Hyun Ji Eo ,Da Som Kim ,Gwang Hun Park

1Special Forest Resources Division,Department of Forest Bio-Resources,National Institute of Forest Science,Suwon 16631,Korea

2Forest Medicinal Resources Research Center,National Institute of Forest Science,Yeongju 36040,Korea

ABSTRACT Objective:To elucidate the potential anti-inflammatory mechanisms of Rhamnus crenata leaf extracts using RAW264.7 cells.Methods:We used 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay to measure cell viability.Nitric oxide (NO)production was measured using Griess reagent.Western blotting and RT-PCR assays were carried out for analyzing the protein and gene expressions of pro-inflammatory mediators,respectively.Moreover,PD98059 (ERK1/2 inhibitor),SB203580 (p38 inhibitor),SP600125(JNK inhibitor),and BAY11-7082 (NF-κB inhibitor) were used to evaluate the anti-inflammatory mechanism of Rhamnus crenata leaf extract.Results:Rhamnus crenata leaf extracts significantly inhibited the production of the pro-inflammatory mediators such as NO,iNOS,COX-2,IL-1β,and TNF-α in lipopolysaccharide (LPS)-stimulated RAW264.7 cells.Rhamnus crenata leaf extracts also suppressed LPS-induced degradation of IκB-α and nuclear accumulation of p65,which resulted in the inhibition of NF-κB activation in RAW264.7 cells.Additionally,the extracts attenuated the phosphorylation of p38,ERK1/2,and JNK in LPS-stimulated RAW264.7 cells.Moreover,HO-1 expression induced by Rhamnus crenata leaf extracts was significantly downregulated by SB230580,PD98059,SP600125 and BAY11-7082.Conclusions:Rhamnus crenata leaf extract may upregulate HO-1 expression through inhibition of p38,ERK1/2,and NF-κB activation,which may contribute to the anti-inflammatory activity of the extracts.Rhamnus crenata leaf extracts may have great potential for the development of anti-inflammatory drugs to treat acute and chronic inflammatory diseases.

KEYWORDS: Anti-inflammatory activity;Heme oxygenase-1;Nrf2;Mitogen-activated protein kinase;Nuclear factor kappa B;Rhamnus crenata

1.Introduction

Inflammation is an innate immune response caused by pathogen infestation or tissue damage and is a complex process carried out by various immune cells[1,2].The prolonged production of inflammatory mediators by macrophages can damage the host cells[3].Accordingly,a continuous inflammatory reaction can cause dysfunction,such as pain,fever,edema,and mucosal damage,and lead to the development of various diseases and cancers[4].The treatment of inflammatory diseases primarily focuses on the suppression of inflammatory mediators,such as nitric oxide (NO),inducible NO synthase (iNOS),cyclooxygenase 2 (COX-2),nuclear factor kappa B (NF-κB),mitogen-activated protein kinase (MAPK),and reactive oxygen species (ROS),as well as the inhibition of complex networks of signaling pathways[5].Nuclear factor erythroid-2-related factor-2 (Nrf2) regulates antioxidant enzymes and detoxification-related enzymes in the body;after binding to antioxidant response elements,it increases the expression of these enzymes and cytoprotective genes[6].Additionally,Nrf2 regulates the expression of heme oxygenase (HO-1)[7],which is involved in the production of carbon monoxide,biliverdin,bilirubin,and iron to reduce the generation of inflammation-inducing cytokines,and has antioxidant and anti-inflammatory effects.

Recently,various studies have been conducted to identify novel plant-based drugs with low toxicity and high anti-inflammatory effects,from various medicinal herbs[8].Rhamnus crenata(R.crenata) Siebold &Zucc,which belongs to Rhamnaceae,is a deciduous shrub or small tree plant that is distributed in China,Japan,Taiwan,and Korea.The leaves,branches,and fruit ofR.crenatahave been reported to have antioxidant and immune properties[9].However,research on the action mechanism underlying the anti-inflammatory activity ofR.crenatais insufficient.Therefore,in this study,the anti-inflammatory activity and action mechanism ofR.crenataextracts were investigated using lipopolysaccharide(LPS)-induced RAW264.7 mouse macrophages.

2.Materials and methods

2.1.Chemical reagents

In this study,a 1∶1 mixture of Dulbecco’s modified Eagle’s medium(DMEM),Ham’s F-12 nutrient mixture,2.50 mML-glutamine,and 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer(DMEM/F-12) were used to culture mouse macrophages,which were purchased from Lonza (Walkersville,MD,USA).LPS and inhibitors (PD98059,SB203580,SP600125,and BAY11-7082)were purchased from Sigma-Aldrich (St.Louis,MO,USA),and antibodies against IκB-α (#4148),phospho-IκB-α (#2859),phospho-ERK1/2 (#4377),ERK1/2 (#9102),phospho-p38 (#4511),p38(#9212),phospho-JNK(#9251),JNK (#9252),NF-κB p65 (#8242),HO-1 (#70021),Nrf2 (#12721),TATA-binding protein (TBP,#8515),and β-actin (#5125) for Western blotting assays were purchased from Cell Signaling Technology (Danvers,MA,USA).

2.2.Sample preparation

TheR.crenataleaves used in this study were collected from Yeosu-si,Jeollanam-do,Republic of Korea,in June 2019 (voucher specimen: FMCBcYS-1906-1) and formally identified by Gyu Young Chung,a professor of Andong National University,Korea.The use ofR.crenataleaves material in the present study complied with international,national,and institutional guidelines.TheR.crenataleaves were stored in Forest Medicinal Resources Research Center (voucher number FMRC-20190902A1).After lyophilization,400 mL of 70% ethanol was added to 20 g of the powdered leaves,stirred,and extracted for 48 h at 25 ℃.After extraction,the sample was filtered through filter paper (No.2,Whatman Co.,Maidstone,England),concentrated in a vacuum evaporator in a bath below 40 ℃,and lyophilized.The sample extract was then dissolved in dimethyl sulfoxide(DMSO) and used in subsequent experiments.

2.3.Cell culture

Mouse RAW264.7 cells were purchased from the Korea Cell Line Bank (Seoul,Korea).After the addition of 10% fetal bovine serum,the cells were cultured in a DMEM/F-12 medium,containing penicillin and 100 μg/mL streptomycin,at 5% CO2.When the density of the cells was more than 80%,trypsin-ethylenediaminetetraacetic acid solution was used for passage.

2.4.Measurement of cell viability

Cell viability was measured using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay.RAW264.7 cells were cultured at a concentration of 1×106cells/well in 12-well culture plates and treated withR.crenataleaf extracts for 12 h.Subsequently,the cells were incubated with 200 μL of MTT solution (1 mg/mL) for an additional 2 h.The resulting crystals were dissolved in DMSO.Formazan formation was determined by measuring the absorbance at a wavelength of 570 nm (Perkin Elmer,Waltham,MA,USA).

2.5.Measurement of NO production

NO production was measured using the method described by Namkoonget al.[10],with some modifications.Mouse macrophage RAW264.7 cells were dispensed into 12-well plates and cultured for 24 h at 37 ℃.Subsequently,the cells were treated with 0,25,50,and 100 μg/mLR.crenataleaf extracts and incubated for 2 h at 37 ℃.Afterward,they were treated with 1 μg/mL LPS and cultured for 18 h at 37 ℃.NO production was measured using Griess reagent (Sigma-Aldrich Co.,St.Louis,MO,USA).Absorbance was measured at 540 nm using a microplate reader (PerkinElmer,Waltham,MA,USA).

2.6.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting assay

Cells were washed with 1× phosphate-buffered saline to extract proteins from the cells.Protease and phosphatase inhibitor cocktails(Sigma-Aldrich Co.,St.Louis,MO,USA) were added to the radioimmunoprecipitation assay buffer (Boston Bio Products,Ashland,MA,USA) to obtain lysed proteins.After quantifying the proteins using the bicinchoninic acid protein assay (Pierce Biotechnology Inc.,Waltham,MA,USA),proteins were loaded onto 10% SDS acrylamide;the resolved proteins were then transferred to a nitrocellulose membrane (GE Healthcare Life Science,Germany),which was blocked with 5% non-fat dry milk for 1 h at 25 ℃.After 1 h,the membrane was incubated with the primary antibody (1∶1 000),dissolved in 5% non-fat milk,at 4 ℃ overnight.The membrane was washed thrice for 5 min with Tris-buffered saline (1×TBS-T) containing 0.005%Tween 20.Then,the membrane was incubated with a secondary antibody (1∶500),dissolved in 5% non-fat milk,for 1 h.After washing thrice for 5 min with TBS-T,the proteins were visualized using the enhanced chemiluminescence Western blotting substrate(Amersham Biosciences Co.,Little Chalfont,England).

2.7.Reverse transcription-polymerase chain reaction (RTPCR) assay

Total RNA was prepared using the RNeasy Mini kit (QIAGEN GmbH.,Hilden,Germany).Then,1 μg RNA was used for cDNA preparation using the Verso cDNA kit (Thermo Fisher Scientific Inc.,Waltham,MA,USA).PCR was performed using a PCR master mix kit (Promega Co.,Madison,WI,USA);the primers used are listed in Table 1.The quality of cDNA was determined through PCR;the amplified PCR products were analyzed through 1% agarose gel electrophoresis using stain Safe Shine Green stain(10 000×Biosesang).The gel was visualized in Chemidoc (Biorad,Chemi Doc MP Imaging system,Hercules,CA,USA).The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (a housekeeping gene) was used as an internal control.

Table 1.Sequences of primers used for RT-PCR.

2.8.Statistical analysis

All data are presented as mean±standard deviation (SD).The differences between treatments were determined by one-way analysis of variance (ANOVA) andP＜0.05 was considered statistically significant.

3.Results

3.1.Inhibitory effects of R.crenata leaf extracts on NO production and proinflammatory expression in LPSstimulated RAW264.7 macrophages

The MTT assay showed thatR.crenataleaf extracts did not exhibit toxicity (Figure 1A).R.crenataleaf extracts also reduced the production of LPS-induced NO in RAW264.7 macrophages in a concentration-dependent manner (Figure 1B).In addition,LPSstimulated RAW264.7 macrophages were used to determine the inhibitory activity of inflammatory mediator genes includingiNOS,COX-2,IL-1β,andTNF-α,which are involved in the pathogenesis of inflammatory diseases.The extracts decreased the expression of these inflammatory mediators in a concentration-dependent manner(Figure 1C).These findings confirm thatR.crenataleaf extracts exhibit anti-inflammatory activity.

3.2.Inhibitory effects of R.crenata leaf extracts on NF-κB signaling activity in LPS-stimulated RAW264.7 cells

Inhibition of NF-κB signaling activity is considered an important target for anti-inflammatory activity;accordingly,we investigated whetherR.crenataleaf extracts affect the LPS-induced NF-κB signaling activation.R.crenataleaf extracts blocked the LPSinduced degradation and phosphorylation of NF-κB inhibitor alpha(IκB-α) in a concentration-dependent manner (Figures 2A and 2B).Additionally,it inhibited the nuclear translocation of p65 (Figure 2C).These results suggest thatR.crenataleaf extracts exert an antiinflammatory effect by inhibiting NF-κB activity.

3.3.Inhibitory effects of R.crenata leaf extracts on MAPK signaling activity in LPS-stimulated RAW264.7 cells

MAPK signaling,such as ERK1/2,p38,and JNK,exists in various conformations.To investigate the effects ofR.crenataleaf extracts on the inhibition of MAPK activity,different concentrations ofR.crenataleaf extracts were used to treat LPS-induced RAW264.7 cells.Phosphorylation of ERK1/2,p38,and JNK was significantly inhibited in a concentration-dependent manner (Figures 3A-3C).These findings suggest that the extract exerts anti-inflammatory effects by inhibiting MAPK signaling pathway.

3.4.R.crenata leaf extracts activate HO-1 expression in RAW264.7 cells

R.crenataleaf extracts dose-dependently increased HO-1 protein levels (Figure 4A),and HO-1 expression was induced 9 h after treatment withR.crenataleaf extracts,with the highest expression being observed after 24 h (Figure 4B),which indicates that HO-1 expression is influenced byR.crenataleaf extracts.To investigate the upstream kinases such as ERK1/2,p38,JNK,and NF-κB associated with HO-1 expression,we pretreated RAW264.7 cells with PD98059(ERK1/2 inhibitor),SB203580 (p38 inhibitor),SP600125 (JNK inhibitor),or BAY11-7082 (NF-κB inhibitor) and then co-treated them withR.crenataleaf extracts.As shown in Figures 5A-5D,the pretreatments of PD98059,SB203580,SP600125,and BAY11-7082 blocked HO-1 expression induced byR.crenataleaf extracts in RAW264.7 cells.These results indicate that the HO-1 protein expression induced byR.crenataleaf extracts is dependent on ERK1/2,p38,JNK,and NF-κB.

3.5.HO-1 expression induced by R.crenata leaf extracts is dependent on Nrf2 activation in RAW264.7 cells

We investigated whether Nrf2 activation is involved in theR.crenataleaf extracts-induced HO-1 expression.When RAW264.7 cells were treated withR.crenataleaf extracts,Nrf2 accumulated in the nucleus in a concentration-dependent manner (Figure 6A),and the highest value was observed after 1 h of treatment (Figure 6B).These findings indicate thatR.crenataleaf extracts may upregulate HO-1 expression through Nrf2 activation.

Figure 1.Effects of Rhamnus crenata leaf extracts (RCL) on cell viability and the production of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated RAW264.7 cells.(A) Cell viability was determined by MTT assay.(B) RAW264.7 cells were pretreated with 0,25,50,and 100 μg/mL of RCL for 6 h and then co-treated with LPS (1 μg/mL) for 18 h.The nitric oxide (NO) production was measured by Griess assay.(C) The gene expressions of proinflammatory mediators were measured by RT-PCR assay.GAPDH was used as an internal control.The density of mRNA bands was calculated using the software Chemidoc.The data are expressed as mean±standard deviation (SD) and analyzed by one-way analysis of variance (ANOVA).#P＜0.05 compared to the cells without the treatment of RCL and LPS,and *P＜0.05 compared to the cells treated with LPS alone.

Figure 2.Inhibitory effect of RCL on the activation of NF-κB signaling pathway in RAW264.7 cells.(A and B) RAW264.7 cells were pretreated with RCL(0,25,and 50 μg/mL) for 6 h,followed by treatment with LPS (1 μg/mL) for 40 min.(C) RAW264.7 cells were pretreated with RCL (0,25,and 50 μg/mL)for 40 min.After the treatment,the cytosol and nucleus were isolated from the cells.The cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis,and Western blotting assay was performed using antibodies against p65,IκB-α,and phospho-IκB-α.β-actin and TBP were used as internal controls.The data are expressed as mean±standard deviation (SD) and analyzed by one-way analysis of variance (ANOVA).#P＜0.05 compared to the cells without the treatment of RCL and LPS,and *P＜0.05 compared to the cells treated with LPS alone.TBP: TATA-binding protein.

Figure 3.Inhibitory effects of RCL on the activation of MAPK signaling pathway in RAW264.7 cells.RAW264.7 cells were pretreated with RCL (0,25,and 50 μg/mL) for 6 h and then co-treated with LPS (1 μg/mL) for 40 min.The cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis,and Western blotting assay was performed using antibodies against phosphorylated p38,extracellular signal-regulated kinase 1/2 (ERK1/2),and c-Jun N-terminal kinase (JNK).The data are expressed as mean±standard deviation (SD) and analyzed by one-way analysis of variance (ANOVA).#P＜0.05 compared to the cells without the treatment of RCL and LPS,and *P＜0.05 compared to the cells treated with LPS alone.

Figure 4.Effects of RCL on heme oxygenase 1 (HO-1) expression in RAW264.7 cells.RAW264.7 cells were treated with 50 μg/mL RCL for the indicated periods and then Western blotting analysis was performed.β-actin was used as an internal control.The data are expressed as mean±standard deviation (SD) and analyzed by one-way analysis of variance (ANOVA).*P＜0.05 compared to the cells without the treatment of RCL.

Figure 5.Effects of inhibitors on RCL-mediated HO-1 expression in RAW264.7 cells.RAW264.7 cells were pretreated with 20 μM each of (A) SB203580,(B) SP600125,(C) PD98059,and (D) BAY11-7082 for 2 h and then co-treated with 50 μg/mL of RCL for 24 h.Western blotting analysis was performed using anti-HO-1 antibodies.The data are expressed as mean±standard deviation (SD) and analyzed by one-way analysis of variance (ANOVA).*P＜0.05 compared with the cells treated with RCL only.

Figure 6.Effects of RCL on Nrf2 activation in RAW264.7 cells.(A) RAW264.7 cells were treated with 0,25,and 50 μg/mL of RCL for 1 h;then,the nuclear proteins were subjected to Western blotting analysis using an anti-Nrf2 antibody.(B) RAW264.7 cells were treated with RCL (50 μg/mL) for 24 h and then Western blotting analysis was performed using an anti-Nrf2 antibody.TBP was used as an internal control.The data are expressed as mean±standard deviation(SD) and analyzed by one-way analysis of variance (ANOVA).*P＜0.05 compared to the untreated control cells.

4.Discussion

In the immune system,macrophages play an important role in regulating immune function and maintaining homeostasis by suppressing the inflammatory reactions caused by various stimuli,such as free radicals and stress[11].In particular,macrophages are involved in the production of NO,a representative inflammatory mediator,which is a highly reactive biomolecule produced fromL-arginine by NOS[12].iNOS and COX-2 are representative proinflammatory cytokines.iNOS is primarily produced by stimulated macrophages,resulting in the production of NO[13,14].COX-2 is an enzyme that converts arachidonic acid into prostaglandins;although COX-1 is primarily found in normal cells,COX-2 is expressed at the site of inflammation and is associated with cancer[15].Since cytokines such as IL-1β,IL-6,and TNF-α are inflammatory cell signaling proteins[16],inhibition of these inflammatory mediators is a promising target for the treatment of inflammatory diseases[17].In this study,the effects ofR.crenataleaf extracts on NO production,cell viability,and expression of pro-inflammatory cytokines were investigated using RAW264.7 cells.

NF-κB is a transcription factor that participates in various processes,such as general immunity,inflammation,and cell growth regulation.Usually,it exists in an inactive state,which is bound to the inhibitory protein IκB in the cytoplasm[18].As p65 and p50,which are bound to the cell,move from the cytoplasm to the cell nucleus,NF-κB activation occurs,and inflammation is promoted[19].When NF-κB signaling is activated,p65 (a component of the NFκB complex) is transferred to the nucleus[20],and its phosphorylation plays a major role in NF-κB activity because it regulates migration from the cytoplasm to the nucleus.Inhibition of phosphorylation is associated with the inhibition of NF-κB activity[21].R.crenataleaf extracts inhibited LPS-induced NF-κB activity and intranuclear migration in RAW264.7 cells.Western blotting analysis revealed that the quantity of protein transferred in the nucleus was significantly decreased in cells treated with 25 and 50 μg/mL ofR.crenataleaf extracts compared to the LPS-treated control group.The MAPK family is a signal transduction mediator in cellular responses to various external stimuli and includes ERK,p38,and JNK[22].In this study,Western blotting results showed thatR.crenataleaf extracts inhibited the LPS-induced phosphorylation of ERK,p38,and JNK in RAW264.7 cells.

HO-1 induction acts against the mechanisms that protect cellular lipids and proteins from oxidative damage,thereby weakening the inflammatory response[23].Nrf2 increases the expression of certain genes after binding to antioxidant response elements[24].Under normal conditions,there are low levels of Nrf2 in the cytoplasm because it forms a complex with Kelch-like ECH-associated protein 1 (Keap1),which degrades it.However,it can separate from Keap1 by external stimulation or oxidative stress and move to the nucleus[25].Of note,Nrf2 and HO-1 are closely related,and HO-1 expression is increased by Nrf2[26,27].In this study,Western blotting was performed to confirm the intranuclear migration of Nrf2;it was found that the amount of Nrf2 increased in the nucleus followingR.crenataleaf extract treatment.These findings indicated thatR.crenataleaf extracts promoted the migration of Nrf2 into the nucleus and increased the expression of HO-1.In conclusion,our results demonstrated thatR.crenataleaf extracts reduce the expression of various inflammatory genes and can regulate inflammation through various pathways,such as the NF-κB,MAPK,and Nrf2/HO-1 pathways.

Conflict of interest statement

The authors declare no conflict of interests.

Funding

This work was supported by the research project of the National Institute of Forest Science (project No.FP0400-2019-01-2022).

Authors’contributions

All authors contributed to the conception and design.HJE mainly engaged in experimental design and operation,data collation,article writing,and commissioning.Material preparation,data collection,and analysis were performed by DSK.GHP helped in experimental design,paper writing and modification,and submission.All authors reviewed and approved the final manuscript.