-1 axis and reducing ROS production.
The ER stress response is associated with several liver diseases, for example, ALI, obesity-related fatty liver and viral hepatitis [11
]. Studies have shown that ER stress is a key strength of mediation in the LPS-induced inflammation and the protective effect of 4-PBA is related to ER stress and autophagy in LPS-challenged mice. Moreover, 4-PBA is a chemical chaperone, currently undergoing clinical trials, that inhibits ER stress, and is promising as a future research candidate for new asthma therapies [49
]. As shown in the results section, p-PERK, GRP78 and CHOP expression was induced by LPS. Hispolon treatment decreased the levels of these, and ER sensor proteins, such as ATF6, caspase-12 and IRE1, can inhibit their activation. These findings indicate that hispolon plays an anti-inflammatory role by ameliorating ER stress.
The activation of autophagy can degrade proteins against ER stress induced toxicity [18
]. Reports have indicated that the ER stress-activated PERK protein may induce LC3, which is involved in the induction of autophagy. In the current study, the upregulation of autophagy-related proteins such as LC3-II and Beclin 1 was observed in lung tissue after 6 h of induction by the intratracheal instillation of LPS. This could suggest that autophagy acts as a compensatory mechanism during lung injury, and the induction of autophagy may improve lung cell survival by providing energy in adverse environments. To date, the exact role of the autophagy process in ALI remains unclear. Specifically, ER stress could be an upstream mediator to regulate autophagy. In addition, autophagy is a multi-faceted process, and changes in autophagy signaling are often observed in cancer and the recruitment of LC3-II-mediated of phagocytic membrane to damaged organelles. Thus, hispolon activated LC3-II in nasopharyngeal carcinomas cells [37
]. Our preliminary data showed that hispolon activated the TLR4/PI3K/Akt/mTOR axis and the protein expression of the LC3-II and Beclin 1 was reduced, suggesting that ER stress is the cause of LPS challenge autophagy activation in mice.
Apoptosis is induced when cells receive internal or external signals. Intrinsic apoptosis is caused by a variety of disturbances to the microenvironment, including DNA damage and ER and ROS stress. The apoptosis induced by ER stress is an example of intrinsic apoptosis signaling [21
]. ER stress and inflammatory signaling are intrinsically linked through a variety of mechanisms, and ER stress increases the induction of inflammatory cytokines in macrophages by LPS by several times [50
]. Our data showed that hispolon suppressed Bcl-2 protein expression and increased Bax and caspase-3 protein expression, well-known apoptosis markers, resulting in the inhibition of severe ER stress and limiting the lung injury triggered by lung cell apoptosis and liver inflammation in ALI mice induced by LPS. In addition, the regulating caspase activation and inhibition mechanisms is a key factor in the treatment of cancer, because these processes can induce apoptosis [52
]. Hispolon induces cancer cells apoptosis through the activation of caspases-8, -9 and -3 and the cleavage of PARP [40
]. Moreover, the results of this study indicate that autophagy and apoptosis may play different roles at different stages of LPS-induced ALI. Autophagy peaked at 2 h. However, apoptosis reached its maximal level at later stages (6 h) [54
]. The relationship between autophagy and apoptosis can be very complex. Both autophagy and the apoptosis of lung cells can be triggered by extracellular stimulation. Some intracellular signaling pathways, including MAPK and NF-κB, are involved in both types of cell death. A recent study revealed the interaction between autophagy and apoptosis-related intracellular signaling pathways in lung cells, suggesting that autophagy may be a survival mechanism against Fas-mediated apoptosis [55
]. In this study, hispolon reduced the protein level of Bax and increased the protein level of Bcl-2, resulting in a decrease in the ratio of Bax to Bcl-2 and reduced apoptosis. Further research is needed to study the possible role of autophagy and apoptosis in ALI.