Anti‑inflammatory role of Prunus persica L. Batsch methanol extract on lipopolysaccharide‑stimulated glial cells.

Glial cells are the resident immune cells of the central nervous system. Reactive glial cells release inflammatory mediators that induce neurotoxicity or aggravate neurodegeneration. Regulation of glial activation is crucial for the initiation and progression of neuropathological conditions. Constituents of the peach tree (Prunus persica L. Batsch), which has a global distribution, have been found to exert therapeutic effects in pathological conditions, such as rashes, eczema and allergies. However, the therapeutic potential of its aerial parts (leaves, fruits and twigs) remains to be elucidated. The present study aimed to evaluate the anti‑inflammatory role of P. persica methanol extract (PPB) on lipopolysaccharide (LPS)‑stimulated glial cells. High‑performance liquid chromatography coupled with tandem mass spectrometry analysis showed that PPB contained chlorogenic acid and catechin, which have antioxidant properties. Western blot and reverse transcription polymerase chain reaction results indicated that PPB reduced the transcription of various proinflammatory enzymes (nitric oxide synthase and cyclooxygenase‑2) and cytokines [tumor necrosis factor‑α, interleukin (IL)‑1β and IL‑6] in LPS‑stimulated BV2 cells. In addition, PPB inhibited the activation of NF‑κB and various mitogen‑activated protein kinases required for proinflammatory mediator transcription. Finally, nitrite measurement and immunocytochemistry results indicated that PPB also suppressed nitrite production and NF‑κB translocation in LPS‑stimulated primary astrocytes. Thus, PPB may be used as a potential therapeutic agent for neurodegenerative diseases and neurotoxicity via the suppression of glial cell activation.

Introduction

Glial cells are immune cells that reside in the central nervous system, and they have an important role in numerous neurodegenerative diseases, including Alzheimer's (AD), Parkinson's and Huntington's disease (1,2). Glial cells are normally in a resting state; however, they are activated in response to an inflammatory stimulus. Despite comprising <10% of the cells in the brain, the microglia regulate the neuroimmune system (3). Reactive microglia are observed in several neuroinflammatory conditions, including ischemia, brain injury and infection (4). These glial cells aggravate neurodegenerative diseases by secreting inflammatory mediators or proinflammatory cytokines that induce neurotoxicity (5–7). In addition, they release soluble toxins, which induce reactive A1 astrocytes that are neurotoxic (8). Similarly, astrocytes, which are the most abundant brain cell types, cause several brain inflammatory conditions by secreting proinflammatory mediators in response to a neuroinflammatory stimulus (9,10). Elevated nitric oxide (NO) levels have been observed in glial cells of patients with AD or ischemia (11,12). In addition, reactive glial cells promote the production of tumor necrosis factor (TNF) α, interleukin (IL) 1β, and IL-6, which contribute to the development of neuroinflammatory conditions and brain damage (13–15). Therefore, reactive glial cell regulation is crucial for the initiation and progression of neurodegenerative diseases and neuronal cell death (4,16,17).

Plant-based anti-inflammatory compounds may be potential sources of safe and effective drugs (18,19). Traditional herbal medicines derived from natural products and their active constituents have been extensively studied, and several ingredients from natural products have been found to be beneficial for the treatment of neuroinflammatory and neurodegenerative diseases, given their ability to suppress glial activation (20–22). Panax ginseng (23), Curcuma longa (24), and Camellia sinensis (25) display neuroprotective effects by suppressing neuroinflammatory cytokine secretion via glial cell activation. In addition, natural products are safe, inexpensive and easy to obtain. Therefore, the discovery of a novel natural product that can regulate glial activation is crucial for the development of neurodegenerative disease treatments (26).

The peach tree (Prunus persica L. Batsch) was initially reported as a deciduous tree native to the northwest region of China. It is now grown worldwide, including the temperate regions of eastern Asia, including Vietnam, China, Japan and Korea (27). Various parts of P. persica have therapeutic effects. The dried seeds of ripened P. persica fruit are widely used in traditional medicine (Persicae Semen) in Korea and China, and persicaside, an alkaloid compound derived from P. persica seeds, inhibits the production of NO (28). P. persica flowers are used for the treatment of rashes and eczema (29). The flavonoid compounds 4-O-caffeoylquinic acid, quercetin-3-O-rhamnoside and kaempferol glycoside, which are P. persica flower derivatives, have anti-inflammatory properties (30). In addition, P. persica fruit has anti-allergenic properties (31). P. persica roots can suppress the growth of liver cancer cells (27), and its leaves have antioxidant and antibacterial properties (32–34). However, the antioxidant properties of P. persica leaves have only been evaluated with in vitro analysis (e.g., 2,2-diphenyl-1-picrylhydrazyl assay) (32,33). Therefore, in vivo biological evidence is required.

The present study aimed to investigate the anti-inflammatory effects of P. persica aerial parts (leaves, fruits and twigs) on glial cells. P. persica methanol extract (PPB) was applied to BV2 cells and primary astrocytes. PPB inhibited the production of proinflammatory mediators and cytokines in lipopolysaccharide (LPS)-stimulated BV2 cells by suppressing NF-κB translocation and mitogen-activated protein kinase (MAPK) signaling pathways. Furthermore, PPB also inhibited NO production and NF-κB translocation in cultured primary astrocytes.

Materials and methods

Materials

DMEM and FBS were purchased from Gibco (Thermo Fisher Scientific, Inc.). Chlorogenic acid, catechin, quercetin, quercetin-3-O-glucoside, formic acid, diclofenac, and LPS (Escherichia coli O55:B5) were purchased from Sigma-Aldrich (Merck KGaA). Antibodies against inducible nitric oxide synthase (iNOS; cat. no. 610431; BD Biosciences), cyclooxygenase-2 (COX-2; 1:2,000; cat. no. sc-376861; Santa Cruz Biotechnology, Inc.), ERK [1:2,000; cat. no. 9102S; Cell Signaling Technology, Inc. (CST)], phosphorylated (p)-ERK (1:1,000; cat. no. 9101S; CST), JNK (1:2,000; cat. no. 9258S; CST), p-JNK (1:1,000; cat. no. 9251S; CST), p38 (1:2,000; cat. no. 9212S; CST), p-p38 (1:1,000; cat. no. 9211S; CST), inhibitor of (I)κBα (1:2,000; cat. no. 9242S; CST), p-IκBα (1:1,000; cat. no. 2859S; CST), and β-actin (1:5,000; cat. no. sc-47778; Santa Cruz Biotechnology, Inc.) were used for western blot analysis. Horseradish peroxidase (HRP)-conjugated secondary antibodies for western blots, goat anti-mouse IgG (1:5,000; cat. no. 1706516; Bio-Rad Laboratories, Inc.) and goat anti-rabbit IgG (1:5,000; cat. no. 1706515; Bio-Rad Laboratories, Inc.) were used. For immunocytochemistry, p65 (1:500; cat. no. 8242S; CST Biological Reagents Co., Ltd.) and glial fibrillary acidic protein (GFAP; 1:500; cat. no. MAB3402; Merck KGaA) were used as primary antibodies, and Alexa Fluor 488 donkey anti-mouse IgG (1:500; cat. no. ab150105; Abcam) and Alexa Fluor 594 donkey anti-rabbit IgG (1:500; cat. no. ab150076; Abcam) were used as secondary antibodies.

Preparation of PPB

In 2008, P. persica was obtained from various regions in Vietnam (Bằng Lũng, Chợ Đồn, and Bắc Kạn). The samples were authenticated by the chief executive of the Institute of Ecology and Biological Resources. It is characterized by 4–7 m tall, dark green, deciduous leaves and flowers in single, semi-double and double form in colors from white to deep red. A voucher specimen (KRIB0018669, accession number of KRIBB) was deposited in the herbarium of the Korea Research Institute of Bioscience and Biotechnology. P. persica dried and refined aerial parts (leaves, fruits and twigs; total weight, 63 g) were extracted with 600 ml of 99.9% (v/v) methanol and were repeatedly sonicated with 1,500 W input and 40 kHz frequency for 15 min at 2-h intervals for 3 days. All procedures were performed at 45°C. The product was filtered through non-fluorescent cotton and concentrated by rotary evaporation using an N-1000SWD device (EYELA Co., Ltd.) under reduced pressure at 45°C. Finally, 8.05 g of PPB was obtained by freeze-drying and then dissolved in DMSO (Sigma-Aldrich; Merck KGaA).

Contents of the marker substances in PPB

Chlorogenic acid, catechin, quercetin and quercetin-3-O-glucoside were selected as marker substances in PPB based on a previous study (35). The extract contents were measured using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). Briefly, to identify the product ions of each substance, the marker substances (100 ng/ml) were individually infused into the mass spectrometer at a flow rate of 10 µl/min. Precursor ions and fragmentation patterns were monitored in a negative-ion mode. Using an API 4000 LC/MS/MS system (AB SCIEX Analytical Instrument Trading Co.) equipped with an electrospray ionization interface, major peaks in the MS/MS scan were used to quantify the substances. The compounds were separated on a reversed-phase column (Kinetex® C18, 2.6-µm particle size, 100×2.1 mm internal diameter; Phenomenex Ltd.) in the mobile phase of a water:acetonitrile mixture at a 3:7 (v/v) ratio including 0.1% formic acid. The column was heated at 30°C, and the mobile phase was delivered at a flow rate of 0.2 ml/min using an HP 1100 series pump (Agilent Technologies, Inc.). The injection volume was 5 µl. The turbo ion spray interface was operated at −4,200 V at 450°C. Nitrogen was used as a curtain gas at 35 psi, and air dried was nebulized as a collision gas at 4 psi and ion source gases 1 (45 psi) and 2 (55 psi). The ion transitions of the precursor to the product ion were monitored in negative mode as deprotonated ions [M-H]− at m/z 353.0 → 190.8 [declustering potential (DP), −60 eV; collision energy (CE), −22 eV] for chlorogenic acid, 289.0 → 244.8 (DP, −90 eV; CE, −20 eV) for catechin, 301.0 → 151.0 (DP, −100 eV, CE, −32 eV) for quercetin, 463.1 → 300.0 (DP, −120 eV, CE, −36 eV) for quercetin-3-O-glucoside, and 296.1 → 251.7 (DP, −100 eV, CE, −32 eV) for diclofenac (internal standard). Quantification was performed by selective reaction monitoring of deprotonated precursor ions and related product ions using the ratio of the area under the peak for each solution. All analytical data were processed using Analyst software (version 1.5.2; Applied Biosystems; Thermo Fisher Scientific, Inc.). Standard marker substance solutions were serially diluted with methanol including the internal standard (IS, 100 ng/ml) to obtain 10–1,000 ng/ml concentrations. Using linear regression, two calibration graphs were derived from the ratios between the areas under the peaks for each substance and those of the IS. PPB (20 mg/vial) was dissolved in 1 ml of methanol and was serially diluted with methanol including the IS by 1,000-fold. The concentrations of each marker substance were predicted using the calibration graph, and their contents within the extracts were calculated.

Cell culture and treatment

The BV2 cells (CLC catalog code, ATL03001) were provided by Dr Dong-Kug Choi (Konkuk University, Chungju-si, Chungcheongbuk-do, Republic of Korea) and were maintained in DMEM containing 5% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin (Welgene, Inc.) in a humidified incubator with air containing 5% CO2. In all of the experiments, 500,000 cells were plated and treated with PPB (20, 100 and 200 µg/ml) for 1 h prior to LPS treatment (200 ng/ml, cat. no. L2880; Sigma-Aldrich; Merck KGaA), and the vehicle was a DMSO only treatment (0.1%). BV2 cells were treated with LPS for 6 and 24 h for reverse transcriptase polymerase chain reaction (RT-PCR) and western blots, respectively.

Hydrogen peroxide (H2O2, 250 µM) was applied to BV2 cells for 24 h to measure cytotoxicity.

Primary astrocytes were prepared from the cortices of postnatal day 2 Sprague-Dawley rats (7–9 g), as previously described by McCarthy and de Villis (36). Postnatal day 1 Sprague-Dawley rats were purchased (Young Bio) and were maintained for 24 h in controlled temperature (20–25°C), humidity (50–55%) and 12-h light/dark cycle with their mother. A total of 6 animals (3 males and 3 females) were used for this study. All animals were handled in accordance with the Principle of Laboratory Animal Care (37) and the study was approved by the Institutional Animal Care and Use Committee of Chung-Ang University (2017-00093).

Briefly, the astrocytes were plated in poly-d-lysine (PDL)-coated T-75-cm2 flasks in DMEM/F12 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Microglia were removed at 7 days in vitro (DIV), and subcultured astrocytes at 15 DIV were used for further experiments. Primary astrocytes were treated with PPB (20, 100 and 200 µg/ml) for 1 h prior to LPS treatment (10 ng/ml), and the control group was treated with vehicle (DMSO; 0.1%).

Cell viability

Cell viability was determined using the MTT assay. Cells were treated with PPB for 24 h and incubated with MTT (100 µg/ml) for 2 h. DMSO was added to dissolve the insoluble formazan crystals that had formed within viable cells, and the absorbance was measured at 540 nm.

Propidium iodide (PI) staining

BV2 cells, which were protected from the light, were fixed with 70% ethanol at for 30 min at 20–25°C and stained using FxCycle PI/RNase staining solution (cat. no. F10797; Thermo Fisher, Inc.) for 30 min at 20–25°C. Nuclei were stained with DAPI (cat. no. D1306; Invitrogen; Thermo Fisher Scientific, Inc.) for 10 min at 20–25°C. Cell images were visualized using a confocal microscope (Zeiss GmbH).

Nitrite measurement

NO release was measured from nitrite levels using a Griess reagent (0.1% naphthylethylenediamine and 1% sulfanilamide in 5% H3PO4). The supernatant was mixed with an equal volume of Griess reagent for 5 min at 20–25°C. Mixture absorbance was measured at 540 nm using a microplate reader (Tecan Group, Ltd.).

Isolation of total RNA and RT-PCR

Total RNA was isolated from LPS-stimulated cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Reverse transcription was conducted for 5 min at 70°C and 5 min at 4°C with total RNA (1 µg) and GoScript Reverse Transcriptase (Promega Corporation). cDNA was subsequently amplified by RT-PCR with specific primers and GoTaq DNA polymerase (Promega Corporation). The PCR primer sets were as follows: iNOS (sense, 5′-GAGGTACTCAGCGTGCTCCA-3′; antisense, 5′-AGGGAGGAAAGGGAGAGAGG-3′), COX-2 (sense, 5′-TGAGTGGTAGCCAGCAAAGC-3′; antisense, 5′-CTGCAGTCCAGGTTCAATGG-3′), TNF-α (sense, 5′-AGGGAGAGTGGTCAGGTTGC-3′; antisense, 5′-CAGCCTGGTCACCAAATCAG-3′), IL-1β (sense, 5′-CAAGGAGAACCAAGCAACGA-3′; antisense, 5′-TTGGCCGAGGACTAAGGAGT-3′), IL-6 (sense, 5′-GGAGGCTTAATTACACATGTT-3′; antisense, 5′-TGATTTCAAGATGAATTGGAT-3′), and GAPDH (sense, 5′-CCAGTAGACTCCACTCACG-3′; antisense, 5′-CCTTCCACAATGCCAAAGTT-3′). Thermal cycling was performed as follows: Initial denaturation (95°C for 2 min), amplification (95°C for 1 min; 55°C for 1 min and 72°C for 1 min, 35 cycles), and final extension (72°C for 5 min). After amplification, the PCR products were resolved by agarose gel electrophoresis on 1% agarose gel containing 1X TAE (Biosesang, Inc.) and 1X Midori green advance (Nippon Genetics Europe GmbH), and visualized using a LAS-3000 device (FujiFilm). The band intensity was determined using ImageJ software (version 1.38; National Institutes of Health).

Western blot analysis

Lysates were extracted from cells using RIPA buffer (Biosesang, Inc.) and total protein was determined by BCA assay. A total amount of 30 µg of proteins were loaded. Proteins were separated using 10% SDS-PAGE and transferred to nitrocellulose membranes (Whatman; GE Healthcare Life Sciences). The membranes were blocked with 5% skimmed milk (Biosesang, Inc.) in TBS-Tween (0.1% Tween-20) for 1 h at 20–25°C. The membranes were incubated with primary antibodies for overnight at 4°C. Then, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at 20–25°C. The bands were developed using enhanced chemiluminescence solution (WEST-ZOL Plus, iNtRON Biotechnology) and visualized with the LAS-3000 device (FujiFilm). The band intensity was analyzed with ImageJ software (version 1.38; National Institutes of Health).

Immunocytochemistry

Cells were fixed with 4% paraformaldehyde for 15 min at 20–25°C and then permeabilized with 0.1% Triton X-100 for 10 min. Coverslips were incubated with blocking buffer (1% BSA in PBS) for 1 h at 20–25°C and then incubated with primary antibodies for overnight at 4°C. Coverslips were incubated with secondary antibodies for 1 h at 20–25°C, and images were visualized using a confocal microscopy (400× magnification; Carl Zeiss).

Statistical analysis

Data were expressed as mean ± SEM from ≥3 independent experiments. Statistical analyses were performed using GraphPad Prism 5.0 software (version 5.01; GraphPad Software, Inc.). The significance of each group was determined using one-way ANOVA followed by Turkey's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results

Fig. 1 illustrates the mass spectra and proposed fragment ions of the four marker substances (left column), chromatograms of the standard solution (250 ng/ml) of each substance (middle column), and PPB solution that was diluted 1,000-fold (right column). PPB contained 0.193% chlorogenic acid, 0.097% catechin, 0.040% quercetin and 0.035% quercetin-3-O-glucoside.

Figure 1.

Contents of substances in PPB. Mass spectra and proposed fragment ions (left), chromatograms of the 250 ng/ml standard solution (middle), and PPB solution diluted 1,000-fold (right). PPB, Prunus persica methanol extract; cps, counts per second.

Effect of PPB on NO release in LPS-stimulated BV2 cells

To examine the anti-inflammatory role of PPB, the nitrite levels in LPS-stimulated BV2 cells was measured. LPS-induced upregulation of NO production was reduced by PPB treatment in a dose-dependent manner (Fig. 2A). The MTT assay showed that PPB did not have a significant effect on cell viability (Fig. 2B). To further examine the presence of cytotoxicity, PI staining was performed as previously described (38) and quantitatively showed that H2O2, but not PPB, induced cellular toxicity (Fig. 2C). These results suggested that PPB suppressed NO release in LPS-stimulated BV2 cells without inducing cellular toxicity.

Figure 2.

PPB suppressed nitric oxide release in LPS-stimulated BV2 cells. (A) Nitrite was measured in LPS-stimulated BV2 cells. (B) Cell viability was measured with the MTT assay. (C) Cell cytotoxicity was measured with PI staining. H2O2 induces cytotoxicity, and PI-positive cells are magnified (arrow). ###P<0.001 vs. Veh; ***P<0.001 vs. Veh + LPS. PPB, Prunus persica methanol extract; LPS, lipopolysaccharide; PI, Propidium iodide; Veh, vehicle.

Effect of PPB on iNOS, COX-2 and proinflammatory cytokine expression

LPS promoted iNOS and COX-2 protein expression, which was inhibited by PPB (Fig. 3A). PPB attenuated LPS-induced elevation of iNOS and COX-2 mRNA (Fig. 3B). Therefore, PPB inhibits iNOS and COX-2 expression in LPS-stimulated BV2 cells. To further investigate the anti-inflammatory role of PPB, TNF-α, IL-1β and IL-6 mRNA expression levels were investigated. PPB alleviated LPS-induced enhancement of proinflammatory cytokines (Fig. 3C). Notably, PPB significantly inhibited iNOS and COX-2 expression at a lower concentration (100 µg/ml) than the concentration that affected TNF-α, IL-1β, and IL-6 expression (200 µg/ml). The present results may indicate that PPB had preferential inhibitory effects on iNOS and COX-2 compared with TNF-α, IL-1β and IL-6 in LPS-stimulated BV2 cells.

Figure 3.

PPB inhibits proinflammatory mediator expression in LPS-stimulated BV2 cells. (A) iNOS and COX-2 protein expression is normalized with β-actin. (B) mRNA expression levels of iNOS and COX-2. iNOS and COX-2 mRNA expression was normalized with GAPDH. (C) TNF-α, IL-1β and IL-6 protein expression was normalized with GAPDH. ###P<0.001 vs. Veh; *P<0.05, **P<0.01, and ***P<0.001 vs. Veh + LPS. PPB, Prunus persica methanol extract; LPS, lipopolysaccharide; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2; TNF, tumor necrosis factor; IL, interleukin; Veh, vehicle.

Effect of PPB on LPS-induced NF-κB pathways

NF-κB is a major transcription factor that regulates the expression of proinflammatory cytokines during inflammation. In response to inflammatory stimuli, IκB is rapidly phosphorylated and degraded, and subsequently dissociates from NF-κB, which translocates to the nucleus, leading to the transcription of proinflammatory mediators and cytokines (39,40). To clarify the underlying mechanism of the anti-inflammatory effects of PPB, the NF-κB signaling pathway was investigated. Normally, the level of IκBα expression is high, whereas that of p-IκBα expression is low. In the present study, LPS exposure decreased IκBα expression and enhanced p-IκBα expression (Fig. 4A). PPB ameliorated the LPS-induced alteration of IκBα and p-IκBα expression. To further examine the effect of PPB on p65 translocation, the expression of p65 was investigated by immunostaining. As expected, PPB suppressed LPS-induced p65 translocation into the nucleus (Fig. 4B). The present results indicated that PPB regulates proinflammatory cytokine expression by suppressing the NF-κB signaling pathway.

Figure 4.

PPB suppresses LPS-induced NF-κB activation. (A) Representative images of p-IκBα and total IκBα protein expression. PPB inhibits LPS-induced p-IκBα enhancement. p-IκBα is normalized with IκBα. (B) Immunostaining of p65. Arrow indicates p65 translocation into the nucleus. PPB (200 µg/ml) inhibited LPS-induced p65 translocation into the nucleus. ###P<0.001 vs. Veh; ***P<0.001 vs. Veh + LPS. PPB, Prunus persica methanol extract; LPS, lipopolysaccharide; NF, nuclear factor; p-, phosphorylated; I, inhibitor; Veh, vehicle.

Effect of PPB on LPS-induced MAPK activation

In microglia, LPS exposure enhances iNOS and TNF-α expression, which is accompanied by the activation of MAPK signaling pathways, including the ERK, JNK and p38 pathways (16). In addition, MAPK is involved in COX-2 and iNOS production in LPS-stimulated microglial cells (41). To further examine the underlying mechanism of PPB, MAPK activation was investigated. PPB repressed LPS-induced phosphorylation of ERK, JN and p38 (Fig. 5). Notably, PPB significantly suppressed the JNK phosphorylation at a lower concentration (20 µg/ml) than that for ERK or p38 (100 µg/ml). The present results indicated that PPB preferentially inhibited JNK in LPS-stimulated BV2 cells. Taken together, the present results demonstrated that PPB exerts an anti-inflammatory effect in LPS-stimulated BV2 cells through the inhibition of NF-κB and MAPK pathway activation.

Figure 5.

PPB inhibits MAPK activation in LPS-stimulated BV2 cells. Representative images of total and p-MAPK. p-MAPK is normalized with total MAPK. #P<0.05, ###P<0.001 vs. Veh; **P<0.01, ***P<0.001 vs. Veh + LPS. PPB, Prunus persica methanol extract; LPS, lipopolysaccharide; p-, phosphorylated.

Effect of PPB on NO release and p65 translocation in LPS-stimulated primary astrocytes

To further evaluate the suppressive effect of PPB on neuroinflammation, NO production in primary cultured astrocytes was investigated. Consistent with the BV2 cell results, PPB also suppressed LPS-induced NO production without inducing cellular toxicity (Fig. 6A and B). In addition, immunostaining results indicated that PPB successfully alleviated LPS-induced p65 translocation into the nucleus (Fig. 6C). The present results indicated the anti-inflammatory role of PPB in glial cell activation by repressing repressed NO production and p65 translocation in primary astrocytes (Fig. S1).

Figure 6.

PPB inhibits nitrite production and p65 translocation in LPS-stimulated primary astrocytes. (A) Nitrite production in primary astrocytes. Astrocytes were treated with PPB and subsequently treated with LPS (10 ng/ml). ###P<0.001 indicates a significant difference with the vehicle without LPS. ***P<0.001 indicates a significant difference in LPS treatment. (B) Cell viability measurement with the MTT assay. (C) Immunostaining of p65. Cultured astrocytes were stained with GFAP, and PPB (200 µg/ml) inhibited LPS-induced p65 translocation into the nucleus. Magnified images indicate p65 translocation into the nucleus (arrow). PPB, Prunus persica methanol extract; LPS, lipopolysaccharide; GFAP, glial fibrillary acidic protein; Veh, vehicle.

Discussion

The present study indicated that PPB presents anti-inflammatory effects on LPS-stimulated glial cells. PPB significantly decreased pro-inflammatory mediators and cytokines at the transcriptional level, which was accompanied by NF-κB and MAPK activation suppression in LPS-stimulated microglial cells. The present in vitro findings suggested PPB repressed NO production and p65 translocation in primary astrocytes.

Although iNOS is rarely expressed under normal conditions, it is highly expressed in activated glial cells under neuroinflammatory conditions (42). In contrast to other NOS enzymes in the brain, iNOS exerts neurotoxic effects through continuous NO production due to its calcium independence (43). Therefore, iNOS regulation is key to suppressing NO production in the presence of a neuroinflammatory stimulus. The inhibitory effect of PPB on NO release was examined via the suppression of iNOS expression. In addition, the expression levels of COX-2, which is the most representative proinflammatory mediator during an inflammatory response, was examined (44).

To clarify the protective role of PPB in glial cells, the LPS-induced neuroinflammation model was used. Peripheral LPS administration has not been effective in the brain because LPS could not penetrate the blood-brain barrier (BBB) (45). However, previous studies have indicated that LPS can induce neuroinflammation in the brain with minimal penetration (46) or BBB disruption (47). In previous studies, LPS treatment was considered a representative neuroinflammation model for several brain inflammatory cells, such as primary cultured astrocytes (48), microglia (49), BV2 cells (16) and C6 glioma cells (50). Therefore, the results of the present study suggested that PPB has suppressive effects in response to neuroinflammation.

Microglial cell activation increases the levels of certain neurotoxic and proinflammatory mediators and induces neuronal cell death, leading to various neuroinflammatory diseases (9,16,17). Thus, the regulation of microglial cell activity alleviates neuroinflammation, which may further inhibit neuronal cell death. NF-κB, a transcription factor, regulates the production of multiple genes related to immunity and inflammation (51). NF-κB activation induces the transcription of proinflammatory genes through a variety of intracellular signaling pathways (52). In the cytoplasm, NF-κB is a heterotrimeric complex consisting of p50, p65, and IκB subunits (39). Activation of this complex results in IκB phosphorylation and degradation, which leads to the translocation of NF-κB dimers (p50/p65 complex) to the nucleus as well as the activation of target genes, including proinflammatory cytokines (40). Thus, regulating NF-κB translocation is important in inflammatory diseases. The inhibitory activity of PPB may affect this signaling by acting as a suppressor of inflammatory mediators. Indeed, PPB inhibits IκB phosphorylation and p65 expression in the nucleus of LPS-stimulated BV2 cells and astrocytes. These findings suggest that the repression of inflammatory mediators by PPB is caused by NF-κB signaling pathway inhibition.

MAPK signaling pathways (i.e., ERK, JNK and p38 MAPK) can regulate NF-κB activity in LPS-stimulated cells and participate in COX-2 and iNOS production (53). In in vivo and in vitro experiments, MAPK signaling pathways induce the activation of transcription factors and proinflammatory cytokines (54,55). Activated ERK pathways induce COX-2 expression and PGE2 production in microglia (56). In primary astrocytes and microglia, LPS not only increases iNOS and TNF-α expression, but also induces MAPK activation (57). Consistent with these studies, the results of the present study showed that PPB inhibited MAPK activation in LPS-stimulated BV2 microglial cells, suggesting that its anti-inflammatory effects were also due to MAPK signaling pathway inhibition.

Epidemiological studies have suggested that phenolic compounds and carotenoid from peach pulps and peel decrease the risk of cardiac disorders, brain disorders and cancer because of their antioxidant and anti-inflammatory effect (58,59). In addition, P. persica flower extract suppressed inflammation in LPS-simulated macrophages (60). It was hypothesized that the antioxidant effect of P. persica is derived from phenolics, flavonoids, and anthocyanins (58). Previous studies evaluating P. persica phenolic and flavonol content showed that chlorogenic acid and catechin were enriched in the peels and pulps of the fruit from P. persica cultivars (35). Likewise, PPB contained chlorogenic acid, catechin, quercetin, and quercetin-3-O-glucoside. This finding suggests that the anti-inflammatory effect of PPB in BV2 cells was due to the presence of phenolics and flavonols. In a previous study, chlorogenic acid suppressed glucose-induced glial cell activation (61). Moreover, neochlorogenic acid, an isomer of chlorogenic acid, inhibited the activation of LPS-induced microglia and p38 MAPK (62). Catechin also inhibited LPS-induced NO production in BV2 cells (63). In addition, the catechin flavonoid, epigallocatechin gallate, exerts a neuroprotective effect by suppressing glial cell activation induced by LPS (25) or β-amyloid (64). The present results indicated that the application of chlorogenic acid, catechin, or their combination may suppress the activation of glial cells. To further confirm whether the anti-inflammatory effect of PPB is due to the presence of chlorogenic acid or catechin, each bioactive molecule should be explored in future studies. In addition, future studies should also investigate whether PPB has a greater anti-inflammatory effect than chlorogenic acid or catechin alone due to the synergistic effect of bioactive molecules. Although results from BV2 cells or primary astrocytes may not reflect in vivo conditions, these findings suggest that PPB is a potential therapeutic agent for neuroinflammatory and neurodegenerative disease treatment via the repression of glial cell activation.