Background
Although inflammation is known to be a defense mechanism against noxious stimuli, abnormal inflammatory response causes a variety of human diseases such as obesity [
1], cardiovascular [
2] and neurodegenerative disease [
3], cancer [
4] and osteoporosis [
5].
Of the various inflammatory mediators, nitric oxide (NO) contributes to anti-inflammatory activity in normal physiological conditions [
6], but excessive NO production is thought to cause chronic inflammation in abnormal situation, which indicates that NO is a major molecule that plays a key role in the pathogenesis of inflammatory disorders [
6]. Since inducible nitric oxide synthase (iNOS) is involved in the synthesis of NO, inhibition of iNOS expression has been regarded as an important molecular target for anti-inflammatory action [
7,
8]. In addition to NO, prostaglandin E
2 (PGE
2) produced by cyclooxygenase-2 (COX-2) is also associated with the progression of the inflammatory diseases induced by chronic inflammation [
9]. Therefore, suppression of NO and PGE
2 production through inhibition of iNOS and COX-2 expression, respectively has been thought to be important targets for the treatment of inflammatory diseases [
6,
10]. The inflammatory mediators such as NO, PGE
2, iNOS, COX-2 and IL-1β have been known to be closely related to the pathogenesis of osteoporosis in the human inflammatory diseases [
11].
For the evaluating the pharmacological activity of plants, the choice of plant species has been usually determined by the fact that it is already used for some purpose.
Vaccinium oldhamii Miquel (
V. oldhamii) native in Korea has been used to treat gonorrhea, vomiting, diarrhea, eruption and inflammation [
12]. The fruit of
V. oldhamii has been reported to exert anti-oxidant and anti-cancer activity [
13]. In addition,
V. oldhamii inhibits α-amylase and acetylcholinesterase [
12,
14]. The fruit of
V. oldhamii is considered to be an important resource for the development of new blueberry cultivars [
13] because it has higher antioxidant activity than blueberries [
15]. The contents of anthocyanin and polyphenol from the fruit of
V. oldhamii have been reported to be higher than those of southern highbush blueberry and northern highbush blueberry [
13]. In addition,
V. oldhami leaves have been reported to inhibit NO production in LPS-stimulated RAW264.7 cells [
16].
Although the anti-inflammatory activity of V. oldhamii have been reported, it is still insufficient. Thus, in this study, we compared the anti-inflammatory activity of the plant parts of V. oldhamii such as stems, leaves and fruits. In addition, we investigated the mechanism of action on anti-inflammatory activity of the stems with the highest anti-inflammatory activity.
Materials and methods
Materials
Dulbecco’s Modified Eagle medium (DMEM)/F-12 1:1 Modified medium (DMEM/F-12) for cell culture was purchased from Lonza (Walkersville, MD, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,2-diphenyl-1-picrylhydrazyl (DPPH), tolfenamic acid (TA), tartrate-resistant acid phosphatase (TRAP) solution and lipopolysaccharide (LPS) for inflammation induction was purchased from Sigma Aldrich (St. Louis, MO, USA). Antibodies against iNOS (#13120), COX-2 (#12282), IκB-α (#4814), p65 (#8242), phospho-ERK1/2 (#4377), ERK1/2 (#9102), phospho-p38 (#4511), p38 (#9212), phospho-JNK (#4668), JNK (#9258), p-ATF2 (#9221), ATF2 (#35031) and β-actin (#5125) were purchased from Cell Signaling (Bervely, MA, USA). Antibodies such as NFATc1 (#556602) and c-Fos (SC-52) were purchased from BD Pharmingen (San Diego, CA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively.
The extraction of
V. oldhami (VO) was carried out according to the literatures with some modification [
13,
16]. VO (voucher number: Jeong 201,802 (ANH)) was generously provided from Forest Medicinal Resources Research Center, National Institute of Forest Science, Yongju, Korea. VO was formally identified by Ho-Jun Son a researcher of Forest Medicinal Resources Research Center, Korea. Five grams of the stems, leaves and fruits from VO were extracted with 100 ml of 70% ethanol for 72 h under stirring at room temperature. After 72 h, the ethanol extracts were filtered and concentrated to approximately 30 ml volume using a vacuum evaporator and then freeze-dried. The ethanol extracts from the stems (VOS), leaves (VOL) or fruits (VOF) of VO were kept in a refrigerator until use.
The analysis of anti-inflammatory compounds from VOS was performed using GC/MS and HPLC. In GC/MS analysis, Agilent 6890 GC interfaced to an Agilent 5973 MS equipped with an EI source and autoinjector (Agilent Technologies, Santa Clara, CA, USA) was used. The GC system was equipped with a HP-5 column (30.0 m × 0.25 mm × 0.25 μm). The oven temperature was 70 °C (5 min) and raised to 290 °C (5 min) at 5 °C/min, and injection volume was 1 μl. The injection was performed in the split mode adjusted to 1:5. The carrier gas was helium at 1.0 ml/min. Inlet, source and quadrupole temperatures were set at 290, 230 and 190 °C, respectively. For MS detection, the electron ionization mode with an ionization energy of 70 eV was used with a mass range at m/z 50–550. Agilent ChemStation software was used for data processing. Anti-inflammatory compounds from VOS were identified by mass fragmentation patterns compared by using Wiley Spectral library search program. In HPLC analysis, Waters 1525 system with a Waters 2487-dual λ absorbance detector was used. The column was equipped with the SUNFIRE C18 column (250 mm × 4.6 mm). The binary mobile phase consisted of 14% methanol (solvent A) and 86% water (solvent B, pH 3.1). The flow rate was kept constant at 1.0 ml/min for a total run time of 60 min. The injection volume of the extract was 5 μl. The elution was monitored at 280 nm. Anti-inflammatory compounds from VOS were identified by the chromatogram of the analytical standards such as (+)-catechin, (−)-epicatechin, proanthocyanidin A2 and cinnamtannin.
DPPH radical scavenging assay
DPPH radical scavenging assay was applied to evaluate anti-oxidant activity of VOS, VOL or VOF. DPPH radical scavenging assay was carried out according to the literatures with some modification [
17,
18]. Briefly, 152 μl of DPPH solution (1 mM DPPH in 95% ethanol) was added with 8 μl of VOS, VOL or VOF containing different concentrations (25 and 50 μg/ml) in 96-well plate. The mixtures were reacted for 30 min in the dark at 37 °C. After reaction, the absorbance was measured at a wavelength of 517 nm using UV/Visible spectrophotometer (Human Cop., Xma-3000PC, Seoul, Korea).
Determination of the contents of total phenolic compounds
The contents of total phenolic compounds were measured using the Folin-Ciocalteu assay [
18]. Briefly, 0.5 ml of VOS (50 mg/ml), VOL (50 mg/ml) or VOF (50 mg/ml) in 1 ml of distilled water was mixed with 0.5 ml of 2 N Folin-Ciocalteu reagent for 5 min, and then added 2 ml of 7% (w/v) sodium carbonate. The mixtures were incubated for 90 min at room temperature. After 90 min, the absorbance was measured a wavelength of 750 nm using UV/Visible spectrophotometer (Human Cop., Xma-3000PC, Seoul, Korea).
Cell culture and treatment
Mouse macrophage cell line, RAW264.7 has long been used for the evaluating anti-inflammatory activity. Thus, RAW264.7 cells were used in this study. RAW264.7 cells was purchased from Korean Cell Line Bank (Seoul, Korea) and maintained at 37 °C under a humidified atmosphere of 5% CO2 using DMEM/F-12 media containing 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. VOS, VOL or VOF was dissolved in dimethyl sulfoxide (DMSO) and treated to cells. DMSO was used as a control and the final DMSO concentration did not exceed 0.1% (v/v).
Cell viability assay
MTT assay was applied to evaluate cytotoxicity of VOS, VOL or VOF. MTT assay was carried out according to the literatures with some modification [
16]. Briefly, VOS, VOL or VOF was treated to the cells cultured on a 96-well plate at a density of 3 × 10
3 cells/well for 24 h. Then, the cells and incubated for 2 h after adding 50 μl of MTT solution (1 mg/ml). Then, cell culture supernatants were removed and DMSO was added to the cells for dissolving the resulting crystals. The formation of formazan was measured by reading absorbance at a wavelength of 570 nm using UV/Visible spectrophotometer (Human Cop., Xma-3000PC, Seoul, Korea).
Determination of NO, PGE2, IL-1β, IL-6 and TNF-α
Determining NO production was performed using Griess assay according to the literatures with some modification [
16]. Briefly, VOS, VOL or VOF was pretreated to the cells cultured on a 12-well plate at a density of 1 × 10
5 cells/well for 6 h. After 6 h, LPS (1 μg/ml) was co-treated to the cells for 18 h to induce inflammatory response. Then, 100 μl of the cell culture supernatants was mixed with 100 μl of Griess reagent (Sigma Aldrich), reacted at room temperature for 15 min, the absorbance was measured at 540 nm using UV/Visible spectrophotometer (Human Cop., Xma-3000PC, Seoul, Korea). The level of PGE
2, IL-1β, IL-6 or TNF-α levels were measured accordingly with the manufacturer’s protocols of Prostaglandin E
2 ELISA Kit (Cayman Chemical, Ann Arbor, MI, USA), Mouse IL-1β ELISA Kit (Invitrogen, Carlsbad, CA, USA), IL-6 (Mouse) ELISA Kit (Cayman Chemical), TNF-α (Mouse) ELISA Kit (Cayman Chemical).
TRAP assay
TRAP assay was carried out according to the literatures with some modification [
5]. To differentiate the effect of VOS on osteoclastogenesis, RAW 264.7 cells at 5 × 10
3 cells per well were seeded on a 96-well plate with RANKL (100 ng/ml) and various concentrations of VOS. Five days later, cells were fixed using a 10% formalin solution and stained for TRAP according to the manufacturer’s protocol. The stained cells were imaged using an inverted microscope (100×) and measured using Image J software (National Institutes of Health, Bethesda, MD, USA). TRAP activity was determined in the supernatants collected from wells using a TRAP solution (Pnpp in 0.5 M acetate, dissolved with tartrate acid solution).
Isolation of nucleus fraction
Nuclear fractions of cells were extracted using a nuclear extract kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s protocols. Briefly, RAW264.7 cells were collected with cold 1 × hypotonic buffer and reacted at 4 °C for 15 min. Then, detergent was added and vortexed for 10 s. The cells were centrifuged at 14,000 g for 1 min at 4 °C and the cell pellets were used for nuclear fraction collection. Nuclear fractions from the cell pellets were extracted using complete lysis buffer by the incubation at 4 °C for 30 min under shaking. After 30 min, nuclear fractions from the cell pellets were centrifuged at 14,000 g for 10 min at 4 °C, and the supernatants (nuclear fraction) were stored at − 80 °C for further analysis.
SDS-PAGE and Western blot
After treatment, the cells were washed twice with cold 1 × phosphate-buffered saline (PBS), and the cellular proteins were extracted using radioimmunoprecipitation assay (RIPA) buffer (Boston Bio Products, Ashland, MA, USA) supplemented with protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor cocktail (Sigma-Aldrich). The concentration of the proteins extracted from the cells was quantified using BCA protein assay (Thermo Fisher Scientific, Waltham, MA USA). The equal protein (30 μg/well) was separated on SDS-PAGE and transferred to PVDF membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The PVDF membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBS-T) by stirring at room temperature for 1 h and then incubated with specific primary antibodies (1:1000) in 5% non-fat dry milk in 0.05% TBS-T at 4 °C for 16 h. After 16 h, the PVDF membranes were washed three times for 5 min with 0.05% TBS-T, and then incubated with horse radish peroxidase-conjugated immunoglobulin G (1:1000) for 1 h at room temperature. Chemiluminescence was detected with ECL Western blotting substrate (Amersham Biosciences, Piscataway, NJ, USA) and visualized in Polaroid film. The density of Western blot bands was calculated using the software UN-SCAN-IT gel version 5.1 (Silk Scientific Inc. Orem, UT, USA).
Reverse transcriptase-polymerase chain reaction (RT-PCR)
After treatment, total RNA was extracted from the cells using a RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and 1 μg of total RNA was synthesized using a Verso cDNA Kit (Thermo Scientific, Pittsburgh, PA, USA) according to the manufacturer’s protocol. PCR was performed using PCR Master Mix Kit (Promega, Madison, WI, USA). The primer sequences used in this study were shown in Table
1. The PCR results were visualized using agarose gel electrophoresis. PCR reaction conditions were used: 1 cycle of (3 min at 94 °C for denaturation), 30 cycles of (30 s at 94 °C for denaturation, 30 s at 60 °C for annealing, and 30 s at 72 °C for elongation), and 1 cycle of (5 min for extension at 72 °C). The density of mRNA bands was calculated using the software UN-SCAN-IT gel version 5.1 (Silk Scientific Inc. Orem, UT, USA).
Table 1
The primer sequences used in this study
iNOS | Forward 5′-ttgtgcatcgacctaggctggaa-3′ Reverse 5′-gacctttcgcattagcatggaagc-3′ |
COX-2 | Forward 5′-gtactggctcatgctggacga-3′ Reverse 5′-caccatacactgccaggtcagcaa-3′ |
IL-1β | Forward 5′-ggcaggcagtatcactcatt-3′ Reverse 5′-cccaaggccacaggtattt-3′ |
IL-6 | Forward 5′-gaggataccactcccaacagacc-3′ Reverse 5′-aagtgcatcatcgttgttcataca-3′ |
TNF-α | Forward 5′-tggaactggcagaagaggca-3′ Reverse 5′-tgctcctccacttggtggtt-3′ |
TRAP | Forward 5′-acttccccagcccttactaccg-3′ Reverse 5′-tcagcacatagcccacaccg-3’ |
NFATc1 | Forward 5’-tgctcctcctcctgctgctc-3′ Reverse 5′-cgtcttccacctccacgtcg-3’ |
c-Fos | Forward 5’-atgggctctcctgtcaacac-3′ Reverse 5′-ggctgccaaaataaactcca-3’ |
MMP-9 | Forward 5’-cgacttttgtggtcttcccc-3′ Reverse 5′-tgaaggtttggaatcgaccc-3’ |
CTK | Forward 5’-aggcggctatatgaccactg-3′ Reverse 5′-ccgagccaagagagcatatc-3’ |
CA2 | Forward 5’-ctctcaggacaatgcagtgctga-3′ Reverse 5′-atccaggtcacacattccagca-3’ |
OSCAR | Forward 5’-ctgctggtaacggatcagctccccaga-3′ Reverse 5′-ccaaggagccagaaccttcgaaact-3’ |
ATP6v0d2 | Forward 5’-atggggccttgcaaaagaaatctg-3′ Reverse 5′-cgacagcgtcaaacaaaggcttgta-3’ |
GAPDH | Forward 5′-ggactgtggtcatgagcccttcca-3′ Reverse 5′-actcacggcaaattcaacggcac-3′ |
Transient transfection and luciferase activity
Transient transfection for luciferase activity was performed using the PolyJet DNA transfection reagent (SignaGen Laboratories, Ijamsville, MD, USA). Cells cultured on 12-well plates at a density of 2 × 105 cells/well were treated with plasmid mixtures containing 1 μg of the NF-κB luciferase constructs (Addgene, Cambridge, MA, USA) and 0.1 μg of pRL-null vector, and then cultured for 24 h. After 24 h, VOS was pretreated to the cells for 6 h, and then LPS (1 μg/ml) was co-treated to the cells for 18 h. After treatment, the cells were then harvested in 1 × luciferase lysis buffer, and luciferase activity was normalized to the pRL-null luciferase activity using a dual-luciferase assay kit (Promega, Madison, WI, USA).
Statistical analysis
All the data are shown as mean ± SD (standard deviation). Statistical analysis was performed with one-way ANOVA followed by Dunnett’s test. Differences with *P or #P < 0.05 were considered statistically significant.
Discussion
Since inflammatory diseases are considered to be one of the major health problems, the development of anti-inflammatory drugs for the treatment of inflammatory diseases has been longstanding. Currently, non-steroid anti-inflammatory drugs (NSAIDs) have been prescribed for the treatment of inflammatory diseases, but the long-term use of NSAIDs is known to cause serious side effects [
29]. Thus, the importance of searching for anti-inflammatory candidates with low side effects has been emphasized. In this study, we demonstrated that stem extracts from
V. oldhami (VOS) inhibits LPS-stimulated inflammatory response in RAW264.7 cells.
Overproduced nitric oxide (NO) by inducible nitric oxide synthase (iNOS) and interleukin 1β (IL-1β) has been reported to be associated with the onset of chronic diseases [
8,
30,
31]. NO can promote osteoclast formation by inducing cell fusion and increasing actin remodeling in mononuclear pre-osteoclast, which eventually results in fusion and formation of multinucleated osteoclasts [
22,
32]. In addition, NO produced by iNOS activates osteoclast, resulting in bone loss [
33]. IL-1β involved in NO production has been reported to directly or indirectly cause osteolysis [
34]. It is known that increased prostaglandin E
2 (PGE
2) produced by cyclooxygenase-2 (COX-2) in excessive inflammation also causes inflammatory-bone resorption [
35], so that inhibition of COX-2 expression can suppress osteoclast-induced bone loss [
36,
37]. Therefore, inhibition of NO production by blocking iNOS and IL-1β expression and PGE
2 production by blocking COX-2 expression may be a useful clinic strategy for treating inflammatory osteoporosis.
In this study, we observed that VOS inhibited LPS-induced NO and PGE2 production through inhibition of iNOS and IL-1β, and COX-2 expression, respectively. In addition, VOS blocked IL-6 and TNF-α expression. In order to confirm the degree of anti-inflammatory activity of VOS, we compared the inhibitory effect of VOS against LPS-induced overproduction of NO with tolfenamic acid (TA) as one of non-steroidal anti-inflammatory drugs. Although VOS had a lower inhibitory activity against LPS-induced NO production than TA, VOS is considered to be a potential source for the development of anti-inflammatory drugs because VOS is a crude extract.
To demonstrate the mechanism of osteoclast suppression of VOS, a RANKL-induced RAW264.7 cells were used [
38]. TRAP secreted only by osteoclast has been considered as a phenotype of osteoclasts [
39]. In the present study, VOS inhibited osteoclast differentiation and its activity. Previous studies have demonstrated that NFATc1 and c-Fos are the master regulator in osteoclastogenesis [
40,
41]. In addition, overexpression of NFATc1 and c-Fos by RANKL induces differentiation of osteoclast precursor cells into osteoclasts [
40,
42]. We observed that VOS inhibited the expression of NFATc1 and c-Fos. Additional, NFATc1 and c-Fos regulate various markers involved in osteoclast such as MMP-9, CTK and CA2. These genes play an important role in the degradation and resorption of the bone matrix [
43]. CA2 is placed on the bone matrix and acidifies the bone surface [
44]. After that, bone resorption markers such as MMP-9, CTK lead to absorb. OSCAR is related to osteoclast differentiation and bone homeostasis [
45]. ATP6v0d2 is an indicator of cell fusion in osteoclastogenesis and important constituent of osteoclast-related proton pump that controls acidification in matrix of bone [
46]. In the present study, VOS inhibited various genes related to osteoclast differentiation. These results indicated that VOS has an inhibitory effect on osteoclast differentiation by suppressing expression of osteoclastogenesis marker genes.
Abnormal activation of nuclear factor kappaB (NF-κB) signaling in excessive inflammatory responses is closely related to onset of various inflammatory diseases such as rheumatoid arthritis, atherosclerosis, chronic obstructive pulmonary disease, asthma, inflammatory bowel disease and ulcerative colitis [
47,
48], and also induces osteoclast formation by increasing expression of NFATc1 [
49]. Thus, inhibition of NF-κB signaling activation may provide an effective approach to inhibit osteoclast-induced bone resorption by excessive inflammatory responses. In current study, the inhibition of LPS-induced NF-κB signaling activation by VOS was confirmed by the inhibition of VOS on IκB-α degradation, p65 nuclear accumulation and NF-κB luciferase activation. These results indicate that VOS may inhibit the abnormal inflammatory response and inflammation-induced osteoclastogenesis via NF-κB signaling.
There is growing evidence that mitogen-activated protein kinases (MAPK), known as excessive inflammatory signaling, also plays a positive role in osteoclastogenesis [
50]. Indeed, the inhibition of ERK1/2, p38 and JNK is known to inhibit the differentiation of osteoclast-precursor cells into osteoclast [
23]. Activating transcription factor 2 (ATF2) activation by its phosphorylation and subsequent nuclear accumulation has been reported to be involved in MAPK signaling-induced production of the inflammatory mediators [
25]. In addition, ATF2, which is activated by MAPK signaling, has been established to be involved in osteoclast differentiation [
26,
27]. Luteolin, a flavonoid compound, has been reported to inhibit osteoclast differentiation through inhibition of ATF2 activation.
In GC/MS and HPLC analysis, we observed that VOS contained several compounds with anti-inflammatory activity such as 4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol [
51], methyl palmitate [
52], n-hexadecanoic acid [
53], sinapyl alcohol [
54], phytol [
55], linolenic acid [
56], stigmast-5-en-3-ol (phytosterols) [
57], β-amyrin [
58], (+)-catechin [
59], (−)-epicatechin [
59] and proanthocyanidin [
60]. Although various compounds with anti-inflammatory activity were analyzed from VOS, it is necessary to investigate which compounds affect the anti-inflammatory activity of VOS through activation tracing separation.
In this study, we confirmed that VOS inhibits MAPK activation through blocking the phosphorylation of ERK1/2, p38 and JNK, and MAPK-induced phosphorylation and nuclear accumulation of ATF2. These results indicate that VOS may inhibit the abnormal inflammatory response and inflammation-induced osteoclastogenesis via MAPK/ATF2 signaling.
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