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Isosinensetin alleviates the injury of human bronchial epithelial cells induced by PM 2.5

. Flavonoids which are extracted from citrus peel and pulp have been reported to have multiple beneficial effects on human health. Isosinensetin (ISO) is a type of flavonoid compound, which has several protective effects including anticancer, antioxidant, antiviral, anti‑inflammatory and bacteriostatic. However, the molecular mechanism of its antioxidant and anti‑inflammatory effects remain unclear. The present study aimed to investigate the intervention effect and possible mechanism of ISO on human bronchial epithelial cells injured by fine particular matter ≤2.5 µm in diameter (PM 2.5 ). In the present study, the cell viability was detected by Cell Counting Kit‑8 method. The levels of pro‑inflammatory cytokines were analyzed by ELISA. The level of reactive oxygen species (ROS) was detected by fluorescence probe. The expression levels of proliferating cell nuclear antigen (PCNA), nuclear factor erythroid 2‑related factor 2 (Nrf2) and nuclear factor кΒ (NF‑кB) proteins were detected by western blotting. The results revealed that ISO evidently increased the viability of 16‑HBE cells and sharply decreased the levels of pro‑inflammatory factors in cell culture supernatant. ISO significantly inhibited ROS release caused by PM 2.5 . Moreover, the expression levels of PCNA, Nrf2 and NF‑кB proteins were downregulated after ISO incubation. These results indicated that ISO alleviated 16‑HBE‑cell injury by PM 2.5 through the ROS‑Nrf2/NF‑кB signaling pathway.


Introduction
In recent years, environmental pollution has become a major threat to human health. Increased attention has been paid to the effect of environmental factors on the occurrence and development of respiratory diseases. Fine particular matter ≤2.5 µm in diameter (PM 2.5 ) is the main pollutant in the atmosphere. It refers to fine particulate matter with an aerodynamic diameter under 2.5 µm. PM 2.5 inhalation can induce respiratory tract injury directly and aggravate a variety of diseases (1,2). A large number of epidemiological studies have revealed that PM 2.5 exposure is closely related to the morbidity and mortality of cardiovascular and respiratory systems as well as other diseases, such as arterial embolism, asthma, pneumonia, and lung cancer (3,4).
Flavonoids are natural compounds with a wide range of molecular diversity. Fruits, vegetables, herbs and other plant foods are the sources of flavonoids (5). Flavonoids have received increasing attention due to their anti-inflammatory, antimicrobial and anticancer activities (6). The important and beneficial effect of flavonoids is antioxidant activity for human body health, which is related with scavenging of reactive oxygen species (ROS) (7). Flavonoids can adjust the expression of inflammatory mediators including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and cyclooxygenase-2 (COX-2) through regulation of the Toll receptor (TLR)/NF-κB axis (8). The large number of natural compounds containing flavonoids used to treat, manage, and prevent human diseases have been widely studied. A typical example is usage of licorice derived from the dried roots and rhizomes of the Glycyrrhiza species. Licorice has been used to treat diabetes, tuberculosis, and other inflammatory disorders (9). In addition, some flavonoids (quercetin, apigenin and luteolin) have been revealed to reduce the expression and secretion of cytokines (10). However, the anti-inflammatory effect of isosinensetin (ISO) has not been reported. Therefore, in the present study, the intervention effect and possible mechanism of ISO on human bronchial epithelial cells injured by PM 2.5 were investigated.

Materials and methods
Materials. The human bronchial epithelial cell line, 16-HBE, was obtained from the American Type Culture Collection
Drug. Isosinensetin (99.5% purity) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (NICPBP; Beijing, China). ISO (2 mg) was firstly dissolved in 0.1 ml absolute ethyl alcohol. The concentrations used were 0, 20, 40, 80 or 160 µg/ml and the drug was stored at 4˚C ( Fig. 1; structure of ISO). NAC (2 mg) was dissolved in 2 ml sterile deionized water and stored at 4˚C. PM 2. 5 sample. The collection and preparation of PM 2.5 sample were conducted according to our previously reported methods (11). Briefly, samples were collected on nitrocellulose filters using a high-volume sampler particle collector in the center of Shenyang city. PM 2.5 was extracted from the nitrocellulose filters by immersing in deionized water and then sonicating for 30 min. The PM 2.5 sample was stored at -80˚C.
Cell culture. 16-HBE cells were cultured in ECM supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 1% penicillin/ECGs at 37˚C in an atmosphere of 5% CO 2 and 95% air. The cells were routinely sub-cultured every 2-3 days and it was confirmed that all of the cells were in the logarithmic phase before subsequent experimentation.
Cell viability assay. 16-HBE cells were seeded at a density of 1x10 4 cells/ml in a 96-well plate. When confluence reached 70-80%, the cells were treated with serum-free medium for 12 h at 37˚C. Subsequently, the cells were incubated with PM 2.5 solution or PM 2.5 solution plus ISO for 6, 12, or 24 h at 37˚C. The cell viability was assessed by the CCK-8 assay. A total of 10 µl of CCK-8 reagent was added to each well, and incubated at 37˚C for 1 h. The absorbance was measured at 540 nm using a microplate spectrophotometer (Thermo MK3; Thermo Fisher Scientific, Inc.).

Contents of pro-inflammatory factors determination.
The concentrations of IL-1β, IL-6 or TNF-α were detected by ELISA kits. 16-HBE cells were seeded at a density of 2x10 5 cells/ml in 6-well plates and cultured as aforementioned, followed by exposure to PM 2.5 solution or PM 2.5 solution plus ISO for 24 h. The supernatants were collected. Inflammatory factors were detected using IL-6, IL-1β and TNF-α ELISA kits (eBioscience; Thermo Fisher Scientific, Inc.) by measuring the absorbance at a 460-nm wavelength.
Assessment of ROS production. 16-HBE cells were cultured and treated as aforementioned. A DCFH-DA probe was used to determine the level of intracellular ROS production. A total of 10 µM DCFH-DA probe was added for 30 min at 37˚C in the dark. After incubation, the cells were washed three times with ice-cold PBS. The fluorescence was observed with an immunofluorescence microscope (excitation wavelength, 488 nm; emission wavelength, 525 nm) (Leica CTR 4000; Leica Microsystems GmbH).
Statistical analysis. All data are presented as the mean ± standard deviation (SD) from three or more independent experiments. Differences between the groups were evaluated for significance by one-way ANOVA followed by Tukey's post hoc test for pairwise comparisons or by Dunnett's test for comparisons vs. a control, where appropriate. The statistical graphs were constructed using GraphPad Prism (version 5; GraphPad Software, Inc.) and photoshop (version 8.0; Adobe systems, Inc). P<0.05 was considered to indicate a statistically significant difference.

PM 2.5 induces damage of human bronchial epithelial cells.
A CCK-8 assay was used to assess the viability of 16-HBE cells in order to determine the effective dose of PM 2.5 on 16-HBE cells after PM 2.5 exposure for 6, 12 and 24 h, respectively. As revealed in Fig. 2, a dose greater than 25 µg/ml PM 2.5 posed a threat to cell viability of 16-HBE cells (P<0.05).
PCNA is a type of nucleoprotein, whose level can reflect the condition of cell growth and proliferation (13). Therefore, the expression of PCNA protein in 16-HBE cells was detected by western blotting. The results revealed that after exposure to 25 and 50 µg/ml PM 2.5 , the expression of PCNA was significantly decreased compared with the control group of 16-HBE cells. 2.5 . In order to investigate the role of ISO in alleviating PM 2.5 -induced cell damage and select an optimal concentration to perform further experiments, the effects of various concentrations of ISO (0, 20, 40, 80 and 160 µg/ml) were investigated regarding the viability of 16-HBE cells exposed to PM 2.5 at 25 µg/ml which caused a 20-30%-decrease in cell viability (Fig. 3). The results indicated that the addition of ISO could attenuate the toxicity caused by PM 2.5 on 16-HBE cells. The optimum concentration observed in cells treated with ISO was 80 µg/ml. Compared with the cells exposed to PM 2.5 for 24h, ISO (80 µg/ml) significantly increased the expression of PCNA protein in 16-HBE cells.  2.5 . ROS acts as a contributor to the toxicity of PM 2.5 and flavonoids are the antioxidants of nature (14). Fluorescence microscopy revealed that ISO significantly inhibited the ROS content caused by PM 2.5 (Fig. 5). The level of ROS was significantly decreased in the PM 2.5 + ISO co-incubation group in comparison with the PM 2.5 group. These data demonstrated that ISO played a restraining role against the toxic effect of PM 2.5 by reducing ROS.

ISO inhibits the ROS generation induced by PM
ISO downregulates the expression of inflammatory-related proteins. The transcription factor NF-κB may be related with the anti-inflammatory properties of PM 2.5 exposure. The proteins p65 and IKK are the important subunits of the NF-κB family and their expression is correlated with NF-κB activity (15). Our results revealed that the expression levels of p65 and IKK in the PM 2.5 group were markedly higher than that in the NC group. However, the expression levels of p65 and IKK were significantly decreased after treatment with 80 µg/ml ISO.
Nrf2 protein is a critical signal molecule that regulates immunity and inflammation (16). The expression of Nrf2 is associated with inflammatory molecules. The results revealed that ISO downregulated the expression of Nrf2 protein. These data illustrated that ISO played a restraining role against the toxic effect of PM 2.5 through the ROS-Nrf2/NF-κB signaling pathway (Fig. 6).
Moreover, the results revealed after inhibiting ROS generation through ROS inhibitor NAC treatment, the expression levels of Nrf2, p65 and IKK proteins were downregulated compared with the PM 2.5 group. However, there was no statistical significant difference between the NAC + PM 2.5 group and NAC + PM 2.5 + ISO group (Fig. S1).

Discussion
In the present study, it was firstly indicated that ISO had protective effects on oxidative damage and decreased the expression levels of inflammatory factors induced by PM 2.5 . ISO could alleviate the injury of human bronchial epithelial cells induced by PM 2.5 . PM 2.5 , as one of the toxic and harmful components in the air, is correlated with various systemic diseases, including respiratory and cardiovascular diseases (17). Fine particles can reach and be retained at the alveolar walls of the lungs, which results in allergies, asthma and lung emphysema. In previous studies, it has been demonstrated that PM 2.5 exposure caused significant formation of ROS and an inflammatory reaction. The mechanisms mainly involved PM 2.5 triggering systemic oxidative stress and inflammation (18)(19)(20).
Various studies have revealed that flavonoids are the main bioactive components in plants which possess health-promoting properties. Numerous flavonoids such as hesperidin, quercetin and apigenin have anti-nociceptive and anti-inflammatory properties (21,22). Nobiletin, as a classical polymethoxyflavone, has been reported to have an anticancer effect by inhibiting the migration ability of breast cancer cells (23). ISO is a compound of flavonoids, found in fruits and vegetables. In the present study, our results indicated that ISO could alleviate the injury of human bronchial epithelial cells and had a protective effect on 16-HBE by reducing the release of inflammatory cytokines and relieving the oxidative stress under PM 2.5 exposure.
It has been reported that the activators of Nrf2 and NF-κB proteins can act with strong antioxidative and anti-inflammatory activities after harmful chemical stimulation in a cell culture model (24,25). Nrf2 is a transcription factor which plays a vital role in activating antioxidant response by decreasing ROS production (26). NF-κB plays an important role in regulating the inflammatory response. The increase of    16-HBE cells were exposed to PM 2.5 (25 µg/ml) for 24 h, and the ROS level was examined by DCFH-DA probe. (C) 16-HBE cells were exposed to PM 2.5 (25 µg/ml) for 24 h, then treated with ISO (80 µg/ml) for 24 h, and the ROS level was examined by DCFH-DA probe. (D) Quantification of 16-HBE cells exposed to PM 2.5 (25 µg/ml) or PM 2.5 (25 µg/ml) plus ISO (80 µg/ml) for 24 h. P indicates 25 µg/ml PM 2.5 solution treatment. P + I indicates 25 µg/ml PM 2.5 solution + 80 µg/ml ISO treatment. * P<0.05 compared with the NC group; # P<0.05 compared with the P group. ISO, isosinensetin; ROS, reactive oxygen species; PM 2.5 , fine particular matter ≤2.5 µm in diameter; DCFH-DA, 2',7'-dicholorofluorescein-diacetate; NC, negative control. Figure 6. ISO treatment inhibits increase in production of related proteins induced by PM 2.5 exposure. (A) 16-HBE cells were cultured, exposed to PM 2.5 (25 µg/ml) or exposed to PM 2.5 (25 µg/ml, 24 h) and treated with ISO (80 µg/ml) for 24 h, and the expression levels of Nrf2, p65 and IKK proteins were detected by western blotting. Quantification of (B) Nrf2, (C) p65 and (D) IKK protein expression in 16-HBE cells. P indicates 25 µg/ml PM 2.5 solution treatment. P + I indicates 25 µg/ml PM 2.5 solution + 80 µg/ml ISO treatment. * P<0.05 and ** P<0.01 compared with the NC group; # P<0.05 compared with the P group. ISO, isosinensetin; PM 2.5 , fine particular matter ≤2.5 µm in diameter; Nrf2, nuclear factor erythroid 2-related factor 2; IKK, IκB kinase. ROS can lead to continuous activation of NF-κB and promote the release of cytokines (27). Our data revealed that ISO could downregulate the expression of Nrf2 and NF-кB in 16-HBE cells induced by PM 2.5 . Moreover, after inhibiting the release of ROS induced by PM 2.5 , the expression levels of Nrf2, p65 and IKK proteins exhibited no obvious differences between the PM 2.5 group and PM 2.5 + ISO group. These results indicated that ISO could alleviate the injury of 16-HBE cells induced by PM 2.5 through the ROS-Nrf2/NF-кB signaling pathway. However, a limitation of the present study was that further investigation of the mechanism of Nrf2/NF-кB after PM 2.5 or ISO treatment was not performed in 16-HBE cells. It will be conducted in subsequent experiments.
The apparent effect of ISO has been revealed to be presumably dependent on the concentration. Nonetheless, the anti-inflammatory and oxidative mechanisms of ISO have not yet been clearly determined. Among the different molecules produced intracellularly, the transcription factor Nrf2 plays a vital role in the prevention of cell dysfunction in response to oxidative stress and in protection against exposure to toxins and carcinogens through the ARE-mediated expression of a battery of cytoprotective genes (28). It is anticipated that in future studies, ISO may be used to evaluate the therapeutic interventions on the cellular injury induced by PM 2.5 .