Bergenin-activated SIRT1 inhibits TNF-a-induced pro-inammatory response via blocking NF-kB signaling pathway

Background Bergenin, a kind of polyphenol compound, has been showed to exhibit antiulcerogenic, anti-inammatory, antitussive and burn wound healing properties, However, its therapeutic effect on TNF-a induced pro-inammatory response in airway and its potential mechanisms of actions were still unclear Aim of this study This study aimed to investigate the anti-inammatory effects and possible mechanism of bergenin in TNF-a-stimulated 16-HBE cells. Materials and methods CCK8 was used to determine the cytotoxicity. Cytokines expression were analyzed by qRT-qPCR and ELISA. Immunouorescence, western blotting and SIRT1 activity assay were employed to investigate the possible molecular mechanism. Our results showed that bergenin treatment obviously decreased the mRNA and protein expressions of IL-6 and IL-8 in TNF-a-stimulated 16-HBE cells. Bergenin blocked TNF-a-mediated the activation of NF-kB signaling and NF-kB nuclear translocation. Interestingly, RT ‐ qPCR and western blotting revealed that bergenin did not affect the expression of sirtuin-1 (SIRT1), but signicantly increased the activity of SIRT1. Furthermore, bergenin-mediated SIRT1 activation was further conrmed by the resulted that bergenin decreased acetylation levels of NF-kB-p65 and p53. The inhibitory effects of bergenin on the mRNA and protein expression levels of IL-6 and IL-8 were reversed by addition of SIRT1 inhibitor. In addition, combination of bergenin and dexamethasone showed additive effects on the reduction of IL-6 and IL-8. These ndings demonstrated that bergenin could suppress TNF-α-induced pro-inammatory response via augmenting SIRT1 activity to block NF-kB signaling pathway, which may contribute to provide benecial effect for the treatment of airway inammation in asthma.


Introduction
Asthma is a chronic in ammatory disease of airway in which characterized by abnormal immunein ammatory responses, increased mucus secretion and airway remodeling [1,2]. The clinical symptoms of this disease presented as recurrent episodes of wheezing, changeable degree of air ow obstruction, breathlessness and airway hyperresponsiveness [3,4].The epidemiological statistics showed that prevalence of asthma was increased in last decades, which constituted great threat to human health [5][6][7]. It should point out that airway in ammation disorder elicited by various stimulus contributed to asthma progression. Clinically available medications for asthma associated with in ammation included inhaled glucocorticoids, which has been shown that asthma individual became insensitive and sideeffects [8,9]. Therefore, there is urgent need to develop an alternative agent for the treatment of asthma.
Tumor necrosis factor (TNF) -α is a multifunctional pro-in ammatory cytokine that has been found to be secreted in various of immune cells and bronchial epithelial cells [10,11]. Secreted TNF-α bind to its receptors (TNF-R p55 and p75) via an autocrine or a paracrine manner, and thereby lead to NF-κBtriggered exacerbated pro-in ammatory cytokines production [12,13]. Accumulating evidence has indicated that TNF-α played a critical pathogenesis role in the development of asthma. Previous studies reported that elevated levels of circulating TNF-α has been found in allergen-induced asthma patients [14]. In addition, levels of TNF-α in asthmatic patients' variety of specimens, such as bronchoalveolar lavage uid (BALF), peripheral blood monocytes, alveolar macrophages, sputum and bronchial biopsies tissues, are much higher than healthy individual [15][16][17]. Furthermore, it has been found that the high levels of TNF-α are positively correlated with airway hyperresponsiveness in asthmatic patients [18]. Mechanistically, airway bronchial epithelial cells with TNF-α stimulation led to upregulated intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1), facilitating in ammatory cells airway in ltration [19,20]. Besides, TNF-α, a key mediator in cytokine dependent in ammatory event, causes the recruitment of neutrophils and eosinophils, which led to asthma exacerbation [21]. Thus, TNFα may be attractive therapeutic target for the treatment of asthma with in ammation. However, it has been reported that severe persistent asthmatic patients with TNF-α monoclonal antibody golimumab treatment did not show signi cant improvements in their FEV1 and asthma exacerbations [22].
The polyphenol compound bergenin has been showed to exhibit antiulcerogenic, anti-in ammatory, antitussive and burn wound healing properties [23]. It has been found that bergenin could ameliorates LPS-induced acute lung injury in mice by the inhibition of NF-κB-mediated pro-in ammatory response [24]. In addition, bergenin reduced the expressions of pro-in ammatory IL-6 and TNF-α in experimental colitis mice [23]. However, the effects and the underlying molecular mechanism of bergenin on TNF-αmediated pro-in ammatory response in asthma has not yet to be revealed. Therefore, we sought to investigate the effects of bergenin on TNF-α-mediated pro-in ammatory cytokine release and associated signaling events in airway bronchial epithelial cells. Our ndings would provide new insights into the mechanism of bergenin and offer an effective candidate agent for asthma treatment.

Cell viability
Cytotoxicity of bergenin was estimated by Cell Counting Kit-8 (CCK-8; Dojindo, Tokyo, Japan) according to the manufacturer's instructions. Brie y, 16-HBE cells were seeded in the 96-well plates (5 × 106 cells/well) for overnight adherence. Then, different concentrations of bergenin (0-300 µM) was added to cells. After 24 h incubation, 10 µL of CCK-8 working solution was added to each well and incubated at 37 °C for another 1 h. The OD value of each well was measured at 450 nm using a microplate reader (Thermo Fisher Scienti c).

RNA extraction, RT-PCR and qPCR
The effects of bergenin on the mRNA levels of in ammatory cytokines were analyzed by qRT-PCR. Firstly, total RNA was extracted from the 16-HBE cells using TRIzol (Takara) according to the manufacturer's protocol. Then, 1∝ g of total RNA was reversed transcribe into cDNA using RT First Strand Kit (Takara).
qPCR was applied to quantify the interesting genes with speci c primers using SYBR® Premix Ex Taq™ II (Takara). And qPCR primer sequences were shown in Table 1. The qPCR conditions were as follows: 95 °C for 2 min, 40 cycles at 95 °C for 10 sec and then 60 °C for 30 sec. The related expression levels of interesting genes were normalized to internal control GAPDH gene using the 2 −△△Ct method. Milano, Italy). After blocking in 5% non-fat milk, membranes were incubated with speci c primary antibodies at 4 °C overnight. Primary antibodies purchased from Cell Signaling Technology were as following: phosphorylated NF-κB p65, total NF-κB p65, IKB-α, acetly-p53, acetly-p65, GAPDH. membranes were washed with 1 ⋅ TBS/Tween -20 buffer (0.1%, v/v) for three times, and then incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Enhanced chemiluminescence reaction kit (ECL, Millipore, Billerica, MA, USA) were used to visualize protein bands.
ImageJ software (version 1.43) was used to quanti ed the intensity of protein bands.

Enzyme-linked immunosorbent assay (ELISA)
TNF-α stimulated 16-HBE cells were treated with indicated concentration of bergenin (0-200 µM). After 24 h incubation, culture supernatants were harvested and centrifuged at 10,000 × g for 15 min. Then, supernatants were stored and frozen at -80 °C. The levels of IL-6, IL-8 in the culture supernatants were analyzed using a Human ELISA Kit (Multisciences, Biotech Co., Ltd, Norman, OK) according to the manufacturer's protocols.
2.6 Immuno uorescence staining 16-HBE cells were seeded on glass coverslips inside a 48-well plate and stimulated with TNF-α (20 ng/ml) in the absence or presence of bergenin (0-200 µM). After 24 h, cells were with PBS for three times and xing with 4% paraformaldehyde for 15 min at room temperature. Then, cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min, and rinsed again with PBS for three times. After blocking with 5% BSA (in PBS) for 30 min, cells were incubated with primary antibody against NF-κB p65 for overnight incubation at 4 °C. Next, cells were incubated with FITC-linked anti-rabbit (green, 1:100; Beyotime, Shanghai, China) secondary antibody, and 4′,6-diamino-2-phenylindole (DAPI, Beyotime biotechnology, China) was utilized to stained the cells nuclei. The uorescent signals were detected by a laser scanning confocal microscope (Olympus, Tokyo, Japan).

SIRT1 activity assay
In order to measure SIRT1 deacetylase activity, the extraction of nuclear proteins was performed. SIRT1 Deacetylase Activity Assay Kit (Genmed Scienti cs Inc., Arlington, MA, USA) was utilized to measured SIRT1 deacetylase activity according to the manufacturer's instructions. The optical densities (OD) of each well at 405 nm was recorded on a microplate reader (Thermo Fisher Scienti c).

Statistic analysis
Data are expressed as the mean ± SEM. SPSS v. 21.0 software was used for data analyses. Statistical signi cance between two groups was determined by Student's t-test. P-value less than 0.05 was considered a signi cant difference.

Cytotoxicity of bergenin
In order to choose appropriate concentration for the present study, we rst set out to determine the cytotoxicity of bergenin on 16-HBE cells. A series concentration of bergenin ranges between 0-300 µM were added to 16-HBE cells for 24 h incubation. The CCK-8 results indicated that the cell viability of 16-HBE cells was not affected by these concentrations of bergenin (Fig. 1B). Therefore, we chose maximum concentration of bergenin was 200 µM for the further experiments.
3.2 Bergenin inhibited TNF-α-stimulated expression of proin ammatory cytokines in 16-HBE cells Accumulated evidences indicated that TNF-α-triggered abnormal pro-in ammatory response played a critical role in the pathogenesis of asthma [25]. Especially, pro-in ammatory cytokines including IL-6 and IL-8 have been shown to contribute to asthma exacerbation. Therefore, we investigated whether bergenin affected TNF-α-induced expression levels of IL-6 and IL-8 in 16-HBE cells. As shown in Figs Therefore, these results demonstrated that bergenin has the capacity to decrease expression of IL-6 and IL-8 in TNF-α-stimulated 16-HBE cells.

Bergenin inhibited TNF-α-mediated activation of NF-κB signaling
NF-κB signaling is well-known for TNF-α-initiated pro-in ammatory cytokines expression [26].Given the inhibition of TNF-α-induced expression of IL-6 and IL-8 by bergenin, we speculated that bergenin might suppress TNF-α-mediated activation of NF-κB to exert inhibitory effects on the expression of IL-6 and IL-8. As a hallmark of NF-κB activation, degradation of IkBα was clearly suppressed by bergenin treatment (Fig. 3A). Meanwhile, in the same sample TNF-α-induced phosphorylation of p65 NF-κB was clearly inhibited by bergenin treatment (Fig. 3A). To further con rm the inhibition of NF-κB activation by bergenin, immuno uorescence was employed to investigate the effect of bergenin on NF-κB nuclear translocation. The results showed that bergenin treatment signi cantly blocked TNF-α-induced p65 NF-κB nuclear translocation (Fig. 3C). Therefore, these results so far indicated that the inhibitory effects of TNFα-induced expression of IL-6 and IL-8 by bergenin might be associated with its NF-κB-inhibiting activity.
Previous results reported that SIRT1 as a deacetylase is responsible for p65 NF-κB acetylation, and subsequently decreased NF-κB nuclear translocation [27]. As the above results revealed that bergenin inactivated NF-κB in response to TNF-α stimulation, it was necessary to clarify whether the inhibitory effects on NF-κB activation by bergenin was associated with its biological property by modulation of SIRT1. Firstly, we employed qRT-PCR and immunoblotting to investigate the effects of bergenin on the expression of SIRT1. Interestingly, bergenin treatment did not affect the mRNA and protein expression levels of SIRT1 ( Fig. 4A and 4B). While, TNF-α-mediated reduction of SIRT1 activity was dose-dependently increased by bergenin treatment (Fig. 4C). Simultaneously, the activation of SIRT1 activity by bergenin was further con rmed by the results of decreased acetylation level of p53 (Fig. 4D). Moreover, the acetylation of p65 NF-κB was also signi cantly decreased by bergenin treatment (Fig. 4E). So far, we can suppose that the inhibition effect of NF-κB by bergenin treatment might be due to its SIRT1-activated property. Therefore, we investigated the expression of pro-in ammatory cytokines to con rm this speculation in begernin-treated cells with or without SIRT1 inhibitor salermide treatment. As shown in Fig. 5G and 5H, inhibition effects of begernin on TNF-α-induced IL-6 and IL-8 were reversed by addition of salermide. Taken together, these results suggested that begernin treatment has capacity to increase SIRT1 activity, and thereby contribute to suppress NF-κB-mediated expression of pro-in ammatory cytokines in response to TNF-α stimulation.

Combination of dexamethasone and bergenin treatment effectively reduced TNF-α-induced pro-in ammatory cytokines expression
Inhaling glucocorticoids such as dexamethasone are widely applied to in treatment of asthmatic patient with airway in ammation [28]. However, a growing body of evidence indicated that many patients with asthma gradually became insensitive to glucocorticoids treatment. The above results suggested that bergenin could decrease TNF-α-induced pro-in ammatory cytokines. Therefore, we wondered whether bergenin has the potential to increase the glucocorticoids sensitiveness. As expected, we found that combination of bergenin and dexamethasone treatment caused an additive effect on reduction of IL-6 and IL-8 in TNF-α-stimulated 16-HBE cells ( Fig. 5A and 5B). These results indicated that bergenin may serve as an adjunct to pharmacotherapy for glucocorticoid-insensitive asthma.

Discussion
In the present study, we investigated the anti-in ammatory effects of bergenin using TNF-α-stimulated human bronchial epithelial cells model. Our results showed that bergenin signi cantly decreased TNF-αmediated production of IL-6 and IL-8 at mRNA and protein in 16-HBE cells, which was found to as a result of its NF-κB inhibition property. Further study demonstrated that the NF-κB inhibition property of bergenin was associated with increased SIRT1 activity. Moreover, the combination of bergenin and dexamethasone treatment could exert additive reduction effects on TNF-α-mediated expression of IL-6 and IL-8.
Bronchial epithelium cells served as a barrier to maintain lung homeostasis and protected against external harmful stimulus. However, persistent damages to the bronchial epithelium driven dysregulation of pro-in ammatory response, which was supposed to be involved in the context of airway remodeling and progression to asthma.
Diverse research suggested that the early immune response in the airway is regulated mainly by the proin ammatory cytokine TNF-α [25,29]. Both of abnormal in ammation and airway remodeling are key histopathologic features in asthma [30]. Upon TNF-α stimulation, the neutrophils and eosinophils airway recruitment, and the increased release of cytokines have been demonstrated to contribute to early onset asthma and asthma exacerbation [21,31]. In addition, TNF-α could upregulate the expression of transforming growth factor (TGF) -β1, which has been shown to be implicated in airway remodeling in severe asthma [32]. These evidences all indicated that the pathogenesis role of TNF-α during the progression of asthma. For these reasons, TNF-α has been suggested as an important target for asthma control. Therefore, in this study, we employed 16-HBE cells with TNF-α stimulation to investigate whether bergenin could exert inhibitory effects on TNF-α-induced pro-in ammatory response.
Our study showed that bergenin has an inhibitory effect on TNF-α-mediated elevation of IL-6 and IL-8 in 16-HBE cells. Accumulated evidences have provided a correlation between these pro-in ammatory mediators and asthma in both mouse models of allergic asthma and patients with asthma. The amounts of IL-6 has been found to be increased in the sputum and BAL uid of patients with asthma [33,34]. IL-6 levels signi cantly correlated with disease severity and lower forced expiratory volume in 1 second (FEV1) in children and adults with asthma [35,36]. Blockade of IL-6 attenuated airway recruitment of eosinophil and neutrophil in allergen-challenged mice [37]. In addition, IL-8, a powerful chemoattractant of neutrophil, has also been thought to play an indispensable role in initiating and/or exacerbating of asthma [38]. Kinetics of IL-8 showed that level of IL-8 was signi cantly increased in acute exacerbation of asthma, but was decreased at resolution [39]. It is postulated that the increased IL-8 was responsible for virus-driven asthma exacerbations and correlated with more neutrophils in the sputum of asthma patients [40]. IL-8-recruited neutrophil was believed to lead to development of severe clinical forms of neutrophilic asthma, which was intractable for asthma treatment due to frequent steroid resistance [41].
Thus, IL-8 could be developed as attractive target for neutrophilic asthma. Based on these evidences, down-regulation of IL-6 and IL-8 by bergenin treatment may sever as effective strategy for asthma management The transcription factor NF-κB was well-known for pro-in ammatory cytokines initiation, and has been demonstrated to be closely associated with the development of asthma [42]. Aberrant activation of NF-κB caused the proliferation of airway smooth muscle cells, resulting in airway narrowing and hyperresponsiveness in asthmatic [43]. Increased NF-κB nuclear translocation has been observed in the airway recruited in ammatory cells of asthmatics [44]. Besides, NF-κB subunit (including p50 or c-Rel) de ciency in mice abrogated the development of allergic asthma, which directly supported that functional role of NF-κB in the pathogenesis of allergic airways disease [45,46]. Upon allergens airway exposure, released of many pro-in ammatory mediators including IL-6 and IL-8 were regulated, at least in part, by NF-κB [47,48]. The results of the present study demonstrated that bergenin suppressed degradation of IkBα, phosphorylation of p65 NF-κB and NF-κB nuclear translocation. Therefore, we can suppose that the decreased of IL-6 and IL-8 secretion by bergenin was due to inhibition of NF-κB activation.
Interestingly, our results showed that expression levels of SIRT1 was not affected by bergenin, but the decreased SIRT1 activity mediated by TNF-α stimulation was signi cantly reversed by bergenin. SIRT1, belonging to a member of NAD+-dependent class III histone/protein deacetylases (HDACs), has been reported to be implicated in deacetylate a variety of substrates, and thus affected a series of cellular pathophysiology process [49]. Research showed that the suppression of SIRT1 activity has been shown to be involved in promoting the progression of asthma [50]. This implied us that increased the SIRT1 activity by novel agents might appears to be a very promising strategy for the treatment of severe uncontrolled asthma. In addition, previous study indicated that activated SIRT1 could attenuate NF-κB-driven in ammatory response upon LPS or TNF-α stimulation [27,51]. Our above-mentioned results showed that bergenin inhibited NF-κB activation. Therefore, we investigated the effect of increased SIRT1 activity by bergenin on the post-translational modi cation of NF-κB-p65 in TNF-α-stimulated 16-HBE cells. As expected, acetylation status of NF-κB-p65 was decreased by bergenin treatment. Moreover, the SIRT1 inhibitor salermide reversed the anti-in ammatory effects of bergenin in TNF-α-stimulated 16-HBE cells. Therefore, we can suppose that the increased SIRT1 activity by bergenin could deacetylate NF-κB-p65; in turn the inhibition of NF-κB reduced TNF-α-induced production of IL-6 and IL-8.
Inhaled or oral glucocorticoids has been extensively applied to control excess airway in ammation of asthmatic in clinic. However, it has been shown that increased number of patients with asthma gradually fail to respond to glucocorticoids. The molecular mechanism underlying of glucocorticoid resistance or insensitivity was due to excessive activation of MAPK and NF-κB signaling pathway [52]. Furthermore, increased NF-κB activation is closely correlated with the severity of asthma [53]. Therefore, it was proposed that inactivation of NF-κB could provide bene cial effects in the treatment of asthma. In accordance with this, our data indicated that combination of bergenin and dexamethasone exerted additive effects on TNF-α-mediated production of IL-6 and IL-8. Thus, we can suppose that the additive anti-in ammatory effect may be a result of the inhibition property on NF-κB by bergenin. Therefore, we believed that bergenin can contribute as an adjunct to the treatment of glucocorticoid resistance in asthma.

Conclusions
In summary, bergenin can signi cantly attenuate TNF-a-induced pro-in ammatory response in 16-HBE cells. The underlying mechanism of its anti-in ammatory property was associated with increased SIRT activity, and thereby contribute to suppression of NF-κB-p65 acetylation and concomitant inhibition of production of IL-6 and IL-8. Therefore, bergenin might be contribute to therapeutic potential for the prevention and management of airway in ammation in asthma.    were measured by ImageJ software. Data were presented as means ± SEM of three independent experiments. ###P < 0.001 compared to untreated cells; * * * P < 0.001 compared to TNF--treated alone. (C) Immuno uorescence confocal microscopy was used to monitor the TNF-α-stimulated p65 NF-κB (Green) nuclear translocation in the absence or presence of bergenin (0 -200μM) treatment. Nuclei were counterstained with DAPI (Blue). #P < 0.05 ##P < 0.01 ###P < 0.001 compared to untreated cells; * * * P < 0.001compared to TNF-treated alone. Figure 5