Background
Macrophages, known as one of the immune cells, secrete a variety of pro-inflammatory mediators such as nitric oxide (NO), prostaglandin E
2 (PGE
2), inducible nitric oxide (iNOS) and cyclooxygenase-2 (COX-2), as well as pro-inflammatory cytokines including interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) [
1]. The appropriate cytokine secreted by macrophages acts to protect the body from external harmful factors, but excessive cytokines are known to cause chronic inflammation associated with inflammatory human diseases such as atheriosclerosis, arthritis, cardiovascular disease and other deadly diseases [
2,
3]. Therefore, regulation of pro-inflammatory cytokines and mediators has been regarded as complementary strategy to the inflammatory human diseases.
Heracleum moellendorffii Hance (
H. moellendorffii) growing in the field and mountains of Korea, China and Japan has been used as edible wild herb in Korea [
4].
H. moellendorffii leaves have been reported to exert detoxification, antioxidant and anti-melanogenic activities [
4‐
6] and
H. moellendorffii roots have been used as traditional herbal medicine treating inflammatory human diseases such as arthritis, backache and fever [
4]. In a previously reported study of the anti-inflammatory activity of
H. moellendorffii, dehydrogeijerin isolated from
H. moellendorffii leaves has been reported to block the expression of the pro-inflammatory mediators via the inhibition of MAPK signaling activation [
7]. However, there is no studies on the anti-inflammatory activity and its potential mechanism of
H. moellendorffii roots. In this study, we aimed to investigate anti-inflammatory activity of
H. moellendorffii roots in LPS-stimulated RAW264.7 cells, and to elucidate the potential mechanism.
Methods
Materials
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), tolfenamic acid (TA), N-Acetylcysteine (NAC) and LPS were purchased from Sigma Aldrich (St. Louis, MO, USA). Antibodies against IκB-α, p65, phospho-ERK1/2, ERK1/2, phospho-p38, p38, phospho-JNK, JNK, HO-1, Nrf2, β-actin and TBP were purchased from Cell Signaling (Bervely, MA, USA).
Sample preparation
After H. moellendorffii (voucher number: FMCHm-2019-0521-001~003) was collected and identified by Forest Medicinal Resources Research Center, National Institute of Forest Science (Yongju, Korea), H. moellendorffii was generously provided. Twenty gram of H. moellendorffii roots was immersed in 400 ml of 70% ethanol and then extracted for 72 h with stirring at room temperature. After 72 h, the extracts were filtered and concentrated using a vacuum evaporator and then lyophilized. The ethanol extracts of H. moellendorffii roots (HM-R) were stored − 80 °C until use. HM-R was dissolved in dimethyl sulfoxide (DMSO) before the experiment to treat the cells. DMSO was used as a control in all experiments and the concentration of DMSO treated in the cells did not exceed 0.1% (v/v).
Cell culture
RAW264.7 cells (American Type Culture Collection, Manassas, VA, USA) were maintained at 37 °C under a humidified atmosphere of 5% CO2 using Modified Eagle medium (DMEM)/F-12 1:1 Modified medium (Lonza, Walkersville, MD, USA) containing 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin.
Cell viability assay
The cytotoxicity of HM-R against RAW264.7 cells was evaluated using MTT assay. After the cells (3 × 103 cells/well) were plated on a 96-well plate for 24 h, HM-R was applied to the cells for 24 h. Then, 50 μl of MTT solution (1 mg/ml) was added to the cells and incubated for 2 h. Then, cell culture supernatants were removed and DMSO was added to the cells. The absorbance was measured at 570 nm using UV/Visible spectrophotometer (Human Cop., Xma-3000PC, Seoul, Korea).
NO and PGE2 determination
RAW264.7 cells (1 × 105 cells/well) in 12-well plate for 24 h were pretreated with HM-R for 2 h and co-treated with LPS (1 μg/ml) for 18 h. After the treatment, the cell culture supernatants were collected for the analysis of NO and PGE2 production. For measurement of NO production, the cell culture supernatants and Griess reagent (Sigma Aldrich) were mixed at a 1:1 ratio and reacted at the room temperature for 15 min, and the absorbance was measured at 540 nm using UV/Visible spectrophotometer (Human Cop., Xma-3000PC, Seoul, Korea). PGE2 production was analyzed using Prostaglandin E2 ELISA Kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s protocols.
Isolation of nuclear fraction
After the treatment, nuclear protein from RAW264.7 cells was isolated using a Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s protocols. The isolated nuclear protein was stored at − 80 °C until analysis.
SDS-PAGE and Western blot analysis
To extract protein from RAW264.7 cells, RAW264.7 cells were washed three times with cold 1 × phosphate-buffered saline and lysed at 4 °C for 30 min using cold radioimmunoprecipitation assay buffer (Boston Bio Products, Ashland, MA, USA) containing protease inhibitor (Sigma-Aldrich) and phosphatase inhibitor (Sigma-Aldrich). After centrifugation at 15,000 rpm for 10 min, the supernatant was recovered for protein quantitation using BCA protein assay (Thermo Fisher Scientific, Waltham, MA USA). The protein was separated on SDS-PAGE for about 1 h at 150 V and subsequently transferred to PVDF membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA) for 2 h at 100 V. After blocking the PVDF membranes using 5% non-fat dry milk in tris-buffered saline containing 0.05% Tween 20 (TBS-T) by stirring at room temperature for 1 h, the specific primary antibodies in 5% non-fat dry milk dissolved with TBS-T buffer were treated with PVDF membranes and reacted with stirring at 4 °C overnight. Then, PVDF membranes were washed three times with TBS-T buffer, and then treated with the secondary antibodies in 5% non-fat dry milk dissolved with TBS-T buffer for 1 h at room temperature. Chemiluminescence was detected with ECL Western blotting substrate (Amersham Biosciences, Piscataway, NJ, USA) and visualized using LI-COR C-DiGit Blot Scanner (Li-COR Biosciences, Lincoln, NE, USA).
Reverse transcriptase-polymerase chain reaction (RT-PCR)
RNA isolation from RAW264.7 cells and cDNA synthesis from isolated RNA were performed using a RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and a Verso cDNA Kit (Thermo Scientific, Pittsburgh, PA, USA) according to the manufacturer’s protocol, respectively. PCR was performed using PCR Master Mix Kit (Promega, Madison, WI, USA). The sequence of specific primers used for PCR analysis was as follows: iNOS: forward 5′-ttgtgcatcgacctaggctggaa-3′ and reverse 5′-gacctttcgcattagcatggaagc-3′, COX-2: forward 5′-gtactggctcatgctggacga-3′ and reverse 5′-caccatacactgccaggtcagcaa-3′, IL-1β: forward 5′-ggcaggcagtatcactcatt-3′ and reverse 5′-cccaaggccacaggtattt-3′, IL-6: forward 5′-gaggataccactcccaacagacc-3′ and reverse 5′-aagtgcatcatcgttgttcataca-3′; GAPDH: forward 5′-ggactgtggtcatgagcccttcca-3′ and reverse 5′-actcacggcaaattcaacggcac-3′. The PCR bands were visualized using agarose gel electrophoresis.
NF-κB luciferase activity
Transient transfection was performed using the PolyJet DNA transfection reagent (SignaGen Laboratories, Ijamsville, MD, USA) according to the manufacturer’s protocol. Briefly, NF-κB luciferase construct (Addgene, Cambridge, MA, USA, 1 μg/well), pRL-null vector (0.1 μg/well) and PolyJet DNA transfection reagent were mixed for 15 min at room temperature. RAW264.7 cells were treated with the mixtures and incubated for 24 h. The measurement of NF-κB luciferase activity was performed using a dual-luciferase assay kit (Promega, Madison, WI, USA). pRL-null luciferase activity was used to normalize NF-κB luciferase activity.
Analysis of bioactive components
The analysis of anti-inflammatory compounds from HM-R was performed using HPLC. In HPLC analysis, Waters 1525 system with a Waters 2487-dual λ absorbance detector was used. The column was equipped with the Waters SPHERISORB 10 μm Silica (250 mm × 4.6 mm). The mobile phase consisted of 10% ethanol and 90% hexane. The flow rate was kept constant at 1.0 ml/min for a total run time of 10 min. The injection volume of HM-R was 10 μl. The elution was monitored at 254 nm.
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
Many synthetic drugs have been developed to treat inflammatory diseases, but long-term use of such synthetic drugs is known to cause a variety of side effects such as gastric ulcer, bleeding, cardiac abnormalities, bone marrow depression, renal dysfunction and bronchospasm in long-term use [
24]. Thus, there is a need to develop more effective and safe anti-inflammatory drugs [
25]. In this trend, medicinal plants which have been used for the treatment of inflammatory diseases in the past, have been considered important resources for the development of traditional knowledge-based anti-inflammatory drugs [
25].
As traditional herbal medicine,
Heracleum moellendorffii roots (HM-R) have traditionally been used to treat inflammatory human diseases such as arthritis, backache and fever [
4]. However, the anti-inflammatory activity of HM-R has not been scientifically investigated. In order to develop anti-inflammatory drugs using traditional knowledge about the treatment of inflammatory diseases of HM-R, scientific evidence of HM-R’s anti-inflammatory activity and related mechanisms is need. Thus, we investigated anti-inflammatory activity and mechanism of action of HM-R in this study.
Although proper NO plays a major role in maintaining immunity and homeostasis, various human diseases related to inflammation are caused by excessive NO [
26]. In addition, excessive PGE
2 during inflammatory response is known to cause not only tissue damage, but also inflammatory diseases such as rheumatoid arthritis and chronic hepatitis [
27]. In this study, we observed that HM-R blocked LPS-induced NO and PGE
2 overproduction in RAW264.7 cells. Since NO and PGE
2 are synthesized by iNOS and COX-2, respectively, the regulation of iNOS and COX-2 expression has been regarded to be important for suppression of excessive NO and PGE
2 production [
28]. Thus, the effect of HM-R on iNOS and COX-2 expression was investigated and we observed that HM-R inhibited LPS-mediated overexpression of iNOS and COX-2, which indicating that the inhibition of iNOS and COX-2 expression by HM-R may contribute to the attenuation of NO and PGE
2 production. In addition, appropriate pro-inflammatory cytokines such as IL-1β and IL-6 contributes to the recovery of infection, but excessive accumulation of pro-inflammatory cytokines is known to cause chronic inflammation. Thus, the regulation of pro-inflammatory cytokines has been considered to be a complementary strategy for controlling the inflammatory disease process [
2]. In this study, we observed that HM-R significantly inhibits IL-1β and IL-6 expression in LPS-stimulated RAW264.7 cells. These findings indicate that HM-R may exert anti-inflammatory activity. In order to confirm the degree of anti-inflammatory activity of HM-R, we compared the inhibitory effect of HM-R against LPS-induced overproduction of NO with tolfenamic acid (TA) as one of non-steroidal anti-inflammatory drugs. At the same concentrations (12.5 μg/ml) of HM-R and TA, HM-R showed lower inhibitory activity against LPS-induced NO production than TA, but 25 μg/ml of HM-R showed similar inhibitory activity compared to TA (12.5 μg/ml). Although HM-R had a lower inhibitory activity against LPS-induced NO production than TA, HM-R can be considered to be a potential source for the development of anti-inflammatory drugs because HM-R is a crude extract.
The elucidation of mechanism for pharmacological activity is important for the development of related drugs. LPS-induced inflammation is caused by inflammatory cascade signaling pathway, in which NF-κB has been known as a major transcription factor that regulates that production of pro-inflammatory mediators [
29,
30]. Under inflammatory stimuli, NF-κB activation occurs through the phosphorylation and degradation of IκB-α, and subsequent p65 nuclear translocation. Nuclear p65 activates transcription of pro-inflammatory mediators [
29,
30]. Consequently, HM-R blocked LPS-induced degradation of IκB-α and nuclear accumulation of p65, which resulted in the suppression of NF-κB activation. Similar to NF-κB signaling, LPS-activated MAPKs such as ERK1/2, p38 and JNK also play an important role in the generation of pro-inflammatory mediators [
29,
31]. Furthermore, it has been known that MAPK is crucial for NF-κB activation and the binding of NF-κB to pro-inflammatory genes [
32,
33]. In this study, HM-R significantly decreased the phosphorylation of ERK1/2, p38 and JNK. These findings indicate that HM-R may exert anti-inflammatory activity through the inhibition of NF-κB and MAPK signaling activation.
It is known that heme oxygenase-1 (HO-1), which catalyzes the degradation of heme into biliverdin, iron and carbon monoxide has anti-oxidant, anti-inflammatory and anti-proliferative functions [
34,
35]. In fact, the anti-inflammatory activity of HO-1 has been demonstrated by various studies. It has been reported that overexpression of HO-1 prior to inflammatory stimulation inhibited expression of inflammatory mediators such as NO and IL-6 [
36,
37]. In addition, severe inflammation appeared in mice model deficient in HO-1 [
38]. These previous experimental evidence suggest that HO-1 may be a potential molecular target for treating inflammation [
21]. NF-E2-related factor 2 (Nrf2), known as the upstream mediator of HO-1, is present in the cytoplasm under unstressed condition, while accumulated nuclear Nrf2 under the oxidative stress causes the expression of HO-1 [
39]. In this study, we confirmed that nuclear accumulation of Nrf2 and HO-1 expression were increased in HM-R treated RAW264.7 cells. We also found that nuclear accumulation of Nrf2 and the increased expression of HO-1 by HM-R were reduced in NAC-treated RAW264.7 cells. These results indicate that HM-R may induce HO-1 expression through ROS-dependent Nrf2 activation, which contributes to anti-inflammatory activity.
In the analysis of anti-inflammatory compounds from HM-R using HPLC, falcarinol (Molecular formula: C
17H
24O, Molecular weight: 244.378) also known as panaxynol was analyzed. The previous study has reported that HM-R contains falcarinol [
22]. In addition, falcarinol was reported to exert anti-inflammatory effect through Nrf2/HO-1 signaling activation [
23].
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