Effect of Berberine on Proinflammatory Cytokine Production by ARPE-19 Cells following Stimulation with Tumor Necrosis Factor-α

Abstract

Purpose.: Berberine (BBR) is a well-known drug used in traditional medicine and has been shown to possess anti-inflammatory properties. Whether it can affect the production of inflammatory cytokines by RPE cells is not yet clear and was therefore the subject of our study.

Methods.: ARPE-19 cells were cultured with TNF-α in the presence or absence of BBR to different time points. Concentrations of IL-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1) in the supernatant were measured by using an ELISA. The mRNA expression of these cytokines was measured by real-time PCR. Phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal–regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK) was measured by Western blot assay. The signal transduction mechanisms involved in cytokine production were evaluated using various inhibitors for p38, ERK1/2, and JNK.

Results.: TNF-α significantly increased the expression of IL-6, IL-8, and MCP-1 in ARPE-19 cells at both the protein and mRNA levels. It promoted the phosphorylation of p38, ERK1/2, and JNK. Inhibitory experiments showed that IL-6 was modulated by p38, whereas IL-8 and MCP-1 were modulated by p38, ERK1/2, and JNK signal pathways. BBR inhibited the expression of IL-6, IL-8, and MCP-1 remarkably at both protein and mRNA levels and down-regulated the phosphorylation of p38, ERK1/2, and JNK upon stimulation with TNF-α.

Conclusions.: The present results suggested that BBR significantly inhibits the expression of inflammatory cytokines in ARPE-19 cells and that the inhibitory effect is mediated by down-regulation of the p38, ERK1/2, and JNK pathways.

Introduction

The neuroectodermally derived retinal pigment epithelium plays a critical role in inflammatory and noninflammatory retinal diseases, including proliferative vitreoretinopathy, ^1–4 age-related macular degeneration, ^5–8 and autoinflammatory diseases. ^9–12 Previous studies ^13,14 showed that an immortalized human RPE cell line (ARPE-19) has structural and functional properties characteristic of RPE cells. It has been shown that RPE cells can secret a number of cytokines, including IL-6, IL-8 (also known as CXCL8), and monocyte chemoattractant protein-1 (MCP-1; also known as CCL2) in response to TNF-α stimulation. ^15–17  

IL-6 is a proinflammatory cytokine and has been shown to play a critical role in the occurrence of intraocular inflammatory diseases. ^18–21 MCP-1 and IL-8 are thought to be the major mediators for neutrophil and monocyte infiltration during inflammatory retinal disease. ^22–24  

Berberine (BBR), an alkaloid derived from Chinese goldthread (bark of cork tree), has long been used in the treatment of diarrhea and other gastrointestinal disorders. ^25–29 Recent studies have shown that it has antitumor, ^30–32 antimicrobial, ^33,34 and even antibiotic effects. ³⁵ A recent study has suggested that BBR may be effective in the treatment of experimental type I diabetes ³⁶ and experimental autoimmune encephalomyelitis (EAE), ^37,38 raising the possibility of its use in the treatment of autoimmune or autoinflammatory diseases. In view of the inhibitory effect of BBR on inflammation, we examined whether it could also affect cytokine production by ARPE-19 cells, and if so, which mechanisms were involved. Our study showed that BBR could significantly inhibit the expression of IL-6, IL-8, and MCP-1 in association with down-regulated phosphorylation of the p38, extracellular signal-regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK) pathways. 

Materials and Methods

BBR and a cell counting kit were purchased from Sigma-Aldrich (Cell Counting Kit-8 [CCK-8]; Sigma-Aldrich, St. Louis, MO). BBR had a purity of 99% and was dissolved in 50 mM dimethyl sulfoxide (DMSO) as stock solution. The final concentration of DMSO was kept at ≤0.05% in the culture medium. The reagent of Guanidinium thiocyanate-phenol-chloroform extraction was purchased from Invitrogen (TRIzol; Invitrogen, Carlsbad, CA); the reverse transcription system was from Promega (GoScript; Promega, Madison, WI); SYBR Green master mix for real-time PCR was from Applied Biosystems (Applied Biosystems, Foster City, CA). Antibodies against p38, ERK1/2, and JNK were obtained from Cell Signaling (Cell Signaling Technology, Beverly, MA); the enhanced chemiluminescence (ECL) kit for Western blot assay was from Pierce (Pierce Chemical, Rockford, IL). The inhibitors SB203580 (p38 inhibitor), PD98059 (ERK1/2 inhibitor), and SP600125 (JNK inhibitor) were all from Cell Signaling. Some inhibitors were dissolved in DMSO and stored at −20°C until used. Recombinant human TNF-α was obtained from R&D Systems (Minneapolis, MN); ELISA development kits for IL-6, IL-8, and MCP-1 were from R&D Systems (Duoset; R&D Systems). 

Cell Culture

ARPE-19, a human retinal pigment epithelial cell line, was obtained from the American Type Culture Collection (ATCC). Cells were cultured in medium (Dulbecco's modified Eagle's medium: nutrient mixture F12 [DMEM/F12], 1:1; Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (FBS; Invitrogen), 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were incubated in a humidified 5% CO₂ atmosphere at 37°C and passaged every 5 to 7 days. After reaching confluence, the cells were detached with trypsin-EDTA solution, diluted 1:3 to 1:4, and plated for subculture. The ARPE-19 cells used in the experiments were confluent. 

Cell Viability Assay

To investigate the effects of BBR on the viability of ARPE-19 cells, we used the CCK-8 assay, which is based on the conversion of a water-soluble tetrazolium salt, WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] to a water-soluble formazan dye upon reduction in the presence of an electron carrier by dehydrogenases. ³⁹  

ARPE-19 cells were plated in 96-well plates at a density of 1 × 10⁴ cells per well. After 4 days of incubation, the medium was withdrawn. The cells were washed twice with PBS and incubated in serum-free medium for 24 hours. BBR was added to the wells at different final concentrations (5, 10, 25, 50, 100, and 200 μM) and cultured for 24, 48, or 72 hours, and then 10 μl of WST-8 was added to each well. The optical density was read at 450 nm using a microplate reader (Molecular Devices, Sunnyvale, CA). Cells cultured without BBR were used as a control. All groups were tested in triplicate. The results from the CCK-8 assay were confirmed by trypan blue dye exclusion assay as previously described. ^40–42  

Treatment of ARPE-19 Cells with BBR after Stimulation with TNF-α

ARPE-19 cells were seeded in 6-well culture plates at a density of 8 × 10⁵ per well and maintained in DMEM/F12 medium containing 10% FBS for 4 days to become confluent. Before stimulation, cells were serum starved for 24 hours in serum-free medium. ARPE-19 cells were preincubated in serum-free medium with 25 μM BBR. Then, recombinant human TNF-α (10 ng/mL) (R&D Systems) was added to the medium. The cells were cultured for different time periods, and the supernatants were collected to measure the concentrations of IL-6, IL-8, and MCP-1. The cells were subsequently harvested for RNA and/or protein analysis. All groups were tested in triplicate. 

Treatment of ARPE-19 Cells with Inhibitors of p38, ERK1/2, and JNK after Stimulation with TNF-α

For the experiments investigating the effect of MAPK inhibitors on TNF-α–induced cytokine expression, ARPE-19 cells were seeded in 6-well culture plates to become confluent. After being serum starved for 24 hours, these cells were preincubated for 30 minutes in serum-free medium with various MAPK inhibitors, including 10 μM SB203580, 10 μM PD98059, and 10 μM SP600125. ^43–45 Subsequently, recombinant human TNF-α (10 ng/mL) (R&D Systems) was added to the medium. After 24 hours of incubation, the supernatants were collected and stored at −70°C until assay. IL-6, IL-8, and MCP-1 were measured using human ELISA development kits (Duoset; R&D Systems). Each stimulation experiment was repeated three times. 

Enzyme-Linked Immunosorbent Assay

The concentrations of IL-6, IL-8, and MCP-1 were determined using the human ELISA development kits (R&D Systems) according to the manufacturer's instructions with detection limits of 9.4 pg/mL, 15.6 pg/mL, and 31.2 pg/mL, respectively. 

Total RNA Extraction and Real-Time PCR

Total RNA was isolated by using TRIzol (Invitrogen) following the manufacturer's instructions. The purified RNA was reverse transcribed to single-stranded cDNA using the reverse transcription system (GoScript; Promega). mRNA expression was determined by real-time PCR using the SYBR Green master mix under standard thermocycler conditions (Applied Biosystems). Data were collected and quantitatively analyzed on a sequence detection system (ABI Prism 7500; Applied Biosystems). The human β-actin gene was used as an endogenous control for sample normalization. Results are presented as fold increases relative to the expression of human β-actin. The sequences of the PCR primer pairs were as follows: human β-actin, forward, 5′-GGA TGC AGA AGG AGA TCA CTG-3′, and reverse, 5′-CGA TCC ACA CGG AGT ACT TG-3′; human IL-6, forward, 5′-AGT GAG GAA CAA GCC AGA GC-3′, and reverse, 5′-CAG GGG TGG TTA TTG CAT CT-3′; human IL-8, forward, 5′-GAC ATA CTC CAA ACC TTT CCA CCC-3′ and reverse, 5′-CCA GAC AGA GCT CTC TTC CAT CAG-3′; and human MCP-1, forward, 5′-CTC ATA GCA GCC ACC TTC ATT C-3′ and reverse, 5′-TCA CAG CTT CTT TGG GAC ACT T-3′. 

Western Blot Analysis

ARPE-19 cells were washed with cold PBS three times and then scraped from the well. The cells were lysed with lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 2 mM EDTA, and 100 μM phenylmethylsulfonylfluoride. The cell lysate was centrifuged, and supernatant was collected. Protein concentration was determined by bicinchoninic acid (BCA) protein assay. All samples were diluted in Laemmli loading buffer and boiled for 5 minutes. Forty-microgram samples were loaded on a 10% SDS-polyacrylamide gel. Prestained markers were used to estimate molecular weight. Proteins were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA), blocked by 5% skim milk or 5% bovine serum albumin (BSA)–Tris-buffered saline supplemented with Tween 20 (TBST), and incubated overnight at 4°C with antibodies against p38 MAPK, JNK, ERK1/2, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphorylated p38 MAPK, phosphorylated JNK, or phosphorylated ERK1/2 followed by a horseradish peroxidase–conjugated secondary antibody at 37°C for 1 hour. The membranes were further developed by ECL (Pierce Chemical). 

Statistical Analysis

One-way ANOVA and paired-sample t-test were applied using SPSS 12.0. Data were expressed as means ± SD. A P value of <0.05 was considered significant for all experiments. 

Results

Effect of BBR on Viability of ARPE-19 Cells

The chemical form of BBR used in this study was BBR hydrochloride (Fig. 1A). Confluent ARPE-19 cells were cultured with different concentrations of BBR to evaluate its effect on the viability of these cells. The results showed that concentrations of 5, 10, 25, or 50 μM BBR at 24, 48, or 72 hours did not influence the viability of ARPE-19 cells compared to the viability of controls. BBR at a concentration of 100 or 200 μM, however, significantly inhibited cell viability (Fig. 1B). Trypan blue dye exclusion assay was used to verify the viability range of increasing doses of BBR on ARPE-19 cells (Fig. 1C). Meanwhile, the same experiment was conducted in subconfluent cells, which showed that at a concentration of 25 μM, BBR cells also remained viable. A concentration of 25 μM was therefore used in subsequent experiments to study the effect of BBR on the production of IL-6, IL-8, and MCP-1 by ARPE-19 cells and on the phosphorylation of p38, ERK1/2, and JNK pathways. 

Figure 1.

 

(A) Chemical structure of BBR. (B) Cell viability as measured by CCK-8 in ARPE-19 cells cultured in the presence of various concentrations of BBR for 24, 48, and 72 hours. (C) The results were confirmed by trypan blue dye exclusion assay at 48 hours. *P < 0.05 and **P < 0.01 for comparison with control and BBR-treated ARPE-19 cells. Each point corresponds to the mean ± SD of three independent experiments.

BBR Inhibits the Production of IL-6, IL-8, and MCP-1 by ARPE-19

In order to examine the effect of BBR on cytokine production, we first tested the expression of IL-6, IL-8, and MCP-1 by ARPE-19 cells after coculture with TNF-α for different time periods. The results showed that TNF-α could significantly increase the protein levels of IL-6, IL-8, and MCP-1 at 8, 12, 24, and 48 hours following stimulation (Figs. 2A, 2B, 2C). The mRNA expression of these three cytokines was increased at 1, 2, and 4 hours following TNF-α stimulation (Fig. 3). 

Figure 2.

 

BBR inhibits the production of cytokines by ARPE-19 cells. ARPE-19 cells in serum-free medium were preincubated for 30 minutes with BBR (25 μM) or 0.05% DMSO. After TNF-α (10 ng/mL) was added to the medium, the cells were incubated for 2, 4, 8, 12, 24, or 48 hours. The release of IL-6 (A), IL-8 (B), and MCP-1 (C) in the culture medium was examined using ELISA. (D) Cells were incubated with or without 0.05% DMSO for 24 hours. The data are expressed as means ± SD of three independent experiments. *P < 0.05 and **P < 0.01 compared to the value without BBR.

Figure 3.

 

BBR inhibits IL-6, IL-8, and MCP-1 mRNA expression by ARPE-19 cells following stimulation with TNF-α. ARPE-19 cells in serum-free medium were preincubated for 30 minutes with BBR (25 μM) or 0.05% DMSO. After TNF-α (10 ng/mL) was added to the medium, ARPE-19 cells were incubated for 1, 2, and 4 hours. The changes in the IL-6 mRNA (A), IL-8 mRNA (B), and MCP-1 mRNA (C) levels after stimulation with TNF-α are shown. The data are expressed as means ± SD of three independent experiments. *P < 0.05 and **P < 0.01 compared to the value without BBR.

BBR at a concentration of 25 μM was then used to evaluate its effect on cytokine production by these cells. The results showed that it significantly inhibited the expression of IL-6, IL-8, and MCP-1 at both the protein and mRNA level at several time points after coculture (Figs. 2, 3). Further experiments showed that 0.05% DMSO, the vehicle used for BBR, did not affect cytokine production (Fig. 2D). 

The Inhibitory Effect of BBR on Cytokine Expression by ARPE-19 Cells Is Mediated by Down-Regulation of the p38, ERK1/2, and JNK Signaling Pathways

The aforementioned experiment demonstrated a stimulatory effect of TNF-α on cytokine production by ARPE-19 cells. A further study was performed to examine the signaling pathways involved. Phosphorylation of p38, ERK1/2, and JNK was increased following stimulation with TNF-α (Fig. 4). Blocking experiments with SB203580 (p38 inhibitor) showed that it significantly inhibited the production of IL-6, IL-8, and MCP-1. PD98059 (ERK1/2 inhibitor) and SP600125 (JNK inhibitor) inhibited the production of IL-8 and MCP-1 (Fig. 5) but did not detectably affect IL-6 production. The combination of these three inhibitors resulted in a stronger inhibitory effect on IL-8 and MCP-1 than in experiments where each inhibitor was used separately. This was not the case for IL-6. In view of the different pathways identified as involved in the expression of these cytokines, a further study was performed to determine how BBR exerted its role in the modulation of these cytokines. The result showed that BBR significantly down-regulated the phosphorylation of the three pathways investigated (Fig. 6). 

Figure 4.

 

Effect of TNF-α on phosphorylation of p38, ERK, and JNK in ARPE-19 cells. ARPE-19 cells were treated with TNF-α (10 ng/mL) for 1, 2, and 4 hours, and cell lysates were subjected to Western blotting for p38, ERK1/2, and JNK phosphorylation. Right: quantification of the optical density of the bands is shown (*P < 0.05 and **P < 0.01, n = 3).

Figure 5.

 

Effect of MAPK inhibitors and BBR (25 μM) on TNF-α (10 ng/mL)-induced production of IL-6, IL-8, and MCP-1 by ARPE-19 cells. ARPE-19 cells in serum-free medium were preincubated for 30 minutes with MAPK inhibitors, including p38 inhibitor (SB; SB203580), ERK inhibitor (PD; PD98059), and JNK inhibitor (SP; SP600125) alone or in combination and BBR (25 μM). After TNF-α (10 ng/mL) was added to the medium, cells were incubated for 24 hours. The concentrations of IL-6 (A), IL-8 (B), and MCP-1 (C) in the culture medium were examined using ELISA. The data are expressed as means ± SD of three independent experiments. *P < 0.05 and **P < 0.01 compared to the positive controls (cells cultured with TNF-α but without inhibitors). #P < 0.05 and ##P < 0.01 compared to the value with combination of these three inhibitors.

Figure 6.

 

Effect of BBR on the MAPK pathway in ARPE-19 cells. ARPE-19 cells cultured in serum-free medium were preincubated for 1 hour with BBR (25 μM) or 0.05% DMSO. After TNF-α (10 ng/mL) was added to the medium, the ARPE-19 cells were incubated for 4 hours and cell lysates were subjected to Western blotting for p38, ERK, and JNK and phosphorylation of these three pathways. Right: quantification of the optical density of the bands is shown (*P < 0.05 and **P < 0.01, n = 3).

Discussion

BBR, a well-known drug with potential anti-inflammatory properties used in traditional medicine, was shown to markedly down-regulate the expression of IL-6, IL-8, and MCP-1 by ARPE-19 cells. Our study suggests that BBR exerts its inhibitory effect on cytokine production by these cells through down-regulating the phosphorylation of p38, ERK1/2, and JNK signaling pathways. 

BBR has been shown to have anti-inflammatory and immunoregulatory properties. ^37,46–49 BBR has been used to treat trachoma, ^50–52 but whether it can also be used for the treatment of intraocular inflammatory disease is not yet clear and was therefore the rationale for starting this in vitro experimental study. For this purpose, we employed ARPE-19, a cell line with structural and functional properties characteristic of RPE cells, to evaluate the effect of BBR on the production of cytokines critical for inflammation at or around the RPE level. ARPE-19 cells readily secreted IL-6, IL-8, and MCP-1 following TNF-α stimulation. This result is consistent with earlier studies by others. ^15–17 The TNF-α stimulation of RPE cells provided a good in vitro model for investigating the effect of BBR on cytokine production by these cells. In the present study, we showed that BBR was able to significantly inhibit the expression of IL-6, IL-8, and MCP-1 at both the protein and mRNA level. The data suggest that BBR regulates the expression of these cytokines mainly at the gene transcription level. These findings are similar to those observed using colonic epithelial cells, ⁵³ antigen-presenting cells ^36,37 (APCs), kidney cells, ⁵⁴ and macrophages. ^48,55 Collectively, these data suggest that the inhibitory effect of BBR on proinflammatory cytokine production may be a universal phenomenon applicable to most cell types. 

The mechanisms involved in the inhibitory effect of BBR on cytokine production have also been studied earlier by others, and the results suggest that BBR could modulate the MAPK, Janus kinase/signal transducers and activators of transcription (JAK/STAT), nuclear factor–κ-binding factor (NF-κB), and AMP-activated protein kinase (AMPK) signaling pathways in experimental type I diabetes, experimental autoimmune encephalomyelitis (EAE), and obese db/db mice. ^36,37,48 In the present study, we focused on the involvement of MAPK in the cytokine modulation in ARPE-19 cells and the influence of BBR on MAPK pathways. We found that p38, ERK, and JNK were all involved in the modulation of the cytokines tested. Interestingly, we showed that IL-6 expression was only modulated by the p38 signaling pathway following TNF-α stimulation. This finding is in agreement with an earlier report using hydrogen peroxide (H₂O₂) stimulation. ⁴³ Our results also confirm earlier reports that showed that IL-8 and MCP-1 production by RPE cells was modulated by p38 and ERK signal transduction pathways. ¹⁵  

It is worthwhile to point out that there are some limitations of our study. Several signal transduction pathways have been shown to be involved in the development of inflammation and the immune response. ^{36,37,48,56–58} The effects of BBR on other relevant pathways were not addressed in this study. Furthermore, we did not investigate whether there was a protective effect of BBR on the breakdown of the RPE barrier, an important event during inflammation at or around the RPE cell layer. 

The ARPE-19 cells we used may have their limitations, and their behavior depends strongly on the specific culture conditions used. The high serum concentrations we used to grow the cells may not have favored the naïve RPE behavior of the cells. Further BBR confirmatory studies are needed using other culture conditions combined with assays evaluating naïve RPE function. Furthermore, it would be interesting to study the effect of BBR on cytokine production by primary human RPE cells. 

The concentration of BBR used in this study is generally higher than that in serum ^59,60 or in the hippocampus ⁶⁰ following intravenous injection or oral administration. It is not yet known whether it is toxic for tissues or organs in vivo at this concentration. Finally, we are not sure whether the observed inhibitory effect of BBR on the cytokine production in vitro can also be extrapolated to the in vivo situation. Oral administration of BBR in a rat model of lipopolysaccharide (LPS)-induced uveitis, however, did show that this treatment resulted in a dose-dependent inhibition of MCP-1 and cytokine-induced neutrophil chemoattractant 1 (CINC-1) expression by iris/ciliary body cells. ⁴⁹ This latter study did not address the effect of BBR on retinal inflammation. Other models of experimental retinal inflammation, such as experimental autoimmune uveitis, laser-induced choroidal neovascularization (CNV), or experimental diabetes, could be used to study the effect of BBR on the RPE-mediated control of posterior segment eye disease. 

In summary, our study revealed an inhibitory effect of BBR on the production of IL-6, IL-8, and MCP-1 by ARPE-19 cells following TNF-α stimulation. Importantly, we found that this effect was mediated, at least partially, by down-regulation of the p38, ERK, and JNK pathways. These results suggest that BBR may possibly be used in modulating the inflammation mediated by these cytokines released from RPE cells. 

Footnotes

 Supported by the National Basic Research Program of China (973 Program) (2011CB510200), the National Natural Science Foundation Project (30973242), the Key Project of Natural Science Foundation (81130019), the Project of Medical Science and Technology of Chongqing, the Key Project of Health Bureau of Chongqing, the Chongqing Key Laboratory of Ophthalmology (CSTC, 2008CA5003), the Program for the Training of a Hundred Outstanding S&T Leaders of Chongqing Municipality, and the Fund for PAR-EU Scholars Program.

Footnotes

 Disclosure: Q. Wang, None; J. Qi, None; R. Hu, None; Y. Chen, None; A. Kijlstra, None; P. Yang, None