Quercetin protects human oral keratinocytes from lipopolysaccharide-induced injury by downregulating microRNA-22

Abstract

Background:

Quercetin exerts anti-inflammatory effects, but whether it can benefit patients with the chronic inflammatory disease of oral lichen planus (OLP), which is a common chronic mucocutaneous disorder with an immune-mediated pathogenesis, is unclear. The present research examined the impacts of quercetin in a cell-based OLP model in which human oral keratinocytes (HOKs) were treated with lipopolysaccharide (LPS).

Methods:

Effects of quercetin on viability, proliferation, and apoptosis of HOKs were assessed using the Cell Counting Kit-8 assay, Western blotting, and flow cytometry, respectively. Effects of treatment on levels of microRNA-22 (miR-22) were measured using stem-loop reverse transcription polymerase chain reaction, while levels of proteins and phosphorylation in the PI3K/AKT and JAK1/STAT3 cascades were analyzed by Western blot.

Results:

Quercetin mitigated LPS-induced reduction in HOK viability and elevation of apoptosis. It also weakened LPS-induced upregulation of miR-22. Quercetin treatment led to significantly higher levels of p-PI3K, p-AKT, p-JAK1, and p-STAT3. These effects of quercetin were enhanced when miR-22 was knocked down and partly reversed when miR-22 was overexpressed.

Conclusion:

Quercetin can mitigate LPS-induced injury in HOKs by downregulating miR-22, thereby activating PI3K/AKT and JAK1/STAT3 cascades.

Introduction

Oral lichen planus (OLP) is a chronic mucosal inflammatory disease present in approximately 0.5–3% of adults, and this prevalence is not expected to decrease in the near future. ¹ OLP transforms into malignancy in 0.4–5% of affected individuals. ² The pathogenesis of OLP remains poorly understood, and it is believed to involve inflammatory response to an autoimmune reaction. ³ A characteristic of the disease is a band of infiltrating T cells in the lamina propria of the oral mucosa. ⁴ OLP, like other inflammation-related disorders, appears to involve microRNAs (miRNAs). ⁵ Levels of miRNA-146a and miRNA-155 are elevated in the peripheral blood mononuclear cells of patients with OLP, and lesions show altered expression of numerous miRNAs. ⁶

The naturally occurring flavonoid quercetin, which was obtained from flowers, leaves, and fruits of many plants, can mitigate OLP and many other diseases by exerting antioxidant, anti-inflammatory, and antiapoptotic activities. ⁷ How exactly quercetin works against OLP is poorly understood. Several studies have shown that quercetin alters miRNA levels in therapeutically useful ways in various cancers. ^8,9 This raises the question of whether quercetin may work against OLP by altering miRNA levels.

The present study aimed to explore in detail how quercetin can treat OLP, with a focus on downstream miRNAs and signaling pathways involved. As a cellular model of OLP, we stimulated inflammatory responses in cultured human oral keratinocytes (HOKs) using Gram-negative bacterial lipopolysaccharide (LPS). ¹⁰ This cell type was chosen because OLP involves degeneration of basal keratinocytes. ¹¹

Materials and methods

Cell culture and treatment

HOKs were purchased from Barfield Biology (Wuhan, China) and cultured at 37°C in 5% carbon dioxide (CO₂) in oral keratinocyte medium (ScienCell Research Laboratories, California, USA). HOKs were treated for 48 h with 5, 10, 50, 100, or 200 ng/mL of LPS (Sigma, St. Louis, Missouri, USA) and 10, 50, 100, 200, or 400 μM of quercetin (Biyuntian, Hangzhou, China).

Transfection with miRNA

Negative control miRNA containing a scrambled sequence with no known homology to the human genome, a microRNA-22 (miR-22) mimic, or an miRNA targeting miR-22 were synthesized by Barfield Biology. Transfections were performed using Lipofectamine™ 3000 reagent (Invitrogen, Carlsbad, California, USA) based on the manufacture’s protocol.

HOK viability assay

HOK viability was assayed using the Cell Counting Kit-8 (CCK-8; Invitrogen). HOKs were grown in 96-well plates at a density of 5 × 10³ cells/well and cultured overnight at 37°C in 5% CO₂. HOKs were incubated with different concentrations (10, 50, 100, or 200 ng/mL) of LPS for 48 h, and cell viability was assessed. And HOKs were incubated with different concentrations (10, 50, 100, 200, or 400 μM) of quercetin for 48 h, and cell viability was also measured. Then, cultures were treated at the same time with both LPS (100 ng/mL) and quercetin (100 μM) for 48 h, and then the medium was replaced with Dulbecco’s modified Eagle’s medium (Grand Island, New York, USA) containing 10% CCK-8 reagent. Cultures were incubated at 37°C for 1 h and then absorbance values of the active cells at 450 nm was measured using a Microplate Reader (Molecular Devices, Biobase, Tokyo, Japan).

Annexin V/propidium iodide (PI) staining

HOKs growing logarithmically were plated in 6-well dishes (2 mL per well, 1 × 10⁵ cells/mL) and cultured for 24 h. HOKs were treated at the same time with both LPS (100 ng/mL) and quercetin (100 μM) for 48 h, rinsed twice in cold phosphate-buffered saline (PBS), harvested by scraping, suspended in PBS, incubated with 5 µL of FITC annexin V and 100 ng of PI based on the manufacture’s protocol, and analyzed using an LSR II/Fortessa flow cytometer (BD Biosciences, Heidelberg, Germany).

Stem-loop reverse transcription (RT) polymerase chain reaction (PCR)

HOKs were treated at the same time with both LPS (100 ng/mL) and quercetin (100 μM) for 48 h and then total RNA was extracted using an miRNeasy Mini Kit (StemCell Technologies, Ottawa, Canada) based on the manufacturer’s protocol. RT was carried out using PrimeScript™ Master Mix (Invitrogen). The reaction system included 2 µL of 5× PrimeScript RT Master Mix, 1 µL of RNA, and 7 µL of RNase-free distilled water. The reaction cycle was 15 min at 37°C and 5 s at 85°C. RT-PCR was carried out using the TB Green™ Premix Ex Taq™ II and YBR Premix Ex Kit (TaKaRa TOMY) with cycling conditions of 95°C for 10 s and 40 cycles of 95°C for 5 s and 60°C for 31 s. PCR reactions were performed in triplicate.

Western blotting

HOKs were denatured in radio-immunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China), centrifuged, and then heated for 7 min at 100°C. Proteins were separated by sodium salt-polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto nitrocellulose membranes (Millipore, Billerica, Massachusetts, USA). Nonspecific binding sites were blocked for 1 h in 5% nonfat milk and then bands were incubated overnight at 4°C with p53, p16, cyclinD1, Bax, cleaved caspase 3, cleaved caspase 9, PI3K, p-PI3K, AKT, p-AKT, JAK1, p-JAK1, STAT3, p-STAT3, and β-actin. rabbit anti-mouse monoclonal antibodies (1:1000 dilution). After washing with PBS Tween-20 for three times, the bands were incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody (1:3000 dilution) for 2 h. Proteins were detected using luminol reagent and peroxide solution (Millipore). Images were acquired for further analysis.

Statistical analysis

Results are reported as mean ± SD. Differences between groups were assessed for significance using the unpaired two-tailed Student’s t test or one-way analysis of variance as appropriate. The value of p < 0.05 was considered statistically significant.

Results

LPS-induced injury to HOKs

Since 48-h incubation with 100 ng/mL of LPS reduced HOK viability by 50%, these conditions were used in subsequent experiments to examine the effects of quercetin (Figure 1).

Figure 1.

LPS injures HOKs. HOKs were incubated with 10, 50, 100, or 200 ng/mL of LPS for 48 h and then cell viability was determined using the CCK-8 assay. Results are the mean ± SD of three independent experiments. *p < 0.05 versus control. LPS: lipopolysaccharide; HOK: human oral keratinocyte; CCK-8: Cell Counting Kit-8.

Quercetin attenuates LPS-induced HOK injury

First, the effects of quercetin on healthy HOKs were examined by treating cultures with increasing concentrations of quercetin and then determining cell viability. Viability was unaffected by concentrations as high as 100 μM, whereas it fell significantly with concentrations of 200–400 μM (Figure 2(a)). Therefore, the maximum dose of quercetin in subsequent experiments was 100 μM.

Figure 2.

Qu attenuates LPS-induced HOK injury: (a) HOKs were treated with increasing concentrations of quercetin for 48 h, and cell viability was determined using the CCK-8 assay; (b) and (c) HOKs were co-treated with 100 μM of quercetin and 100 ng/mL of LPS for 48 h, and levels of p53, p16, and cyclin D1 were assayed by Western blot; (d) cell viability was determined by CCK-8 assay; (e) cell apoptosis was observed by flow cytometry; (f) and (g) levels of Bax and cleaved caspases 3 and 9 were assayed by Western blot. Results are the mean ± SD of three independent experiments. *p < 0.05 versus control, ^# p < 0.05 versus LPS. Qu: quercetin; LPS: lipopolysaccharide; HOK: human oral keratinocyte; CCK-8: Cell Counting Kit-8.

Next, HOKs were treated with 100 ng/mL of LPS in the presence or absence of 100 μM quercetin. LPS induced changes in p53, p61, and cyclin D1 levels (Figure 2(b) and (c)); quercetin significantly reversed the LPS-induced decrease in cell viability (Figure 2(d)); LPS induced increase in apoptotic rate (Figure 2(e)); and LPS induced changes in levels of Bax and cleaved caspases 3 and 9 (Figure 2(f) and (g)). These results confirm that quercetin can effectively decrease LPS-induced HOK injury.

Quercetin downregulates miR-22 in LPS-treated HOKs

To explore how miRNAs may be involved in the observed anti-inflammatory effects of quercetin, we treated HOKs with LPS in the presence or absence of quercetin and assayed levels of miR-22. LPS significantly upregulated miR-22, which quercetin reversed (Figure 3). These results implicate miR-22 in the ability of quercetin to mitigate LPS-induced injury in HOKs.

Figure 3.

Qu reverses LPS-induced upregulation of miR-22. HOKs were treated simultaneously for 48 h with 100 μM of quercetin and 100 ng/mL of LPS, and miR-22 levels were assayed using stem-loop RT-PCR. Results are the mean ± SD of three independent experiments. *p < 0.05 versus control; ^# p < 0.05 versus LPS. Qu: quercetin; LPS: lipopolysaccharide; HOK: human oral keratinocyte; miR-22: microRNA-22; RT-PCR: reverse transcription polymerase chain reaction.

Quercetin may mitigate LPS-induced injury by downregulating miR-22

We conducted further experiments to verify that miR-22 is a target of quercetin. We transfected HOKs with an miR-22 mimic to test the effects of constitutively high levels of this miRNA or with an miR-22 inhibitor to knockdown the miRNA (Figure 4(a)). Transfected HOKs were treated for 48 h simultaneously with quercetin and LPS. Overexpression of miR-22 significantly reversed the effects of quercetin on LPS-induced loss of cell viability (Figure 4(b)); changes in expression of p53, p16, and cyclin D1 (Figure 4(d) and (e)); increase in proportion of apoptotic HOKs (Figure 4(c)); and changes in expression of Bax and cleaved caspases 3 and 9 (Figure 4(f) and (g)). Conversely, miR-22 knockdown significantly enhanced the therapeutic effects of quercetin. These results suggest that quercetin may mitigate LPS-induced injury in HOKs by downregulating miR-22.

Figure 4.

Qu may mitigate LPS-induced injury in HOKs by downregulating miR-22: (a) HOKs were transfected with NC miRNA, miR-22 mimic, and miR-22 inhibitor, and then miR-22 levels were measured using stem-loop RT-PCR; (b) transfected cells were co-treated with 10 μM of Qu and 100 ng/mL of LPS for 48 h, then cell viability was determined using the CCK-8 assay; (c) cell apoptosis was observed by flow cytometry; (d) and (e) levels of p53, p16, and cyclin D1 were assayed by Western blot. Lane 1, control; lane 2, LPS; lane 3, NC + LPS + Qu; lane 4, miR-22 mimic + LPS + Qu; lane 5, miR-22 inhibitor + LPS + Qu. (f) and (g) Levels of Bax and cleaved caspases 3 and 9 were assayed using Western blot. Lanes are the same as in panels d/e. Results are the mean ± SD of three independent experiments. *p < 0.05 versus control; ^# p < 0.05 versus LPS. NC: negative control; Qu: quercetin; LPS: lipopolysaccharide; HOK: human oral keratinocyte; miR-22: microRNA-22; RT-PCR: reverse transcription polymerase chain reaction; CCK-8: Cell Counting Kit-8.

Quercetin-induced down-regulation of miR-22 activates PI3K/AKT and JAK1/STAT3 cascades

We began to explore what downstream signaling cascades may be affected by quercetin. The compound reversed LPS-induced inhibition of PI3K, AKT, JAK1, and STAT3 phosphorylation, and these quercetin effects were neutralized by miR-22 overexpression but enhanced by miR-22 knockdown (Figure 5(a) and (b)). Our data suggest that quercetin activates PI3K/AKT and JAK1/STAT3 cascades by downregulating miR-22.

Figure 5.

Qu activates PI3K/AKT and JAK1/STAT3 cascades through miR-22 in HOKs. Transfected cells were co-treated with 100 μM of Qu and 100 ng/mL of LPS for 48 h, and levels of proteins in the (a) PI3K/AKT and (b) JAK1/STAT3 cascades were assessed by Western blot. Lane 1, control; lane 2, LPS; lane 3, NC + LPS + Qu; lane 4, miR-22 mimic + LPS + Qu; lane 5, miR-22 inhibitor + LPS + Qu. Data are the mean ± SD of three independent experiments. *p < 0.05 versus control; ^# p < 0.05 versus LPS. NC: negative control; Qu: quercetin; LPS: lipopolysaccharide; HOK: human oral keratinocyte; miR-22: microRNA-22.

Discussion

OLP involves inflammation of the oral or skin mucosal epithelial and often the degeneration of basal keratinocytes. ^12,13 Previous studies have demonstrated the therapeutic effects of quercetin on OLP, but they have not provided detailed molecular insights into how these effects arise. Here we used an in vitro inflammation model with HOKs, and we found that LPS treatment induced the decreased cell viability and increased cell apoptosis, suggesting that LPS intervene could lead to the injury of HOKs. In addition, the cell viability was significantly increased and cell apoptosis was greatly decreased in HOKs after treatment at the same time with LPS and quercetin, indicating that that quercetin reverses LPS-induced HOKs injury. Furthermore, LPS treatment induced the decreased expression level of miR-22, and its expression was significantly upregulated in HOKs after treatment at the same time with LPS and quercetin, suggesting quercetin could induce the miR-22 expression. This downregulation of miR-22 is associated with activation of PI3K/AKT and JAK1/STAT3 signaling cascades. Our results provide several testable hypotheses about how quercetin can treat OLP, which may help guide efforts to improve this treatment and develop new ones.

Our in vitro model recapitulates several aspects of OLP. LPS inhibited HOK proliferation by downregulating cyclin D1 and upregulating p53 and p16. Cyclin D1 interacts with cyclin-dependent kinases and increases phosphorylation of retinoblastoma protein to trigger proliferation, ¹⁴ while p16 and p53 can act as antiproliferative tumor suppressors. ^15,16 In fact, the upregulation of p53 has been linked to OLP. ¹⁶ LPS significantly increased the rate of apoptosis in HOKs, which was associated with the upregulation of Bax and cleaved caspases 3 and 9. Quercetin significantly reversed all these effects of LPS. Consistent with our results, another study in HOKs showed that quercetin promotes HOK proliferation and oral reepithelialization by upregulating TGF-β3. ¹⁷

In our system, the effects of LPS were associated with upregulation of miR-22, while the therapeutic effects of quercetin were associated with its downregulation. These results are consistent with previous work, showing that miR-22 acts as a tumor suppressor to inhibit cell proliferation, migration, and cell cycle progression in oral squamous cell carcinoma. ¹⁸ Indeed, miR-22 is located in the unstable genomic region 17p13.3, and it is deregulated in many tumor types. ¹⁹ Our results strengthen and extend previous studies implicating miRNAs in OLP. For example, upregulating miR-125b may mitigate OLP by activating matrix metalloproteinase-2 and the PI3K/AKT/mammalian target of rapamycin (mTOR) cascade. ²⁰

Our experiments suggest that quercetin-mediated downregulation of miR-22 activates the PI3K/AKT and JAK1/STAT3 cascades, which are involved in cell proliferation and apoptosis. ^21,22 Similarly, the neuroprotective effects of quercetin in a mouse model of Alzheimer’s disease are associated with changes in these two pathways, which ultimately alter cytokine levels. ²³ The previous study and our results showed that quercetin could activate the PI3K/AKT and JAK1/STAT3 signaling pathway to improve some disorders. In addition, a previous study reported that quercetin suppresses the tumorigenesis of oral squamous cell carcinoma by downregulating miR-22. ²⁴ These research demonstrated that quercetin has an antitumor role in many cancers via regulating the expression of miR-22. Moreover, we will confirm our conclusions in vivo to use animal OLP model. In addition, this study showed that quercetin could inhibit the expression level of miR-22 to decrease the cell viability of HOKs, but the detailed mechanism of quercetin action in regulating the miR-22 expression remains unclear. Therefore, we will further discuss the interaction between quercetin and miR-22 in vitro. Further studies should verify and extend our results in more sophisticated OLP models and patient-derived tissues. This work should also explore what other miRNAs and downstream signaling cascades may be affected by quercetin.

Conclusions

In summary, quercetin can mitigate LPS-induced injury in HOKs by downregulating miR-22 and thereby activating PI3K/AKT and JAK1/STAT3 pathways. These results of the present study provide a reference for clinical treatment of OLP.