Citrus Peels Phenolic Derivatives Alleviate Benzo[α]pyrene-Induced Inflammatory Injury of Human Intestinal Epithelial Cells Through NLRP3 and AhR Signaling Pathways


 Benzo[α]pyrene (BaP) is ubiquitous in foods, and possesses a fatal cytotoxicity. In current study, ten Citrus peels (Chenpi) phenolic derivatives (CPDs) were isolated in a cell model of human intestinal epithelial (Caco-2) cells under BaP-exposure by a bio-assay guided method. Among them, methyl (3,4,5-trimethoxybenzoyl) valylphenylalaninate (Citrus peels phenolic derivative-2, CPD-2) performed the most protective activity by promoting the antiinflammatory potential on BaP-induced Caco-2 cells. CPD-2 inhibited BaP-induced intracellular ROS over-production and inflammatory epithelial cytokine, IL-4, IL-8, TNF-α, IL-1β and IL-18 over-expression, but not IL-6. CPD-2 also inhibited BaP-induced NLRP3 inflammasome and AhR signaling pathway activation. Overall, CPD-2 attenuates BaP-induced apoptotic death via promoting the antiinflammatory potentials by inhibiting the NLRP3 and AhR signaling pathways activation of Caco-2 cells. Finally, the Citrus peels phenolic derivatives was observed for the first time against BaP-induced inflammation and oxidative stress in human intestinal epithelial cells.


Introduction
Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental pollutants found in cigarette smoke, automobile exhaust and foods cooked at high temperature [1]. Benzo[a]pyrene (BaP) is a representative PAHs that has been suggested to cause human diseases, including cancers, cardiovascular, pulmonary and neurodegenerative diseases [2]. Currently, atherosclerosis is viewed as an in ammatory process driven, at least in part, by reactive oxygen species (ROS). BaP is known to generate superoxide and hydrogen peroxide via one-electron redox cycling of its metabolite, BaP-quinone [3].
Dried fruit peels of Citrus unshiu Markovich, C. reticulate Blanco, and C. tachibana Makino Tanaka, which are collectively known as "Chenpi", have been widely used as traditional medicine in Korea, China, and Japan [4]. In south China, Chenpi was infused by boiling water to extract their effective components and drunk just like tea, which is supposed to be bene cial to the health. One sort of the active biological constituents of Chenpi are adrenergic amines (such as synephrine, octopamine, and tyramine), at the same time, the hesperidin exists in Chenpi and is considered as the most functional compound [5,6].
Furthermore, the phenolics are also regarded as signi cant bio-active compounds and have been reported as anti-oxidative and anti-in ammatory agents in Chenpi [7,8]. Of interest, the avonoids in Chenpi, such as nobiletin and tangeretin, which also attracted signi cant interest for their properties of antiviral, antiin ammatory, and antiatherogenic activities [9,10]. Previous studies have also revealed that the avonoids isolated from the Chenpi performed signi cant antiin ammatory activity both in vitro and in vivo [11]. However, very few studies of Chenpi phenolic derivatives (CPDs) have been documented for the effect against BaP-induced in ammatory injury in human intestinal epithelial cells. In current study, we designed experiments to reveal whether the CPDs can inhibit BaP-induced in ammatory damages in human intestinal epithelial cells.

Materials And Methods
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Materials and agents
The "Chenpi" (dried fruit peels of Citrus unshiu Markovich) were acquired from the Jiangmen Palace international Food, inc (Guangdong, China), and was authenticated by professor Guangxiong Zhou, College of Pharmacy, Jinan University. A voucher specimen was deposited in the Herbarium of the College of Pharmacy, Jinan University. PierceTM BCA Protein Assay Kit was obtained from Thermo Fisher Scienti c (Rockford, IL, USA). CCK8 assay kit and RIPA buffer were purchased from Beyotime (Shanghai, China). Penicillin-streptomycin (Pen Strep), fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). The antibodies were purchased from Cell Signaling Technology (CST, Beverly, MA, USA).
The other chemicals used in the current study were purchased from Aldrich or Adamas without any further puri cation.

General apparatus and isolation of CPDs
The preparative high performance liquid chromatography (HPLC) was performed on a Varian Postar 210 chromatograph equipped with a Varian-306 pump and a Varian UV/vis-152 detector with a C18 reversedphase column (Cosmosil, 10 mm × 250 mm, 5 µm). ESI-MS data were detected on a Finnigan LCQ Advantage Max ion trap mass spectrometer. NMR spectra were detected on Bruker Avance 400 or 500 MHz spectrometer. The chemical shifts were expressed in δ value (ppm). Silica gel (300-400 mesh, Qingdao Marine Chemical Inc., China) was used for column chromatography. All the other reagents were acquired from Guangzhou Jierui Chemical Company (Guangzhou, China).
The cell viability of the Caco-2 cells was evaluated using cell counting kit-8 (CCK-8) (Beyotime Biotech.) as reported previously [13], following the manufacturer's protocols. The Caco-2 cells (1×10 4 cells/mL) were seeded in 96-well plates for 12 h. Then, the Caco-2 cells were treated with 1.0 µmol/L BaP or PBS. After incubation for 12 h, the Caco-2 cells were collected and washed with cold PBS for twice. Then, the HUCECs were treated with the samples or PBS for 24 h. Afterwards, the CCK-8 reagent was added to each well at 10 µL/mL nal concentration and incubated at 37°C for another 4 h. Finally, cell viability of Caco-2 cells was determined with a microplate reader (Bio-Rad).

Cell Apoptosis Assay
Caco-2 cells (2 × 10 5 /well) were plated in 6-well plate. The Caco-2 cells were incubated with 1.0 µmol/L BaP or PBS for additional 12 h, then, Caco-2 cells were collected and washed with cold PBS. After that, the Caco-2 cells were treated with the CPD-2 (10 or 100 µmol/L) or PBS for 24 h. Afterwards, the Caco-2 cells were collected, washed with cold PBS, suspended in binding buffer (100 µL) (BD Biosciences, CA, USA), treated with Annexin V and Propidium iodide (PI), and incubated in dark for 15 min, anther 300 µL binding buffer was added, then ow cytometry analysis was performed within 1 h to measure the apoptosis rate (%).

Intracellular ROS and Epithelial Cytokines Assays
Intracellular reactive oxygen species were quanti ed with Reactive Oxygen Species Assay Kit (Beyotime Institute of Biotechnology) as previously described. Brie y, Caco-2 cells (2 × 10 5 /well) were plated in 6well plate. The Caco-2 cells were incubated with 1.0 µmol/L BaP or PBS for additional 12 h, then, Caco-2 cells were collected and washed with cold PBS. After that, the Caco-2 cells were treated with the CPD-2 (10 or 100 µmol/L) or PBS for 24 h. After that, the Caco-2 cells were collected, and washed triple with serum free medium. Then, the Caco-2 cells were incubated in 200 µL of serum free medium containing DCFH-DA (25 µM) for 30 min at room temperature. The DCF uorescence was quantitated with a Multiskan Go plate reader (Thermo), with an excitation wavelength of 488 nm and an emission wavelength of 525 nm, and normalized with the total protein content.
After treatments with BaP or CPD-2, the cell supernatant was collected, and the concentration of interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-1β (IL-1β), TNF-α, and IL-18, was quanti ed by using commercial quantikine ELISA kit according to the manufacturer's instructions. All the experiments were performed at least three times.

Western blotting
Caco-2 cells (2 × 10 5 /well) were plated in 6-well plate. The Caco-2 cells were incubated with 1.0 µmol/L BaP or PBS for additional 12 h, then, Caco-2 cells were collected and washed with cold PBS. After that, the Caco-2 cells were treated with the CPD-2 (10 or 100 µmol/L) or PBS for 24 h. After that, the Caco-2 cells were collected, washed with cold PBS, the total cellular proteins were collected after lysed in RIPA buffer and the protein concentrations were measured with BCA protein assay kit. The western blot analysis was carried out with the method reported previously [14]. The β-actin or GAPDH were used as internal standard of process control. Blot bands densitometry was analyzed with Image J software.

Statistical analysis
Data were analyzed using one-way analysis of variance (ANOVA). Tukey's multiple comparison test was used to detect differences between means, and statistical signi cance was set at p < 0.05. All statistical analyses were performed using SPSS software 21.0 (SPSS Inc., Chicago, IL, USA). Graphs were generated using INSTAT software (GraphPad Software, San Diego, CA, USA).

Isolation and structure identi cation of CPDs
Eventually, ve novel Citrus peels (Chenpi) phenolic derivatives (CPDs) were isolated in a cell model of human intestinal epithelial (Caco-2) cells under BaP-exposure by a bio-assay guided method, named (3,4,5- . Furthermore, ve known CPDs were also isolated and structure identi ed, named 1-  13 13 13 13  After the ten CPDs were isolated, the cytotoxicity in Caco-2 cells was rst evaluated (24, 48 and 72 h). The results showed that all the CPDs performed no cytotoxicity in Caco-2 cells up to 1.0 mM (72 h).
We then detected the protective activity of the CPDs against BaP-induced Caco-2 cells cell viability decline, and the results were list in Table 1. The BaP-exposure signi cantly decreased the cell viability.
When compared to the control group, the BaP-exposure decreased the cell viability by 18.4% (p < 0.05), which indicated that the BaP-exposure induced fatal damages to the Caco-2 cells. Interestingly, BaPexposure induced cell viability decrease was attenuated by most of the CPDs. Among them, the CPD-1, CPD-2 and CPD-3 displayed better protective activity than the resveratrol (positive control). Furthermore, the CPD-2 mostly promoted the viability of Caco-2 cells. In current study, cytotoxicity of CPD-2 in Caco-2 cells was further explored. The results indicated that CPD-2 did not exhibit cycotoxicity up to 1.25 mM (72 h, Figure 2A). Furthermore, CPD-2 performed the protective activity against BaP-exposure induced cell viability decrease in a time-and dose-dependent manner ( Figure 2B, 2C).

Effect of CPD-2 on BaP-induced oxidative stress and in ammation in Caco-2 cells
The oxidative stress triggered by BaP induced the increase of ROS production in vitro [15]. As shown in Figure 2D, compared to the control group, BaP-exposure increased the intracellular ROS level by 970.7% (p < 0.01), which indicated that BaP-exposure caused fatal oxidative stress in Caco-2 cells. Remarkably, the BaP-induced ROS over-production was attenuated after the CPD-2 treatments. Compared to the BaP group, CPD-2 decreased the intracellular ROS level by 72.8% (p < 0.05) and 81.7% (p < 0.01), respectively.

Effect of CPD-2 on BaP-induced Caco-2 cells apoptosis
Apoptosis plays a pivotal role in promoting resolution of the acute in ammatory response [17]. The BaPinduced oxidative stress and in ammatory damages can subsequently result in the cell apoptosis [18]. Hence, we detected whether the CPD-2 can attenuate the BaP-induced Caco-2 cell apoptosis, and the results were list in Figure 3A. The BaP-exposure induced 23.1% total apoptosis/necrosis, where 16.1% early apoptosis and 4.34% late apoptosis, and the early apoptosis performed a signi cant contribution. The cell apoptosis provided a visually evidence that the Caco-2 cells suffered fatal injury after the BaPexposure. However, the BaP-induced cell apoptosis was inhibited by the CPD-2 treatment. Compared to the BaP group, the CPD-2 decreased the total apoptosis & necrosis by 3.80%, 6.70% (p < 0.05), respectively. Cell apoptosis provided visually evidence for the protective effect of CPD-2 against the BaPinduced Caco-2 cells apoptosis.
The cellular oxidative stress caused by the ROS over-production, can be part of signal transduction pathway during cell apoptosis. Bcl-2 is identi ed to have anti-apoptotic potential in a variety of cell systems [19]. Bcl-2 maintains the membrane integrity of mitochondria, by directly or indirectly preventing the release of cytochrome C, which, along with apoptotic protease-activating factor-1 (APAF-1) [20]. In the present study, we also evaluated whether the Bcl-2 involved in the protective effect of CPD-2 against the BaP-induced Caco-2 cells apoptosis. As shown in Figure 3B, BaP-exposure induced the release of cytochrome C, and induced the over-expression of APAF-1 and Bax, respectively. At the same time, BaPexposure inhibited the expression of Bcl-2 of Caco-2 cells. Intriguingly, the BaP-induced cytochrome C release, and APAF-1 over-expression were signi cantly suppressed by the CPD-2 treatments. At the same time, the BaP-induced up-regulation of Bax/Bcl-2 ratio was also reversed by the CPD-2 treatments.

Effects of CPD-2 on BaP-induced NLRP3 in ammasome activation
The NOD-like receptor superfamily, pyrin domain containing 3 (NLRP3) in ammasome is composed of NLRP3, apoptosis-associated speck-like protein (ASC) and caspase-1. Caspase-1 is a component of in ammasome with NOD like receptors (NLRs) and their adaptor proteins, and is activated within in ammasome in response to various stressor molecules [21]. The NLRP3 interacts with ASC to activate caspase-1, subsequently leads to maturation and secretion of in ammatory cytokines interleukin (IL)-1β and IL-18, which are involved in ammation response [22,23].
In current study, the BaP-induced in ammation was characterized by the up-regulated levels of in ammatory epithelial cytokines in Caco-2 cells, which were attenuated by the treatments of CPD-2. We evaluated the expression of NLRP3, ASC and caspase-1, to conform that the NLRP3 in ammasome inhibition was also involved in CPD-2 protection against the BaP-induced in ammation in Caco-2 cells. As shown in Figure 4A, the expressions of Caspase-1, ASC and NLRP3 were signi cantly increased. However, the BaP-exposure-induced NLRP3 in ammasome activation was signi cantly inhibited by the CPD-2 treatments. The western blotting results indicated that the NLRP3 in ammasome was involved in the protective effect of CPD-2.

Effects of CPD-2 on BaP-induced AhR signal activation
Free radicals are injurious to cellular lipids, proteins, and nuclear or mitochondrial DNA, inhibiting their normal function, as well as interfering with the signaling pathways within cells. A common cellular mechanism by which BaP exerts adverse effects is its propensity to act directly as free radical generators, promoting oxidative stress and the induction of in ammatory responses. Human cytochrome P450 (CYP450) enzymes are primarily membrane-associated proteins located either in the inner membrane of the mitochondria or the endoplasmic reticulum of cells. CYP1A1 and CYP1B1 are the most important human P450 enzymes involved in the metabolic activation of PAHs [24]. For example, BaP is transformed in vivo to BP-7,8-epoxide by CYP1A1 through the CYP/EH pathway [25]. Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is activated by small molecules provided by the diet, microorganisms, and pollutants. Upon ligand binding, AhR translocates from cytosol to the nucleus, leading to changes in target gene transcription (eg, cytochrome P450 cyp1a1, cyp1b1) and immunotoxicological effects. AhR activation has been shown to be critical in mediating BaP-induced oxidative stress and in ammation [26,27].
To determine whether AhR signaling can also be inhibited by CPD-2 and plays a role in mediating BaPinduced up-regulation of in ammatory epithelial cytokines expression, the transcription of AhR and its major downstream gene cyp1a1 was assessed. As expected, BaP induced signi cantly increased AhR transcription. Similar results were observed for cyp1a1 ( Figure 4B). The BaP-induced expression of AhR and CYP1A1 was attenuated by the CDP-2 treatment was further con rmed by the western blotting ( Figure 4C). De nitely, BaP-induced AhR signaling activation was blocked by the CPD-2 treatments.
To determine whether AhR is involved in BaP-induced in ammatory epithelial cytokines expression, AhR in Caco-2 cells was knocked down by siRNA, and then the cells were exposed to the BaP, and con rmed by western blotting ( Figure 5D). Expression of in ammatory epithelial cytokines was analyzed by ELISA as reported previously [27].
As expected, AhR knockdown signi cantly blocked the BaP-induced in ammatory epithelial cytokines expression as detected by the ELISA, but no change was noted for IL-6 ( Figure 5E). These results imply that the AhR signal pathway plays pivotal role of CPD-2 inhibiting BaP-induced over-release of in ammatory epithelial cytokines in Caco-2 cells.

Discussion
In south China, Chenpi was infused by boiling water to extract their effective components and drunk just like tea, which is supposed to be bene cial to the health. The Chenpi hesperidin is considered as the most functional compound [5,6]. Furthermore, the avonoids in Chenpi, such as nobiletin and tangeretin, which also attracted signi cant interest for their bio-active effects [9,10]. Cardiovascular disease is the leading cause of death globally. Studies elucidated that vascular in ammation, oxidative stress and endothelial dysfunction are pivotal risk factors involved in atherosclerosis [28]. Benzo[α]pyrene (BaP) is one of the main ingredients in cigarettes and barbecue food. As reported, BaP is usually oxidized, which can induce in ammation and oxidative stress [29]. In current study, human intestinal epithelial cells were stimulated with BaP to establish an in ammatory and oxidative stress cell model. After the bio-active compounds were isolated in an activity tracking method in the cell model of Caco-2 cells under BaP-exposure from the Citrus peels (Chenpi) extract, the potential molecular mechanisms were further explored.
The endothelial cells play a crucial role in keeping cardiovascular homeostasis and usually work as the rst defensive line in vessels. Endothelial dysfunction in vessels usually is due to the combined effect of oxidative stress and in ammation [30]. In current study, the BaP-exposure attenuated the viability of Caco-2 cells, while the CPDs treatment reversed its effect, suggesting CPDs may have potential effect against the BaP-induced Caco-2 cells damages.
Endothelial senescence also leads to the specialized cell apoptosis, in ammation and oxidative stress contributing to age-associated diseases, including atherosclerosis [31]. Previous studies demonstrated that the cytotoxic drugs stimulates the NLR protein (NLRP3) in ammasome through the M2 protein, and that caspase-1 and apoptosis-associated speck-like protein containing a carboxy-terminal caspase activation and recruitment domain (ASC) is required. Study has also indicated that BaP induces the in ammasome, leading to the activation of caspase-1 and the production of in ammatory epithelial cytokines [32]. In the present study, the treatment of CPD-2 suppressed the apoptosis of Caco-2 cells induced by BaP-exposure. Furthermore, the BaP-exposure caused an increase expression of NLRP3, ASC and caspase-1, which can aggravate severity of in ammation in Caco-2 cells. Interestingly, CPD-2 suppressed the expression of NLRP3, ASC and caspase-1 of Caco-2 cells under BaP-exposure. These data suggested CPD-2 may ameliorate the BaP-induced endothelial in ammation by inhibiting the NLRP3 in ammasome activation.
The release of cytochrome C from mitochondria has been shown as another important effector molecule in the mediation of apoptosis [20]. The Bcl-2 maintains the membrane integrity of mitochondria, by directly or indirectly preventing the release of cytochrome C, which, along with APAF-1. Bcl-2 protects cells against diverse cytotoxic insults, for example, ROS and cytotoxic agents [33]. In current study, the CPD-2 suppressed the up-regulation of Bax/Bcl-2 ratio, and prevented BaP-induced cytochrome C over-release. At the same time, CPD-2 also inhibited the activation of APAF-1. This partly explains the underlying mechanism of CPD-2 against the BaP-induced Caco-2 cells apoptosis.
BaP, released from various industrial activities, is rst oxidized by CYP1A1 to phenols [26]. CYP1A1 is one of the most important human P450 enzymes involved in the metabolic activation of PAHs. AhR is a transcription factor that is critical in xenobiotic metabolis [27]. In current study, we provided evidence that AhR is a key molecular regulator for BaP-induced the in ammatory epithelial cytokines over-expression. In particular, we found that BaP-induced the activation of AhR signaling as determined by expression of AhR and CYP1A1. Intriguingly, the increased expression was signi cantly inhibited by impaired in cells with AhR knockdown or CPD-2 treatments. These data indicate that AhR signal pathway is also essential in attenuating BaP-induced in ammatory epithelial cytokines expression in Caco-2 cells.

Declarations
Ethics approval and consent to participate Not applicable. quantitated with a Multiskan Go plate reader and normalized with the total protein content; (E) Detection of in ammatory cytokine levels of Caco-2 cells. Levels of IL-4, IL-6, IL-8, TNF-α, IL-1β and IL-18 in culture supernatants were detected by ELISA (n = 3). Data are presented as means ± SEM (n = 3). **p < 0.01, *p < 0.05 compared to the BaP group; #p < 0.05, ##p < 0.01 compared to the Control group. The expression of proteins related to apoptosis were evaluated by western blot analysis. Data are presented as means ± SEM (n = 3). **p < 0.01, *p < 0.05 compared to the BaP group; #p < 0.05, ##p < 0.01 compared to the Control group. treatments; (C) Proteins expression of AhR and CYP1A1 were evaluated by western blot analysis; (D) AhR knockdown was con rmed by western blotting; (E) ELISA analysis for the expression of in ammatory epithelial cytokines in BaP-induced Caco-2 cells with or without AhR knockdown. Data are presented as means ± SEM. Each point represents at least three independent experiments. * or # indicate statistically signi cant differences: **p < 0.01, *p < 0.05 compared to the BaP group; #p < 0.05, ##p < 0.01 compared to the Control group.

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