Effect of berberine on lipopolysaccharide-induced monocyte chemotactic protein-1 and interleukin-8 expression in a human retinal pigment epithelial cell line

Purpose In this study, we elucidated the effects of berberine, a major alkaloid component contained in medicinal herbs, such as Phellodendri Cortex and Coptidis Rhizoma, on expression of monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8) in a human retinal pigment epithelial cell line (ARPE-19) caused by lipopolysaccharide (LPS) stimulation. Methods ARPE-19 cells were cultured to confluence. Berberine and LPS were added to the medium. MCP-1 and IL-8 mRNA were measured by real-time polymerase chain reaction. MCP-1 and IL-8 protein concentrations in the media were measured using enzyme-linked immunosorbent assay. Results After stimulation with LPS, MCP-1 and IL-8 mRNA in ARPE-19 cells reached maximum levels at 3 h, and MCP-1 and IL-8 protein in the culture media reached maximum levels at 24 h. Berberine dose-dependently inhibited MCP-1 and IL-8 mRNA expression of the cells and protein levels in the media stimulated with LPS. Conclusions These findings indicate that berberine inhibited the expression of MCP-1 and IL-8 induced by LPS.


Materials and methods
Cell culture ARPE-19, a human RPE cell line, was purchased from American Type Culture Collection (No. CRL-2302, Rockville, MD, USA). The cells were maintained in Dulbecco's modified essential medium and Ham's F12 (DMEM/F12; 1:1; Gibco-BRL, NY, USA) supplemented with 10% fetal bovine serum (FBS); penicillin, 100 U/ml; and streptomycin, 100 lg/ml, to obtain confluent cells. All cells were cultured at 37°C under 10% CO 2 and 90% moist air. Media were changed twice a week. The viability of ARPE-19 cells after incubation was assessed using trypan blue dye.
Total RNA extraction and MCP-1 and IL-8 mRNA expression by real-time polymerase chain reaction Berberine was purchased from Sigma Chemicals (St. Louis, Mo., USA). The molecular weight of the alkaloid was 371.8, and the chemical structure is shown in Fig. 1. Berberine was dissolved in 50 mM dimethyl sulfoxide (DMSO) just before use. The final concentration of DMSO was kept at B0.05% in the culture media to avoid its inhibitory effects on the proliferation of the ARPE-19 cell line.
ARPE-19 cells were planted and cultured in 6-well culture plates (averaging 2.0 9 10 6 cells/well) for 14 days in DMEM/F 12 medium containing 10% FBS. After the cells were washed twice with serum-free medium, ARPE-19 cells were preincubated for 2 h in serum-free medium with 0.05% DMSO or 0.2, 1, 5, and 25 lM of berberine for 30 min. Then, LPS (Escherichia coli, serotype 055:B5, Sigma-Aldrich, St Louis, Mo., USA) was added to the medium and incubated. Total RNA was extracted from the cells using RNeasy Protect Mini Kit (Qiagen, Hilden, Germany) and treated with RNase-free DNase Set (Qiagen, Hilden, Germany) to remove any residual genomic DNA. One microgram of each total RNA was reverse transcribed using random hexamers, Mul-tiScribe reverse transcriptase (Applied Biosystems, Branchburg, NJ, USA), and thermal cycler (Gene Amp PCR System 2400; Applied Biosystems, Fig. 1 Chemical structure of berberine Norwalk, CT, USA). Condition of the reverse transcription included incubation at 25°C for 10 min, reverse transcription at 48°C for 30 min, and reverse transcriptase inactivation at 95°C for 5 min.
cDNA was used to detect real-time PCR products for MCP-1 and IL-8 using TaqMan Universal Master Mix (Applied Biosystems, Branchburg, NJ, USA) and ABI PRISM TM 7700 Sequence Detector (Applied Biosystems, Foster, CA, USA) with TaqMan Ò Pre-Developed Assay Reagents (Applied Biosystems, Foster, CA, USA) for human MCP-1 and IL-8. The thermal profile for each primer consisted of 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. To compare expression patterns, mRNA template concentrations for glyceraldehyde-3phosphate dehydrogenase (GAPDH) and the target genes were calculated using the standard curve method. The expression levels of MCP-1 and IL-8 mRNA were normalized by GAPDH mRNA level in each sample, and the changes were expressed as an n-fold relative to the value of cells untreated with LPS.
MCP-1 and IL-8 protein concentrations by enzyme-linked immunosorbent assay (ELISA) ARPE-19 cells were seeded in 24-well culture plates (averaging 2.0 9 10 5 cells/well) and incubated for 14 days in DMEM/F 12 medium containing 10% FBS. The cells were washed twice with serum-free medium and incubated in serum-free medium for 2 h. ARPE-19 cells were preincubated in serum-free medium with 0.05% DMSO or 0.2, 1, 5, and 25 lM of berberine for 30 min. Then, LPS was added to the medium, and it was incubated for 24 h. Thereafter, the supernatant was collected and stored at -70°C until assay. MCP-1 and IL-8 protein concentrations were determined using ELISA (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK) and were calculated based on standard curves using concentrations of recombinant MCP-1 and IL-8 in the ranges of 51-2000 and 25-1000 pg/ml, respectively. All assays were performed in duplicate.

Statistical analysis
The results were expressed as mean values ± standard errors. Statistical analysis was performed using the Scheffe's procedure for multiple comparisons of mean value. P \ 0.05 was considered statistically significant.

Discussion
Dunn et al. [13] have demonstrated changes in gene expression and function with time of confluence in ARPE-19 cells with a mature epithelial phenotype require as much as 2-4 weeks of culture. In the present study, therefore, ARPE-19 cells cultured for 14 days after planting were used.  In the our present findings, 0.2, 1, and 5 lg/ml LPS dose-dependently stimulated MCP-1 mRNA and IL-8 mRNA, and 5 lg/ml LPS showed had no significant difference compared with 1 lg/ml LPS in ARPE-19 cells. Therefore, the inhibitory effects of berberine were determined after stimulation of 1 lg/ml LPS in the present study. In the present study, after stimulation with LPS (1 lg/ml), MCP-1 and IL-8 mRNA in ARPE-19 cells reached peak levels at 3 h, and MCP-1 and IL-8 protein in the culture media reached peak levels at 24 h. Therefore, the inhibitory effects of berberine on the MCP-1 and IL-8 mRNA were determined after stimulation of LPS at 3 h, and on the MCP-1 and IL-8 protein were determined after stimulation of LPS at 24 h.
Therefore, the treatment of these eye diseases requires the suppression of MCP-1 and IL-8 expression.
In China, berberine (5-20 mg/kg per day) has long been used in the treatment of diarrhea and gastrointestinal disorders [27,28]. In Japan, berberine alone is not used clinically. Mixed extracts from multiple herbs, which contain several alkaloids, are administered orally. For example, Oren-gedoku-to (Huang-Lian-Jie-Du-Tang in Chinese), which contains extracts from Scutellariae sp. radix, Coptidis sp. rhizoma, Gardeniae sp. fructus, and Phellodendri sp. cortex, is prescribed for treatment of gastritis. Orengedoku-to (1.5 g) usually contains berberine (240 mg). Kong et al. [42] reported that oral administration of berberine (0.5 g twice per day) for 3 months lowers serum low-density lipoprotein cholesterol in hypercholesterolemic people. The alkaloid also reported has the other pharmacological actions, including glucose-lowering potential [44][45][46][47].  We previously reported that berberine inhibited the in vivo expression of MCP-1 and IL-8 induced by LPS, decreasing the inflammatory cell infiltration and the levels of protein and cells in the aqueous humor in rat [36]. In the present study, we found that the alkaloid significantly inhibited the in vitro expression of MCP-1 and IL-8 induced by LPS in a human retinal pigment epithelial cell line. Therefore, an effect of berberine in the chemokine-mediated disorders may be expected.