Quzhou Fructus Aurantii Extract suppresses inflammation via regulation of MAPK, NF-κB, and AMPK signaling pathway

The anti-inflammatory activity of Quzhou Fructus Aurantii Extract (QFAE) has been reported recently. Thus, present study aims to explore the mechanism of anti-inflammation of QFAE in vitro and in vivo to develop a lung phylactic agent. The anti-inflammatory mechanism of QFAE in RAW 264.7 cells and acute lung injury (ALI) mice model was determined by cytokines analysis, histopathological examination, Western blot assay, immunofluorescence, and immunohistochemistry analysis. The results showed that QFAE restrained mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling pathways in LPS-induced RAW 264.7 cells, whereas AMP-activated protein kinase (AMPK) signaling pathways were activated, as revealed by prominent attenuation of phosphorylation of ERK, JNK, p38, p65, IκBα, RSK and MSK, and overt enhancement of phosphorylation of ACC and AMPKα. The levels of pro-inflammatory cytokines TNF, IL-6, and IL-1β were suppressed, whereas the level of anti-inflammatory cytokine IL-10 increased after pretreatment with QFAE in vivo and in vitro. Moreover, QFAE prevented mice from LPS-provoked ALI, bases on alleviating neutrophils, and macrophages in bronchoalveolar lavage fluid (BALF) and mitigatingpulmonary histological alters, as well as hematological change. The MAPK and NF-κB signaling pathways in LPS-stimulated ALI mice were dampened by QFAE pretreatment, whereas AMPK signaling pathways were accelerated, as testify by significant restraint of phosphorylation of ERK, JNK, p38, p65, and IκBα, and distinct elevation of phosphorylation of ACC and AMPKα. The remarkable anti-inflammatory effect of QFAE is associated with the suppression of MAPK and NF-κB signaling pathways and the initiation of AMPK signaling pathway.


Animals and treatment.
A total of 60 male specific pathogen-free ICR mice weighing approximately 18-22 g (6-8 weeks old) were obtained from Shanghai Slack Laboratory Animal Co., Ltd. The animal feeding was followed as previously described 18 .
The mice were randomly divided into six groups (n = 10 each): blank control group (distilled water 21 mL/kg), drug control group (QFAE 21 g/kg), model control group (LPS), treatment groups (QFAE 7 g/kg + LPS, QFAE 21 g/kg + LPS), and positive control group (Dex 5 mg/kg + LPS). On days 0-2, QFAE was administered intragastrically to the drug control and treatment groupsat the indicated doses. The mice in the blank control and model control groups received distilled water. The positive group received Dexamethasone (Dex) intraperitoneally only on day 2. Approximately 1 h after administration on day 2, all the mice (excluding the blank control and drug control groups) were given light anesthesia via intraperitoneal injection with 0.5% pentobarbital sodium and intranasal administration with 20 μg of LPS in 50 μL of sterile saline to induce acute lung injury. The mice in the blank control and drug control groups received 50 µL of sterile saline. On day 3, the mice were sacrificed and blood samples were collected, and the whole lungs of 5 mice in each group were used to collect bronchoalveolar lavage fluid (BALF), the left lungs of other 5 mice were sliced for histopathological examination and immunohistochemistry analysis, and the right lungs of which were used for Western blot analysis.
Blood and BALF analysis. The acquisition and analysis of blood and BALF were followed as previously described 18 . The cell-free supernatant of BALF was used for the analysis of the cytokine levels (TNF-α, IL-6, and IL-10) following the manufacturer's instructions by Mouse Inflammation Kit using a flow cytometer. The production of cytokine level of IL-1β was analyzed with a special ELISA kit according to the manufacturer's instructions.
Histopathological examination. The histopathological examination was followed as previously described 18 . The severity of histological changes in the lungs was scored semiquantitatively from 0 to 4 as previously described 19 .
Immunohistochemistry analysis. The immunohistochemistry analysis was followed as previously described 18 .
Western blot analysis of lung tissues. The steps of Western blot were followed as previously described 18 . Statistical analysis. All data were expressed as mean ± standard error of mean (SEM) and analyzed with SPSS 22.0. Statistical comparisons between the groups were conducted by one-way ANOVA. Statistical significance was defined as P < 0.05.

Results
Component analysis and cytotoxicity of QFAE. The HPLC was applied for the quantitative determination of ingredients in QFAE, including eriocitrin, narirutin, naringin, neohesperidin, hesperidin, and synephrine. The contents of eriocitrin, narirutin, naringin, neohesperidin, hesperidin, and synephrine in QFAE were 0.324, 2.710, 9.972, 8.817, 1.438, and 0.030 mg/mL, respectively ( Fig. 1A-C). The MTT assay was conducted to evaluate the potential cytotoxicity of QFAE on RAW 264.7 cells. The results showed that QFAE was unable to affect the viability of cells at various concentrations from 0 to 40 mg/mL (Fig. 1D).
QFAE regulates MAPK signaling pathways in RAW 264.7 cells. The effect of anti-inflammation of QFAE on MAPK signaling pathways was examined in LPS-induced RAW 264.7 cells to illustrate its potential mechanisms. Western blot results showed that LPS significantly accelerated the phosphorylation of ERK, JNK, and p38, which was prominently attenuated by QFAE pretreatment ( Fig. 2A-D). Furthermore, the influence of QFAE on MAPK signaling pathways was investigated when administered to RAW 264.7 cells alone. Western blot results showed that QFAE had no significant effect on the phosphorylation of ERK, JNK, and p38, thereby indicating that QFAE had no effect on MAPK signaling pathways when administered to RAW 264.7 cells alone (Fig. 2E). The abovementioned findings reveal that the anti-inflammatory effect of QFAE in LPS-induced RAW 264.7 cells is related to MAPK signaling pathways. QFAE adjusts AMPK signaling pathways in RAW 264.7 cells. Next, the effect of QFAE on AMPK signaling pathways was analyzed in LPS-induced RAW 264.7 cells. Western blot results discovered that the phosphorylation of AMPKα and ACC was overtly inhibited due to exposure to LPS, which was evidently enhanced by QFAE pretreatment, as shown in Fig. 3A-C. In addition, the alteration of QFAE on AMPK signaling pathways  www.nature.com/scientificreports www.nature.com/scientificreports/ QfAe coordinates nf-κB signaling pathways in RAW 264.7 cells. NF-κB signaling pathways play a crucial role in LPS-stimulated relevant models. As shown in Fig. 4A-C, the phosphorylation of p65 and IκBα were saliently increased following exposure to LPS alone, which was dramatically reversed by QFAE pretreatment. Moreover, immunofluorescence staining of p65 nuclear translocation demonstrated that pretreatment of QFAE conspicuously suppressed the LPS-induced p65 nuclear translocation, as shown in Fig. 4D. The effect of QFAE on NF-κB signaling pathways when administered to RAW 264.7 cells alone was also observed. The results shown in Fig. 4E illustrated that QFAE had no apparent effect on the phosphorylation of p65 and IκBα, thereby indicating no effect was observed on NF-κB signaling pathways when QFAE was administered to RAW 264.7 cells alone.
The generation of cytokines was mainly regulated by NF-κB signaling pathways. In present study, the pro-inflammatory cytokines IL-6, IL-1β, and TNF-α were conspicuously increased by LPS stimulation in RAW 264.7 cells, while the anti-inflammatory cytokine IL-10 were significantly decreased, as shown in Fig. 4F-I. However, pretreatment with QFAE distinctly prevented the elevated pro-inflammatory cytokines and promoted the decreased IL-10.
These results indicated that QFAE could attenuate the production of inflammatory cytokines provoked by LPS via restraint NF-κB signaling pathways. QFAE mediates the phosphorylation of RSK and MSK in RAW 264.7 cells. The MSK, activated by p38 or ERK via cascade phosphorylation, leads to the transcriptional activation of NF-κB 20,21 . The RSK could be exclusively activated by phosphorylation of ERK in the MAPK signaling pathway and then participates in NF-κB activity 22,23 . The activities of RSK and MSK were examined after LPS induction. Western blot results showed that the phosphorylation of RSK and MSK was increased by LPS exposure but remarkably suppressed by pretreatment with QFAE ( Fig. 5A-C).

QFAE protects against LPS-induced lung injury and inflammation in mice.
The ability of QFAE to prevent mice from LPS-stimulated acute lung injury was analyzed. First, the levels of inflammatory cytokines in the BALF supernatant from ALI mice were detected. As illustrated in Fig. 6A-D, the levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α were markedly increased in BALF with exposure to LPS, while the level of anti-inflammatory cytokine IL-10 was apparently decreased, but a significant reversal by QFAE pretreatment in high dose group. In addition, cell counting and classification in BALF were performed. Neutrophils and macrophages were evidently increased after LPS provocation, but were notably ameliorated by pretreatment with QFAE on the dose of 21 g/kg (Fig. 6D,E). Hematological analysis also indicated that QFAE (21 g/kg) exhibited an arresting influence on decreasing the level of neutrophils enhanced by LPS induction (Fig. 6F). Furthermore, H&E staining was used to determine the degree of lung lesion in LPS-induced ALI mice. The drug control and blank control groups indicated no histopathologic alteration in the lung tissues. Severe pulmonary hemorrhage, evident alveolar collapse, and salient neutrophil infiltration were observed in the model control group. However, QFAE pretreatment (21 g/kg) ameliorated the abovementioned histopathological changes noticeably (Fig. 6G,H). The analogous treatment effects were obtained in the positive control group. The abovementioned findings reveal that QFAE attenuates lung injury in LPS-induced ALI mice and exhibits an excellent anti-inflammatory activity in vivo.   www.nature.com/scientificreports www.nature.com/scientificreports/ QFAE suppresses MAPK signaling pathways in LPS-induced ALI mice. The effects of the anti-inflammation of QFAE on MAPK signaling pathway in the lung tissues of LPS-induced ALI mice were investigated to further elucidate its underlying mechanisms. As shown in Fig. 7A-D, Western blot results revealed that QFAE pretreatment significantly restrained LPS-induced relative phosphorylation of ERK, JNK, and p38. No apparent effect exists on the expression levels of total ERK, JNK, and p38 after treatment with QFAE and LPS. Similar treatment effects were obtained in the positive control group. Moreover, immunohistochemistry analysis of p-ERK also proved that pretreatment of QFAE prevented the LPS-induced phosphorylation in ALI mice (Fig. 7E). The abovementioned results prove that the anti-inflammatory effect of QFAE in LPS-induced ALI mice is related to MAPK signaling pathways. Fig. 8A-E, Western blot results indicated that the relative expressions of p65, p-p65, IκBα, and p-IκBα were dramatically elevated compared with that of loading control α-Tubulin following the exposure to LPS alone in the lung tissues of ALI mice, whereas prominently reversed by pretreatment with QFAE. In addition, immunohistochemistry results indicated that the expression level of IκBα was increased in the inflammatory regions of the lung tissue in the model control group, while the expression level of which in the comparatively normal regions was particularly low. However, no significant difference was observed in the expression of p-IκBα between the inflammatory regions and the normal regions in the model control group (Fig. 8F). On this basis, correlation statistics were used to analyze the correlation between IκBα, p-IκBα, and MPO. The results showed that IκBα/α-Tubulin was www.nature.com/scientificreports www.nature.com/scientificreports/ conspicuously correlated with MPO/β-actin, but the association of p-IκBα/α-Tubulin and MPO/β-actin was at the low level. Thus, no significant difference was observed between the expression of IκBα and MPO, and a marked difference exists between the expression of p-IκBα and MPO (Fig. 8G,H). Simultaneously, p65 and p-p65 obtain similar results. Therefore, IκBα/MPO, p65/MPO, p-p65/α-Tubulin, and p-IκBα/α-Tubulin were adopted to comprehensively analyze the relative phosphorylation, thereby indicating that QFAE regulates NF-κB signaling pathways by inhibiting the expression levels of p-IκBα and p-p65 caused by LPS in ALI mice.

QFAE activates AMPK signaling pathways in LPS-induced ALI mice. The effect of QFAE on AMPK
signaling pathways in LPS-induced ALI mice was analyzed. Western blot results revealed that LPS notably suppressed the relative phosphorylation of AMPKα and ACC in the lung tissue, while pretreatment with QFAE (21 g/kg) distinctly raised its relative phosphorylation, thereby indicating that QFAE exerts an anti-inflammatory role by activating the AMPK signaling pathways (Fig. 9A-C). Similar effects of Dex were revealed in the positive control group.

Discussion
The QFA, also known as Changshanhuyou traditionally, as a conventional medicine with a long history in Changshan County, Quzhou City, Zhejiang Province, should be harvested in July when the peel is still green. Previous studies have shown that the ingredients of the QFA are mainly consistent with that of Fructus Aurantii 24-27 , primarily including nobiletin, eriocitrin, narirutin, naringin, neohesperidin, hesperidin, and synephrine, which indicated that QFA could be used as a substitute for Fructus Aurantii. In our pre-experiment, we conducted an assay of maximum administration dosage (MAD) of QFAE on ICR mice. The results revealed that MAD was 100 g/kg, and there was no adverse reaction on mice. In another study, SD rats were administered intragastrically with 40 g/kg/d QFAE (equivalent to a mouse dose of 60 g/kg/d), and were administered for 30 days in a row. The results showed that there were no abnormalities in body weight, food intake, hematology, biochemistry and pathology indexes of rats. So the doses of QFAE, used in this study, are safety for mice in vivo. Our www.nature.com/scientificreports www.nature.com/scientificreports/ prior research has detected the content of naringin using as a criterion for the quality control and quantification of QFAE 18 . In present experiment, the components of eriocitrin, narirutin, neohesperidin, hesperidin, and synephrine were additionally tested to provide a basis for comprehensive evaluation of the quality control of QFAE. Moreover, the abovementioned monomers are widely distributed in citrus plants, which have been proven to keep anti-inflammatory and anti-oxidative activities [28][29][30][31][32] . Thus, the anti-inflammatory effects of QFAE may be related to the abovementioned compounds.
The ALI, a kind of damage of alveolar epithelial and capillary endothelial cells caused by various direct or indirect injury factors, is characterized by diffused pulmonary interstitial and alveolar edema, neutrophil accumulation, pulmonary hypertension, pulmonary infiltrates, alveolar rupture, and pulmonary hemorrhage, thereby leading to acute hypoxic respiratory insufficiency and, in severe cases, ARDS 1,6,33 . ALI/ARDS is closely related to sepsis 34,35 , pneumonia 36 and trauma 37 , with severe incidence and mortality 38 . In our research, LPS was applied for the provocation of ALI in mice to reproduce the major pathological features. Histopathological examination illustrated that massive inflammatory cells aggregated in the lungs of ALI mice along with alveolar rupture and pulmonary hemorrhage, thereby indicating the success of ALI model. The protective effect of QFAE against ALI mice was proven by biochemical and histological examinations, immunohistochemistry, and protein expression analysis.
Cytokines are the key mediators participating in inflammation and host defense, which could be controlled by the post-transcriptional and have relation to activation of multiple signaling cascades during inflammation progression 39 . Pro-inflammatory cytokines are indispensable for immune system, and although they may produce severe inflammatory lesion, tightly regulating their production and excretion is necessary 40,41 . The IL-10 is a potent anti-inflammatory factor in bacterial endotoxemia and crucial for maintaining homeostasis, which could be stimulated by LPS as a negative feedback by dampening a pro-inflammatory response and preventing inflammation caused by tissue injury 42,43 . Our prior and present study showed that the pro-inflammatory www.nature.com/scientificreports www.nature.com/scientificreports/ cytokines TNF-α, IL-6, and IL-1β exhibited an excess accumulation induced by LPS in vivo and in vitro, while the anti-inflammatory cytokine IL-10 was an overt abrogation. However, an evident moderation of TNF-α, IL-6, and IL-1β and a prominent elevation of IL-10 were observed by pretreatment with QFAE, thereby indicating that QFAE has an effective anti-inflammatory activity in vivo and in vitro.
The diversity of intracellular signaling pathways is comprised in the regulation of inflammatory cytokines. MAPK pathway consists of ERK, JNK, and p38 signaling pathways, which could be activated by several pro-inflammatory cytokines (e.g., TNF-α and IL-1β) or in answer to extracellular stress (e.g., oxidative stress and genotoxicity) 44 . NF-κB, an important regulator involved in the regulation of quantity of genes that govern diverse immune and inflammatory responses, is activated by various stimuli that cover oxidative stress, endotoxin, and inflammatory cytokines, can be further enhanced by TNF-α and IL-1β cytokines, and then activates other cells to synthesize various cytokines, such as IL-6 and IL-8, to form a positive feedback response 45,46 . AMPK is a cellular energy regulator and its activation can significantly suppress the levels of pro-inflammatory cytokines, promote the levels of anti-inflammatory cytokines, and exert anti-inflammatory effects, which are mostly accompanied by inhibition of the NF-κB pathways. Meanwhile, its activity is also regulated by cytokines [47][48][49] . Specifically, the production of pro-inflammatory cytokines is primarily managed by NF-κB signaling pathways and associated with MAPK signaling pathways, and the generation of anti-inflammatory cytokines is correlated with AMPK signaling pathways.
According to the abovementioned results, we focused our attention on MAPK/NF-κB/AMPK signaling pathways that are associated with respiratory inflammation in response to LPS. Previous studies have shown that ERK, JNK, and p38 are closely bound up with various inflammatory diseases, such as acute lung injury, and can be activated by LPS, TNF, and other irritants. When MAPK is activated, phosphorylated ERK, p38, and JNK are transferred to the nucleus alone or in combination to induce expression of relevant inflammatory factor target genes and promote inflammatory response 10,50,51 . Pretreatment of QFAE to LPS induced RAW 264.7 cells dramatically suppressed MAPK signaling pathways as evaluated by the decrease on the phosphorylation of ERK, JNK, and p38. The prevention of QFAE on MAPK signaling pathways was also verified in LPS-stimulated ALI mice by the distinct inhibition of phosphorylation of ERK, JNK, and p38.
AMPK is a key regulator of energy homeostasis. Activation of AMPK directly phosphorylates downstream target ACC, inhibits metabolic pathways, such as fatty acid, cholesterol and protein syntheses, and increases the level of ATP in cells 52 . Although previous studies have confirmed that AMPK has anti-inflammatory effects in lung inflammatory diseases, its specific mechanism remains obscured 53 . In response to various stimulation, the activated AMPK may directly or indirectly regulate the activity of NF-κB through downstream SIRT, Fox 3a, p53, and PGC-1a proteins, thus inhibiting inflammatory factors and playing an anti-inflammatory role 54 . Pretreatment of QFAE to LPS induced RAW 264.7 cells apparently promoted AMPK signaling pathways by upgrading the www.nature.com/scientificreports www.nature.com/scientificreports/ phosphorylation of AMPK and ACC. The acceleration of QFAE on AMPK signaling pathways was also testified in LPS-stimulated ALI mice by the salient motivation of phosphorylation of AMPK and ACC.
NF-κB exists in the cytoplasm and binds to its inhibitory protein IκB as a trimer form with no transcriptional activity in the resting state. Extracellular stimulation signals are transmitted into the cells via membrane receptors to liberate IκB-inhibited NF-κB and activate it, then rapidly enter the nucleus to specifically bind to the κB sequence of the corresponding target gene regulatory element on DNA and up-regulate the expression of these target genes by individual or synergistic action, thereby exerting relevant physiological or pathological effects. Specifically, when p65 and IκBα complexes are stimulated, IκBα is phosphorylated and then degraded, resulting in p65 dissociation and entry into the nucleus to phosphorylate and induce transcription and expression of inflammatory mediators and immune-related genes 47,48,55,56 . In this study, the phosphorylation of IκBα and p65 was significantly accelerated in LPS stimulated RAW 264.7 cells, thereby indicating the activation of NF-κB signaling pathways, which was remarkably restrained by QFAE treatment.
As previously mentioned, in the analysis of the ability of QFAE to protect ALI mice through NF-κB signaling pathways, pretreatment with QFAE overtly suppressed the relative tubunlin expressions of p65, p-p65, IκBα, and p-IκBα in the lung tissues of ALI mice, whereas the relative phosphorylation, p-p65/p65 and p-IκBα/IκBα, was nonsignificantly different. Furthermore, the immunohistochemistry results in the model control group indicated that the expression of IκBα was increased in the inflammatory areas of the lung tissue, whereas the expression of that in the relatively normal regions was quite low. Moreover, in the model control group, the expression of p-IκBα in the inflammatory and normal regions has no significant difference. These results all indicated the excitation of NF-κB signaling pathways in the lung tissue of ALI mice presented as disassembly and phosphorylation of p65-IκBα complexes due to LPS induction. Additionally, MPO is a particular marker released by neutrophils, macrophages, and monocytes and can reflect the degree of inflammation in the inflammatory process 57,58 . An apparent inhibition was observed on the expression of MPO in the lung tissues of ALI mice pretreated with QFAE. Thus, we proposed a correlation analysis method between IκBα, p-IκBα, and MPO. Canonical correlation analysis is a statistical analysis tool used to identify and quantify the correlation between two sets of variables, which is applied in various fields of research, such as biomedicine, ecology, and genetics 59 . We found the expression of IκBα was prominently correlated with the expression of MPO, but the association of p-IκBα and MPO was particularly low. Similar results of p65 and p-p65 were obtained. The outcomes of immunohistochemistry and correlation analysis indicated that the expressions of IκBα and p65 are closely related to the degree of inflammation, and there was no significant relationship between the expressions of p-IκBα and p-p65 and the degree of inflammation. Therefore, in our study, there was no regular change in the relative phosphorylation of p-p65/ p65 and p-IκBα/IκBα, and the relative expressions of p-p65/α-Tubulin and p-IκBα/α-Tubulin were adopted to synthetically evaluate the effect of QFAE in NF-κB signaling pathways in ALI mice.
In addition, RSK and MSK were detected in the present research. The 90 kDa ribosomal S6 kinase (RSK) family of proteins are downstream effectors of ERK/MAPK signaling cascade, which could be directly activated by ERK1/2 22,23,60 . The mitogen-and stress-activated protein kinases (MSKs) that belong to the RSK family of protein kinases are activated by p38 and ERK via cascade phosphorylation, which are regulated by upstream activators and activate downstream targets 20,21,61 . Previous studies have shown that activated RSK and MSK mediated phosphorylation of p65, indicating that RSK and MSK are particular proteins involved in MAPK and NF-κB pathways 62,63 . In this regard, the ability of QFAE to inhibit LPS-induced phosphorylation of RSK and MSK in vitro and in vivo was investigated. Specifically, QFAE markedly restrained the phosphorylation of RSK and MSK in LPS-stimulated RAW 264.7 cells, as well as the phosphorylation of ERK, p38, and p65. However, our in vivo study showed no significant difference in phosphorylation of RSK and MSK, while the phosphorylation of ERK, p38, and p65 were still reversed by pretreatment with QFAE in ALI mice. This condition resulted in a conundrum that needs to be analyzed.
In conclusion, QFAE had an effective anti-inflammation in LPS-induced RAW 264.7 cells and lung phylactic activity in LPS-stimulated ALI mice that saliently alleviated the pro-inflammatory cytokines TNF-α, IL-6, and IL-1β, while noticeably raised the anti-inflammatory cytokine IL-10 in vitro and in vivo. Besides, QFAE protected against ALI in mice via attenuating pulmonary histological alters and hematological changes. The conspicuously protective effect of QFAE was associated with suppression of MAPK and NF-κB signaling pathways and motivation of AMPK signaling pathways. Therefore, QFAE may serve as a potential lung phylactic agent to ALI/ARDS and even other respiratory inflammation cases.