Anti-inflammatory Properties of Ginseng-Derived Exosome-like Nanoparticles in LPS-induced RAW264.7

doi:10.21203/rs.3.rs-2836366/v1

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Anti-inflammatory Properties of Ginseng-Derived Exosome-like Nanoparticles in LPS-induced RAW264.7

https://doi.org/10.21203/rs.3.rs-2836366/v1

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Objectives

Exosomes are extracellular nano-sized vesicles which transport proteins, lipids, nucleic acids and polysaccharides. Panax ginseng C.A. Meyer is known to have various pharmacological activities including anti-inflammatory and antioxidant effects. Furthermore, ginseng derived exosome-like nanoparticles (GDEs) may be considered to contribute to inflammation mechanisms. The main aim of this study elucidated that GDEs exert anti-inflammatory activities, which can molecularly modulate an inflammation mechanism in mammalian cells.

Methods

GDEs were isolated by the method using sucrose cushioning ultracentrifugation and the morphology of GDEs was shown through transmission electron microscope (TEM). The number of particles and size distribution of the GDEs were evaluated using nanoparticle tracking analysis (NTA). To investigate the anti-inflammatory activity of GDEs, the expression levels of inflammatory mediators and cytokines were analyzed in lipopoly-saccarides (LPS)-induced RAW 264.7, murine macrophages. ELISA, qPCR, WB and nitric oxide assay were carried out for analyzing anti-inflammatory effects of the GDEs in mammalian cells.

Results

GDEs showed a spherical shape with an average diameter of 176.2 nm and a substantial number of GDEs was counted as 1.09 x 10¹² ± 1.19 x 10¹¹ particles/ml. The pretreated GDEs in LPS-induced RAW264.7 suppressed the protein expression levels of pro-inflammatory cytokines including interlukin-1beta (IL-1β), interlukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α). In addition, protein expression levels of inflammation-related enzymes, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) as well as production of nitric oxide (NO) were dramatically decreased by the GDEs in LPS-induced RAW264.7. Correspondingly, the GDEs treatment clearly reduced LPS-induced mRNA expression levels of the iNOS and COX-2. In addition, expression patterns of the IL-1β, IL-6, TNF-α were gradually down-regulated by the GDEs in a dosage dependent manner.

Conclusion

These results demonstrated that Ginseng-derived exosome-like nanoparticles inhibited the expression of the inflammation mediators, suggesting that the GDEs possess anti-inflammatory activities and molecularly regulate the inflammatory mechanism in mammalian cells. Collectively, the GDEs has high potential as a novel therapeutic agent to treat chronic inflammatory diseases.

Exosomes

ginseng

ginseng derived exosome-like nanoparticles

anti-inflammatory effect

inflammation

Cell-free therapy, which is based on the use of exosomes, is attracting scientists’ interest and attention as overcoming the limitation of stem cell therapy remarkably ¹. Exosomes are nano-sized extracellular vesicles (30 to 150nm in diameter) originated from intraluminal vesicles (ILVs) in multivesicular bodies (MVBs) which are involved in endosomal sorting complex responsible for transport pathway ². Spherical or cup-shaped exosomes are secreted in all cell types ranging from eukaryotic organisms like animals, plants, protists and fungi as well as prokaryotes ^{3, 4}. Exosomes are considered as a pivotal mediator to transport their genetic materials including DNA, mRNAs, miRNAs and other noncoding RNAs, lipids, proteins and various metabolites in near and long-distance cell to cell communication ⁵. The recent studies demonstrate that the intrinsic properties of exosomes are to have heterogeneity in size, origin and content and to perform multiple functions in maintaining cells by regulating cell proliferation, migration, apoptosis, and angiogenesis ⁶. Moreover, they are regulating immune modulation and inflammation, progressing tumors, and generating nerve systems in mammalian cells and organisms ⁶. Due to these characteristics, exosomes in clinical setting are considered as biomarkers in diagnostic and prognostic application in addition to therapeutic and drug delivery tools ⁷.

About exosomes in plants, their formation and function were considered to be inhibited because plants have cell walls, which are different from mammalian systems. But, plant-derived extracellular vesicles were first discovered in carrot cell culture ⁸. Similar to animal exosomes, they are membranous vesicles secreted by plants and a type of nano-carriers performing intracellular communication, as containing RNAs, proteins, lipids and bioactive constituents ^{9, 10}. The previous studies have reported that plant-derived extracellular vesicles were isolated from the juice, the flesh, and roots of many edible plants such as blueberries, oranges, coconuts, watermelons, sunflower seeds, pears, soybeans tomatoes, etc. and as well as research model plants like Arabidopsis thaliana or tobacco ^{11, 12, 13}.

Even though statements in 2014 and 2018 were announced about minimal experimental necessities for extracellular vesicles-based studies from the international society for extracellular vesicles, the consensus about exosomes derived from plants has not been reached among researchers ^{14, 15}. In plants, specific markers for extracellular vesicle subtypes such as proteins of endosomal origin in mammalian organisms were not established, because the ESCRT genes responsible for plant-derived exosomes have not perfectly elucidated. Also, it is not clear the biogenesis process of plant exosomes passing through the apoplastic space or cell wall and the biological significance of exocyst-positive organelle-mediated secretion of plant derived exosomes ^{9, 16}. In addition, the sizes of plant derived extracellular vesicles are ranging from 50 to 500nm in diameter, which are different from animal exosomes ¹⁷. Because of these reasons, it is difficult to assign the plant derived extracellular vesicles as ‘exosome’ which are widely accepted. Accordingly, many researchers have designated them as ‘plant-derived exosome-like nanoparticles’ or ‘plant-derived nanoparticles’ rather than plant exosomes ^{18, 19, 20}.

Accumulating evidences show that plant-derived exosome-like nanoparticles (PELNs) contain bioactive molecules such as ascorbic acid in strawberry, flavonoid and furanocoumarins in apple, which are toxic against insects and fugal species, and toxic hyperaconitine, mesoaconitine and aconitine that are derived from aconitituber ^{19, 20}. In the therapeutic aspects, many PELNs which are non-toxic and biocompatible show helpful properties regarding treatment in various diseases. Ginger-derived exosome-like nanovesicles (GDNVs) were effective to suppress ulcerative colitis, colon cancer, inflammatory bowel disease, and to maintain the intestinal homeostasis ^{21, 22}. In addition, GDNVs are effective in liver disease, periodontitis and inhibit NLRP3 inflammasome activity in Alzheimer ^{23, 24, 25}. Grape-derived exosome-like nanoparticles (GNVs) had effect on remodeling the intestinal stem cells and protecting mice from sodium dextran sulfate (DSS)-induced colitis ²⁴. Also, PELNs can be used as carriers of nanomaterials, which load therapeutic agents, including drugs, siRNA, miRNA and protein and deliver them to target cells ²⁵. GNVs delivered methotrexate, anti-inflammatory drug to DSS-induced colitis and transported curcumin and Doxorubicin to the tumor site in mice ^{24, 26}.

Ginseng is a traditional medicinal herb used in prevention and treatment of disease. Ginseng in therapeutic aspects has multiple functions in anti-cancer, anti-inflammation, anti-allergy and anti-aging as well as regulating cardiovascular system, relieving stress, enhancing central nervous system and promoting bone remodeling ^{27, 28, 29, 30, 31, 32}. Most effective and bioactive compound is ‘saponin’ which is commonly known as a ginsenoside or a panaxoside, although ginseng contains many other components like polysaccarides, sterols, polyalkenes, and essential oil ³³. Moreover, ginseng-derived exosome-like nanoparticles (GDEs) have been isolated using a few types of methods, sequential sucrose gradient based centrifugations after pellet precipitation of ginseng extracellular vesicles, double purification method using iodixanol, or sucrose gradient-based centrifugations followed by sucrose cushioning ultracentrifugations ^{34, 35}. Isolated GDEs showed several effects as an immunomodulatory mediator and a suppressor in various types of cancers. GDEs significantly promoted the polarization of tumor supportive M₂ to tumoricidal M₁ phenotypes in Tumor-associated macrophages (TAMs) of mouse melanoma cells, and inhibited melanoma growth in mice ³⁵. Also, GDEs exerted to regulate the cell proliferation in skin and to facilitate wound healing in mouse model ³⁶.

Nevertheless, the effect of GDEs, which is related to inflammation is still unclear, although GDEs has been studied in some part of macrophage polarization. Inflammation is an innate immune response against tissue damage or infection. The instinct function of inflammation, is to inhibit the damage of injured tissue and to regenerate the tissue by removing the foreign organisms and antigens ^{37, 38}. Macrophages, which are at the first line of immune response not only detect damaged or pathogen related agents and remove them by phagocytosis, but also secrete pro-inflammatory cytokines and immune mediators which are related to propagation in the inflammatory signal cascades ³⁹. Lipopolysaccharides (LPS) has been isolated from gram-negative bacterial cell wall and used as one of the most potent activators which stimulates macrophages. Toll-like receptor (TLR)4 is activated by binding LPS and start the inflammatory signaling ⁴⁰. LPS-induced macrophages release nitric oxide (NO) by regulating inducible nitric oxide synthase (iNOS) and prostaglandin₂ (PEG₂) by cyclooxygenase-2 (COX-2). Also, they secrete pro-inflammatory cytokines such as interleukin 1-beta, interleukin 6 (IL-6), and tumor necrosis factor-alpha (TNF-α) ^{41, 42}.

In this study, we demonstrated that GDEs molecularly regulated pro-inflammatory cytokines and inflammatory mediators using lipopolysaccharides (LPS)-induced mouse murine macrophage RAW264.7, which has been studied well as inflammation model in vitro. Also, we suggest the potential of GDEs as drug delivery system adding to function as anti-inflammatory agent.

All methods on the roots of Panax ginseng and GDEs were performed in accordance with the relevant guidelines and legislation of Kyung Hee University.

2.1. Isolation of GDEs

The previously reported method was used in order to isolate GDEs ⁴³. So, herein the method is briefly described as follows. The roots of Panax ginseng were purchased from Keumsan, Republic of Korea. 70g of ginseng was ground in a mixer with phospho-buffered saline, (Life Technoloies Corp,USA) at the mid to high speed for 5min. After homogenized ginseng was squeezed, the collected juice was subsequently centrifuged at 2,000 x g for 20 min and 10,000 x g for 60 min at 4°C in order to remove debris and fibers. The supernatant was gently transferred on the top of two sucrose layers, 27% and 68% and ultra-centrifugated at 100,000 x g for 90 min at 4°C. The yellow microvesicles between the two sucrose layers was transferred onto sucrose gradient layers (8%, 30%, 45%, and 60%) and ultra-centrifuged at 200,000 x g for another 90 min at 4°C. The yellow layer between 8% and 30% was collected and filtered with 0.2 µm of a sterilizing grade filter membrane (Pall Corp., USA) to obtain the nanoscale GDEs. The GDEs were used freshly or stored at -80°C. The protein concentration of GDEs was measured by BCA assay kit (Thermo Fisher Scientific Inc., USA) following manufacture’s instruction.

2.2. Characterization of GDEs

Nanoparticle Tracking Analysis (NTA)

The concentration and size distribution of isolated GDEs were measured by a NanoSight NS300 (Malvern Panalytical Ltd., Malvern, UK). Data were analyzed with Nanoparticle Tracking Analysis (NTA) software (ver.3.4). 1:10–1:1000 diluted samples of GDEs were suspended in PBS and determined the size by repeating the measurement three times.

Transmission Electron Microscope (TEM)

A drop of GDEs was deposited onto the surface of formvar-coated copper grids and incubated with 1% uranyl acetate for 2 min followed by washing with absolute ethanol. The samples were left to dry at room temperature and observed using a transmission electron microscope (JEM-1400 Flash, JEOL, Japan)

2.3. LC-MS/MS

The concentrations of Rb1, Rg1, and Rg3 in GDEs were analyzed using LC-MS/MS. Rb1, Rg1, and Rg3 used as standard were purchased from Merck, Germany. 200ul GDEs sample, 100µl BuOH, and 100µl DEPC water were mixed together. Each mixed sample was vortexed for 10 min and centrifugated at 13,000 rpm for 10 min. After each sample was dried in speed vacuum, they were re-conjugated with 10µl distilled water and 10µl IS was added. 5µl out of 20µl mixture was injected into LC-MS/MS system. The LC-MS/MS system consisted of Agilent 1200 series HPLC system (Agilent, Santa Clara, CA) was interfaced with API3200®triple-quadrupole mass spectrometer (Applied Biosystems-SCIEX, Concord, Canada). The HPLC mobile phases were consisted of 0.1% formic acid in water and acetonitrile. The analytes were separated on a reversed -phase Atlantis T3 column (100 x 2.1 mm, 3 µm, Waters Corporation, Milford, MA) using the gradient elution at a flow rate of 0.3ml/min.

2.4. Cell culture

RAW 264.7 cells were purchased from Korean Cell Line Bank. The cells were grown in Dulbecco’s modified Eagle’s medium (Life Technologies Corp., USA) supplemented with 10% fetal bovine serum (FBS), penicillin (100U/ml) and streptomycin (100M/ml) at 37°C and 5% of CO₂ environment. RAW 264.7 cells were sub-cultured in every 2–3 days.

2.5. Cell cytotoxicity

To evaluate toxicity of GDEs in RAW 264.7 cells, EZ-Cytox (Daeil Lab Service, Republic of south Korea) containing WST-1(water soluble tetrazolium) was used. RAW 264.7 cells were grown at the density of 5x10³ cells per well in 96-well plate. After 24 hours of incubation, cells were treated with various concentration of GDEs (0 ~ 40µg/ml) for 24 and 48 hrs. After the treatment, the supernatant of each well was removed and replaced by fresh media and 10% of EZ-Cytox. Additionally, the plate was incubated in the dark at 37°C for further 2 hours. After the incubation, each sample was quantified by a microplate spectrophotometer at the absorbance of 460 nm (Molecular Devices, USA).

2.6. ELISA assay

RAW 264.7 cells (2x10⁴/well) were seeded in 48-well plate and incubated for 18 hours. After removing the media, Various concentration of GDEs (0, 1, 5 and 10µg/ml) mixed in pre-warmed media for 2hrs and LPS (1µg/ml) were subsequently treated in the each well. The cells were incubated for another 7 hours and 24 hours. The cell medium supernatant from the each well was collected and stored at -20°C for the subsequent analysis. An enzyme-linked immunosorbent assay (ELISA) was performed for the quantitative determination of the levels of IL-6 and TNF-alpha in the accordance with the manufacture’s instruction (R&D systems, USA).

2.7. Nitric Oxide assay

RAW 264.7 cells (2x10⁵/well) were cultured in 6-well plate for 24 hours. Each well was respectively changed to L-NMMA (100 µM) or GDEs (0, 1, 5 and 10 µg/ml) with fresh media. After 2 hours, cells were stimulated with LPS (1µg/ml) for further 24 hours. Cell culture supernatant was obtained to measure the level of nitric dioxide with NO detection kit. To determine a proper concentration of L-NMMA (Sigma-Aldrich, USA), different concentrations of L-NMMA (0, 1, 10, 100 and 500 µM) were used in the same way as described in the above NO detection protocol. The NO detection kit (iNtRON biotechnology, Republic of Korea) was strictly used according to manufacture instructions.

2.8. Western blot analysis

RAW 264.7 cells were seeded at a final concentration of 4x10⁵ in 60mm dishes and incubated for 24 hours. The cells were treated with GDEs (0, 1, 5 and 10µg/ml) for 2 hours followed by LPS (1µg/ml) stimulation for 24 hours. The cells were isolated with RIPA buffer (Biosesang, Republic of Korea) containing protease inhibitor cocktails (Sigma-Aldrich, USA) and rested in ice for 30 min. Each supernatant was collected after 12,000 x rpm centrifugation at 4°C for 20 min. Then proteins were quantified by a bicinchoninic acid assay kit (Thermo Fischer Scientific Inc., USA) with a bovine serum albumin as a standard. Protein samples were separated in a 10% sodium salt-polyacrylamide gel, and then transferred onto nitrocellulose membrane (Amersham, Germany). After 1 hour of blocking with 5% non-fat milk in TBST at room temperature, the membranes were incubated overnight at 4°C with antibodies against β-actin (sc-47778; Santa Cruz, USA), iNOS (#13120; Cell Signaling Technology, USA) and COX-2 (#12282; Cell Signaling Technology, USA), which were diluted (1:1000) in blocking buffer (Bio-Rad, USA). After washed three times with 1 x TBST for 30 min, the membranes were subsequently incubated for 1 hour at room temperature with anti-rabbit (sc-2357; Santa Cruz, USA) and anti-mouse (sc-516102; Santa Cruz, USA) secondary antibodies diluted (1:2500) with 5% non-fat milk in 1 x TBST. The membranes were washed three times with 1 x TBST for 30 min, 40 and proteins were detected using an enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc., USA). Protein expression was analyzed by Amersham Imager 600 (GE, USA) and protein band densities were measured using Image J analysis software (National Institutes of Health, USA).

2.9. Quantitative Real-Time PCR

Total RNA was purified from RAW 264.7 cells cultured in LPS (1µg/ml) stimulation for 4h following GDEs treatment for 2h. TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to collect cell lysates.1µg of total RNA was transcribed to cDNA using a reverse-transcription system with random hexamers (Thermo Fischer Scientific Inc., USA). RT-qPCR was performed on StepOnePlus Real-Time PCR system with Power SYBR-PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific Inc., USA). The PCR program was as follow: a denaturation step at 95°C for 10min, 40 cycles at 95°C for 15 min and 60°C for 1min. The data were calculated using the 2 ^–ΔΔCq method and the relative amount was quantified. The gene specific primers were made by Bioneer Corp. in Republic of Korea and are listed in Table 1.

2.10. Statistical analysis.

Data are expressed as the mean ± standard error. Data were compared by one-way or two ANOVA analysis. Statistical analyses were performed using the GraphPad Prism 8 software (GraphPad Software Inc., USA).

3.1. Isolation and characterization of GDEs

GDEs of Panax ginseng were purified with the modified centrifugation-based isolation methods adding the sucrose cushioning. GDEs were obtained from the layer between 8% and 30% sucrose solution after sequential centrifugations of low to high velocity. Figure 1 (A) and (C) showed that size distribution and number of particles of GDEs investigated by Nano tracking analysis (NTA). The number of particles in GDEs was 1.09 x 10¹² ± 1.19 x 10¹¹ particles/ml and the average size of GDEs was 176.2 ± 4.6 nm while mean value was 144.1 ± 3.5 nm. The image of transmission electron microscopy (TEM) in Fig. 1 (B) displayed that GDEs retained spherical shapes.

3.2. The correlation between GDEs protein concentration and number of particles

As reported in the previous study, the purity of exosomes can be reduced during the isolation procedure due to protein contamination ⁴³. The purity is considered an important factor in evaluating the biological activity and availability of exosomes. To evaluate the purity of GDEs, GDEs protein concentration and number of particles were measured. The protein concentration in GDEs was 1.2µg/µl and the number of GDEs was 1.09 x 10¹² ± 1.19 x 10¹¹ particles/ml after averaging three repeated measurements out. The correlation between GDEs protein concentration and the number of particles was calculated by converting the number of particles to the amount of protein concentration. So. the protein concentration of 1µg/ml, 5µg/ml, and 10µg/ml in GDEs mean to contain the number of 9.08 x 10⁸ ± 9.91 x 10⁷ particles/ml, 4.54 x 10⁹ ± 4.95 x 10⁸ particles /ml, and 9.08 x 10⁹ ± 9.91 x 10⁸ particles/ml respectively. When GDEs were treated by the dose depending manner in this study, the protein concentration of GDEs was used instead of the particle numbers of GDEs.

3.3. Analysis of ginsenosides in GDEs by LC-MS/MS

The amount of Rb1, Rg1, and Rg3 in GDEs was analyzed by LC-MS/MS. The chromatography of intensity and retention time were shown in Fig. 2. Rb1 was measured as 2.88µg per 1ml GDEs. Rg1 was detected as 1.01µg per 1ml GDEs. Rg3 was analyzed as 1.13µg per 1ml GDEs. The lower limit of quantitation of Rb1 was 312µg/ml. The values of coefficients of correlation (R) were more than 0.9986. The lower limit of quantitation of Rg1 was 78.1µg/ml. The values of coefficients of correlation (R) were more than 0.9980. The lower limit of quantitation of Rg3 was 625 ng/ml. The values of R were more than 0.9986.

3.4. Cytotoxicity of GDEs and the effect on cell proliferation

WST-1 assay was performed in order to determine the proper concentration of GDEs in RAW 264.7 (Fig. 3). GDEs did not exhibit any significant toxicity in the various concentration of 0.1 ~ 30µg/ml (GDEs 0.1, 0.5, 1, 5, 10, 20, and 30µg/ml compared to control, GDEs 0µg/ml) for 24h and 48h. Rather, WST-1 assay showed that GDEs meaningfully promoted proliferation of RAW 264.7. Compared to control (GDEs 0µg/ml), the viability of RAW 264.7 was increased depending on the GDEs concentration overall. Specially, the proliferation of RAW 264.7 was significantly elevated at GDEs 5, 10 and 20µg/ml-treated group for 24h and at 5, 10, 20 and 30µg/ml-treated group for 48h respectively.

3.5. Effects of GDEs on the secretion of inflammatory cytokines

The effect of GDEs on secretion of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α in LPS-induced RAW264.7 cells were evaluated using ELISA. Cell supernatants were collected and used for cytokine analysis after LPS-stimulation for 6h and 24h incubation followed by pre-treatment with GDEs (0, 1, 5, and 10µg/ml) for 2h (Fig. 4). The expression level of IL-1β at 6h was very low (0 ~ 4pg/ml) overall (Fig. 4A). Otherwise, LPS significantly increased production of IL-1β at 24h. However, the treatment of 1, 5, and 10µg/ml GDEs suppressed the expression of IL-1β in the dose-dependent manner. LPS also augmented the production of IL-6 (Fig. 4B). At 6h, 10µg/ml of GDEs inhibited the expression of LPS-induced IL-6 while 1µg/ml, 5µg/ml of GDEs slightly stimulated LPS-induced IL-6. IL-6 production at 24h was markedly decreased depending on the concentration of GDEs. LPS up-regulated the production of TNF-α. The expression of TNF-α at 6h was suppressed by 5µg/ml and 10µg/ml of GDEs treatment compared to LPS only treatment while 1µg/ml GDEs treatment rather increased the expression of TNF-α (Fig. 4C). At 24h, the secretion of TNF-α was inhibited by GDEs depending on the concentration. These results indicate that GDEs suppress the secretion of IL-1β, IL-6, and TNF-α in LPS-induced RAW264.7 cells.

3.6. Effect of GDEs on the production of NO

Nitric oxide (NO) is an important inflammatory signal molecule produced by inducible NO synthase (iNOS). L-NMMA is an inhibitor which suppresses iNOS and is used as a positive factor to inhibit NO production in LPS-induced RAW264.7. To examine a proper concentration of L-NMMA, LPS was treated after 2h pre-treatment of L-NMMA in the cells and supernatants were collected after further 24h. As shown in Fig. 5A, LPS dramatically increased the production of NO compared to LPS-untreated group (control). L-NMMA inhibited the production of NO which was induced by LPS in dose dependent manners. Specially, high concentration of L-NMMA (100 and 500µM) suppressed NO production effectively controlling iNOS. Figure 5B displays a role of GDEs in NO production. LPS markedly increased the production of NO compared to compared to control. 1,5 and 10µg/ml of GDEs suppressed NO which was induced by LPS. Specially, NO production by10µg/ml of GDEs was lower than NO level in the cells which were treated with LPS and L-NMMA 100µM. This demonstrated that 10µg/ml of GDEs regulated iNOS and inhibited NO production more effectively than 100µM L-NMMA.

3.7. Effects of GDEs on the protein expression of COX-2 and iNOS

The protein expression levels of inflammatory enzymes, COX-2 and iNOS were assessed by western blot analysis. RAW 264.7 cells were stimulated by LPS following 2h pre-treatment of GDEs (0, 1, 5, 10µg/ml) and cell lysate was collected after 22h. Protein expression of COX-2 and iNOS by LPS treatment was remarkably up-regulated compared to control (untreated group). GDEs gradually suppressed the LPS-induced increase on COX-2 protein expression in a dose-dependent fashion (Fig. 6A and 6C). Also, LPS increased the protein expression of iNOS compared to control. GDEs inhibited the LPS-induced increase on iNOS protein expression (Fig. 6B and 6D). Specially, iNOS expression was significantly decreased when 10µg/ml of GDEs was treated in LPS-induced cell.

3.8. mRNA expression of inflammatory genes

To investigate mRNA transcriptional expression of inflammation-related genes, quantitative real-time PCR was performed. RAW 264.7 cells were stimulated with LPS for further 4h (1µg/ml) after the 2h treatment of GDEs (0, 1, 5, and 10µg/ml) and total RNA was collected. The graphs of Fig. 7 show that mRNA expression levels of TNF-α, IL-1β, IL-6, COX-2, and iNOS which were treated with GDEs and LPS. The numerical values of the groups which were treated with LPS or both LPS and GDEs mean the relative amount compared to control group which was set as 1 (Fig. 7). LPS markedly increased mRNA expression of TNF-α, IL-1β, IL-6, COX-2, and iNOS compared to control (untreated group). GDEs reduced the increased mRNA expression levels of TNF-alpha (Fig. 7A). The expression levels of other cytokines, IL-1β and IL-6 were inhibited in LPS induced cells by GDEs depending on the concentration (Fig. 7B and 7C). In the iNOS expression, 5 and 10µg/ml of GDEs diminished the increased mRNA transcriptional level which was induced LPS while 1µg/ml of GDEs activated the mRNA expression of iNOS (Fig. 7D). The mRNA expression of COX-2 was inhibited by GDEs 10µg/ml, whereas transcription levels by 1µg/ml and 5µg/ml of GDEs were up-regulated compared to LPS only treated group (Fig. 7E).

Inflammatory response regulated by immune cells is a host’s defense mechanism against foreign bodies or damages. Macrophages among immune cells are at the first line of inflammation not only eliminating debris and combating microbes through phagocytosis but also, contributing tissue development, wound healing, and angiogenesis ⁴⁴ RAW264.7, a murine macrophage is commonly used in order to investigate the immunity mechanism regulated by governing the role of macrophage in vitro. Commercially available lipopolysaccharide (LPS), derived from gram-negative bacteria is utilized to mimic the bacterial invasion in the body immune system initiating the inflammatory processes. So, RAW264. 7 cells stimulated by exogenous LPS have been considered the inflammation model secreting pro-inflammatory cytokines and inflammatory mediators ^{45, 46}. GDEs had a spherical shape with an average diameter of 176.2 nm and 1.09 x 10¹² ± 1.19 x 10¹¹ particles were detected per ml. LPS which were treated in RAW 264.7 increased the secretion of cytokines such as IL-1β, IL-6 and TNF-α. GDEs down-regulated the increased secretion of IL-1β, IL-6 and TNF-α proteins. Also, the protein expression levels of inflammation-related mediators, COX-2 and iNOS as well as NO were dramatically reduced by GDEs in LPS-induced RAW264.7. In addition, GDEs treatment diminished LPS-induced mRNA expression levels of the iNOS and COX-2. Moreover, the expression of the IL-1β, IL-6, TNF-α were gradually down-regulated by the GDEs in a dosage dependent manner.

The ginseng has many bioactive constituents such as saponin, polysaccharides, peptides, polyacetylenic alcohol and fatty acid, that exert prominent pharmacological properties. The ginseng saponins, known as ginsenoside or panaxoside are a type of saponins with triterpenoid dammarane structure. Ginsenosides are tremendously heterogeneous because higher plants generally produce structure-similar but bioactivity-different secondary metabolites in one plant. ^{47 44}. Ginsenoside can be divided into three categories: oleanolic, panaxadiol, and panaxatriol ⁴⁵. Well known protopanaxadiols include Rb1, Rb2, Rg3, Rh2, and Rh3 and, also well-known protopanaxatriols include Rg1, Rg2, and Rh1 ⁴⁸. In this study, GDEs isolated from Panax ginseng C. A. Meyer were analyzed by Liquid Chromatography Triple Quadrupole Mass Spectrometry (LC-MS/MS). After membranous vesicles were broken by butanol, organic chemical compounds and chemical residues were extracted. The compounds of ginsenoside Rg1, Rg3, and Rb1 were identified, and their amount was quantified. The previously reported GDEs was isolated from 45% sucrose interface using the method, sequential sucrose gradient-based centrifugations after pellet precipitation. In those GDEs, high concentrated ginsenoside, Rg3 was contained, that was detected by electrospray ionization scanning ³⁵. In this way, it was elucidated that GDEs have the ginsenosides. In this study, it was demonstrated that GDEs contained Rb1 and Rg1 as well as Rg3. There is a possibility that additional bioactive molecules may be found through further studies.

In this study, the cytotoxicity of GDEs was measured in RAW 264.7 cells for 24h and 48h to determine the proper concentration for treatment (Fig. 3). Various concentrations of GDEs were treated, and no cytotoxicity was found. Otherwise, the viability of RAW264.7 treated by GDEs was increased compared to untreated RAW264.7. This showed that GDEs activated murine macrophages and contributed to increase the cell proliferation. The similar data were reported that the proliferation of RAW 264.7 was stimulated by xantan gum with anti-inflammatory effect for 24 ~ 72h ⁴⁹. Moreover, bioactive fraction of aronia melanocarpa, which attenuated LPS-induced inflammation in human bronchial epithelial cells (BEAS-2B) elevated the cell proliferation in low concentration (10–25µg/ml) for 24 ~ 72h ⁵⁰.

In addition, the excessive secretion of pro-inflammatory cytokines in macrophages is an important factor in the upregulation of inflammatory process. Increased TNF-α has been known to trigger the activation of inflammatory genes that cause inflammatory bowel disease and psoriasis. Overexpressed IL-1β was found in multiple sclerosis and encephalomyelitis. IL-6 is strongly increased in autoimmune disease such as rheumatoid arthritis, allergies, and asthma ^{51, 52}. According to this study, data demonstrated that GDEs which were treated in LPS-induced RAW264.7 suppressed the protein expression of TNF-α, IL-1β, and IL-6 (Fig. 4). In addition, mRNA expression levels of LPS-induced TNF-α, IL-1β, and IL-6 were suppressed by GDEs depending on the concentration (Fig. 6). Specially, GDEs suppressed the protein secretion and mRNA expression of TNF-α, IL-1β, and IL-6 although GDEs were exposed for short period, for 4h in ELISA and for 6h qPCR. This demonstrates that GDEs, which act rapidly and interfere the inflammatory signaling and are biocompatible to the natural body system. Cytokine secretion and inflammatory mediators have the interdependency. That is, the excessive accumulation of pro-inflammatory cytokines amplifies inflammation due to a complex array of inflammatory cascade mechanism. The earlier released cytokines affect inflammatory genes in the downstream. For example, IL-1β promotes the secretion of TNF-α and IL-6. TNF-α induces the release of IL-6. Meanwhile, the PEG₂ stimulates the expression of IL-6 ^{53, 54, 55}. Therefore, suppressing the production of pro-inflammatory cytokines may influence to inhibit other dependent cytokines. Accordingly, this analysis shows that GDEs have anti-inflammatory effect regulating the transcriptional and translational levels of TNF-α, IL-1β, and IL-6. Also, GDEs may inhibit the connection between inflammatory cytokine and mediators which affect one to the others as well as stimulating each other in the inflammatory signal pathway.

Regarding NO production, GDEs significantly inhibited NO production in LPS-induced RAW 264.7 (Fig. 5B). LPS-induced macrophages release NO, which are produced by iNOS to catalyze the oxidative deamination of L-arginine into nitric oxide ⁵⁶. The excessive NO production is cytotoxic and provoke deleterious consequences such as septic shock and inflammatory disease ^{57, 58, 59}. NO production in 10µg of GDEs which was treated in RAW 264.7 was lower than the level of LPS and L-NMMA-treated cells. This demonstrated that the suppressive effect of GDEs on iNOS could be better than L-NMMA, that is the inhibitor of iNOS. In addition, mRNA expression of iNOS in GDEs (5µg/ml and 10µg/ml) and subsequent LPS-treated group was lower than LPS only treated group even though 1µg of GDEs increased the mRNA expression of iNOS (Fig. 7D). About protein expression of iNOS, GDEs diminished the expression of LPS-induced iNOS compared to LPS-only treated RAW264.7 in the dosage- dependent manner (Fig. 5B and 5D). There are two isoforms of cyclooxyganse, COX-1 and COX-2. COX-2 is a rate limiting enzyme involved in the conversion arachidonic acid to prostaglandin H₂ (precursor to PEG₂). COX-2 in activated macrophages is overexpressed and upregulates the biosynthesis of prostaglandins ⁶⁰. In this data, 10µg/ml of GDEs attenuated the mRNA level of COX-2 while 1 and 5µg/ml of GDEs increased the expression of COX-2 (Fig. 6). Shown in the result of WB, GDEs treated in LPS-induced RAW264.7 also inhibited protein expression of COX-2 compared to LPS only treated cell in dosage-dependent manner. Taken together, GDEs that regulate iNOS as well as NO and COX-2 inhibit the action of inflammatory mediators in inflammatory response (Fig. 8).

Because ginseng has natural and safe substances, people have used it as pharmaceutical materials for thousands of years. Considering the anti-inflammatory effect of non-steroidal anti-inflammatory drugs (NSAIDs), NSAIDs mitigate pain, inflammation and fever by inhibiting COX-1 and COX-2 either selectively or non-selectively. However, NSAIDs also increase the risk of gastrointestinal bleeding, myocardial infarction, and stroke. So, patients with high risk to NSAIDs, over 65 years old-patients who have cardiovascular diseases or autoimmune diseases need much attention in use of NSAIDs ⁶¹. Due to the side effect of these kinds of drugs, many complementary and alternative medicines such as ginseng are in the limelight. As shown in this study, GDEs containing various ginsenosides have anti-inflammatory effect and seem to have the potential as substitute or adjuvant for NSAIDs. GDEs used in this study already have been verified to be produced cost effectively with high yield and purity. Stability of freeze-dried GDEs was observed by quantifying their protein and RNA which were stored at room temperature up to 60 days ⁴³. Manipulating these advantages, GDEs may help alleviating inflammatory diseases in clinical field. On top of that, GDEs can be used as nano-carriers for drug delivery system. Ginseng have the instinct nature such as biocompatibility, low toxicity and low immunogenicity as well as delivering the natural compound to specific cells ^{62, 63}. GDEs carrying the instinct nature of ginseng also have the potential that can be used as a next-generation drug cargo.

In this study, spherical GDEs were isolated and characterized in their size and number of particles. GDEs containing Rb1, Rg1, and Rg3 can regulate inflammation by attenuating the production of pro-inflammatory cytokines and inflammatory mediators in LPS-induced RAW 264.7. This suggests that GDEs can be used as an anti-inflammatory substitute or adjuvant for NSAIDs and have a potential to be used as nano-carrier of drug delivery system.

Author contributions statement: Y.-K.K. and Y.-H.L. wrote the paper. Y.-K.K. and Y.-H.L. contributed to data acquisition, data analysis and interpretation. Y.-K.K., Y.-H.L., and S.-W.L. provided their expertise and contributed to the figures. Y.-K.K., Y.-H.L., and S.-W.L. provided their expertise and contributed to revisions. All authors have read and agreed to the published version of the manuscript.

Funding: This research was supported by the National Research Foundation of Korea Grant (NRF/2020R1F1A1070072).

Acknowledgments: None.

Conflicts of Interest: The authors declare no conflict of interest.

Availability of Data and Materials: The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

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Anti-inflammatory Properties of Ginseng-Derived Exosome-like Nanoparticles in LPS-induced RAW264.7

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Isolation of GDEs

2.2. Characterization of GDEs

2.3. LC-MS/MS

2.4. Cell culture

2.5. Cell cytotoxicity

2.6. ELISA assay

2.7. Nitric Oxide assay

2.8. Western blot analysis

2.9. Quantitative Real-Time PCR

2.10. Statistical analysis.

3. Results

3.1. Isolation and characterization of GDEs

3.2. The correlation between GDEs protein concentration and number of particles

3.3. Analysis of ginsenosides in GDEs by LC-MS/MS

3.4. Cytotoxicity of GDEs and the effect on cell proliferation

3.5. Effects of GDEs on the secretion of inflammatory cytokines

3.6. Effect of GDEs on the production of NO

3.7. Effects of GDEs on the protein expression of COX-2 and iNOS

3.8. mRNA expression of inflammatory genes

4. Discussion

5. Conclusion

Declarations

References

Table 1

Additional Declarations

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