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Cuttlebone Shows Anti-inflammatory Activity Via Suppression of NF-κB Activation in LPS-induced RAW 264.7 Macrophages
Yakhak Hoeji 2021;65(1):41-45
Published online February 28, 2021
© 2021 The Pharmaceutical Society of Korea.

Geum Seon Lee* and Tae Jin Kang**,#

*Department of Counseling Psychology, Sahmyook University
**Institute of Chronic Diseases and Department of Pharmacy, Sahmyook University
Correspondence to: Tae Jin Kang, Ph.D., Department of Pharmacy, Sahmyook University, Seoul 01795, Republic of Korea
Tel: +82-2-3399-1608, Fax: +82-2-3399-1617
E-mail: kangtj@syu.ac.kr
Received January 7, 2021; Revised February 4, 2021; Accepted February 9, 2021.
Abstract
Our previous study reported that cuttlebone (CB) extract shows wound healing activity and enhances cell migration. In the present study, we examined the anti-inflammatory effect of CB in RAW 264.7 cells activated with lipopolysaccharide (LPS) for induction of inflammation. The expression of inflammatory mediators, including proinflammatory cytokines, was measured using enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR) tests. The results showed that CB suppressed nitric oxide (NO) production in macrophages stimulated with LPS. Production of pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, and IL-6, decreased in LPS-induced RAW 264.7 cells after treatment with CB in a dose-dependent manner. Additionally, gene expression was inhibited by CB in a dose-dependent manner in LPS-stimulated macrophages. Furthermore, CB inhibited nuclear factor kappa B (NF-κB) p65 activation in macrophages activated with LPS. Therefore, these results suggest that CB exerts its anti-inflammatory effects via regulation of the NF-κB signaling pathway.
Keywords : cuttlebone, inflammation, macrophages, cytokines, NF-κB
Introduction

Inflammation is a complex response in living tissue that neutralizes harmful pathogens and protects our bodies, without which infection and wounds cannot heal. Additionally, inflammation heals and repairs the damaged areas.1,2) Inflammation may be acute or chronic; acute inflammation can present in minutes to hours, whereas chronic inflammation can develop gradually. Cells involved in the inflammatory response are diverse, although macrophages play the most important role. Macrophages are antigen presenting cells and the first to recognize pathogens. Macrophages protect healthy cells from attack by foreign substances, such as bacteria and viruses. Additionally, macrophages secrete inflammatory mediators, such as inflammatory cytokines, nitric oxide (NO), vascular amines, and prostaglandin E2. The various functions of macrophages help the body to maintain homeostasis through immune regulation.3-5)

The nuclear factor kappa B (NF-κB) signaling pathway is typically associated with the inflammatory response and secretion of mediators.6) NF-κB is a protein complex found in cytosol, although activated NF-κB translocates into the nucleus. Therein, NF-κB binds to the promoter region and induces the expression of a variety of genes leading to the expression of inflammatory mediators.7,8) Therefore, studies on the inhibition of inflammatory mediators in activated macrophages are related to the control of the inflammatory response.

Cuttlebone (CB), also known as cuttlefish bone, has been used as a traditional medicine. Our previous study shows that CB enhances wound healing and cell migration in a burned rat model, suggesting that CB aids in healing of the damaged and inflamed areas.9-11) Thus, we aimed to investigate the role of CB in the control of inflammatory mediators via the NF-κB pathway in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells.

Methods

Reagents and kits

Cuttlebone (Gyeongdong market, Seoul, Korea), HCl, NaOH, ethyl alcohol, NaNO2 (Daejung, Siheung, Korea), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS, Hyclone, Logan, UT, USA), penicillin/streptomycin (P/S, Hyclone, Logan, UT, USA), Cytokine kit (R&D systems, Minneapolis, MN, USA), total RNA purification kit (GeneAll, Seoul, Korea), cDNA synthesis kit, Taq polymerase (Bioneer, Daejeon, Korea), and NF-κB kit (Cell Signaling, Danvers, MA, USA) were used in these experiments. N-(1-naphthyl) ethylenediamine (NED) and sulfanilamide were obtained from Sigma-Aldrich (Seoul, Korea). The other materials used in this experiment were ultrapure grade.

Preparation of CB extract

CB extract was prepared as described previously.9,11) CB powder was mixed with 6 N HCl for de-mineralization at 25°C. Next, secondary de-mineralization was conducted by treating with 2 N HCl. CB was washed with distilled water and precipitated with 4% NaOH solution. The prepared sample was washed with 95%ethyl alcohol and dried in a vacuum oven.

Cell culture and treatment

RAW 264.7 cells, which is a mouse macrophage cell line, were grown in DMEM supplemented with 10% FBS and 1% P/S. Macrophages were treated with CB and LPS in 24-well plates (5×105 cells/well) and incubated in 5% CO2 at 37°C for 4 or 24 h.

Nitric oxide (NO) measurement

Nitric oxide production was measured by Griess reaction using the standard NaNO2 curve. Briefly, the accumulation of nitrite, an estimate of nitrate (NO3) production, was determined colorimetrically after mixing the culture medium or sample with freshly prepared Griess reagent (0.1% NED and 1% sulfanilamide). The concentration of nitrite was estimated by comparing the absorbance readings at 540 nm (SpectraMax Microplate Reader, The Molecular Devices, San Jose, CA. USA).

Cytokine measurement

Cell culture supernatants were obtained for mouse tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, and IL-1β using an ELISA kit according to the manufacturer’s instructions. Briefly, 96-well plates (Nunc, Penfield, NY, USA) were coated overnight at 4°C with capture antibodies for TNF-α, IL-6, and IL-1β. After washing, the plates were blocked for 1 h at 25°C with blocking buffer. Cytokine standards were added to wells with cell culture supernatants. Then, the detection antibody was added and incubated for 1 h at 25°C. Streptavidin-HRP was added for 30 min, then the plates were incubated with substrate solution for 15 min at 25°C. Stop solution was added and absorbance was measured at 450 nm.

RT-PCR

The attached cells were treated with RiboEx and total RNA was isolated. Reverse transcription was conducted using 1 μg of total RNA and a cDNA synthesis kit. Cyclic temperature reverse transcription (CTRT) was performed 12 times. RT-PCR was performed for 35 cycles of denaturation (94°C, 30 s), annealing (each temperature, 45 s), and extension (72°C, 45 s) with a final extension at 72°C for 10 min (Table 1). The PCR products were observed by electrophoresis on 1.5% agarose gel with ethidium bromide (EtBr) under a UV-transilluminator.

Sequence of the primers used in this study

Target Primer
TNF-α Sense 5'-TTG ACC TCA GCG CTG AGT TG-3'
Anti-sense 5'-CCT GTA GCC CAC GTC GTA GC-3'

IL-1β Sense 5'-GAG GAT GAG ATG AGC ACC-3'
Anti-sense 5'-CTC TGC AGA CTC AAA CTC CAC-3'

IL-6 Sense 5'-GTA CTC CAG AAG ACC AGA GG-3'
Anti-sense 5'-TGC TGG TGA CAA CCA CGG CC-3'

GAPDH Sense 5'-GAG TCT ACT GGC GTC TTC AC-3'
Anti-sense 5'-CCA TCC ACA GTC TTC TGA GT-3'


Measurement of phospho-NF-κB p65 protein by ELISA

The macrophages were treated with CB and LPS in 6-well plates and incubated for 4 h. The cell lysates were collected by chilled cell lysis buffer and incubated on ice for 5 min. Cell lysates were centrifuged, and the supernatants used for the ELISA. The levels of phospho-NF-κB p65 protein were assayed using a commercial kit according to the manufacturer’s instructions.

Statistical analysis

All data are expressed as the mean±SD. Statistical analysis of data was performed using Student’s t-test. Statistical significance was considered at p<0.05 or p<0.01.

Results

Effect of CB on NO production in macrophages

NO is used as a host-defense mechanism by macrophages to fight off pathogens when they enter our bodies and maintain homeostasis.12) Thus, to evaluate the effect of CB on NO production by macrophages stimulated with LPS, we measured NO released during inflammation. NO production decreased in a dose-dependent manner by CB in LPS-induced RAW 264.7 cells (Fig. 1).

Fig. 1. Effect of CB on NO production in RAW 264.7 cells. Cells were treated with LPS and CB at 37°C incubator. After 24 h, cell culture supernatant was collected for assay. Data are representative of at least three independent experiments in duplicate. Data are expressed as means±SD. *<0.05, **<0.01 versus LPS alone based on Student’s t-test.

Effect of CB on pro-inflammatory cytokine expression in macrophages

The production of pro-inflammatory cytokines was measured in the cell supernatants. Macrophages were treated with LPS and CB for 24 h and the supernatants were collected. The protein production and mRNA expression were measured using ELISA and RT-PCR, respectively. The production of TNF-α, IL-1β, and IL-6 was reduced in LPS-stimulated macrophages with CB in a dose-dependent manner (Fig. 2). Additionally, the gene expression levels decreased in CB-treated RAW 264.7 cells (Fig. 3).

Fig. 2. Effect of CB on production of pro-inflammatory cytokine in RAW 264.7 cells. Cells were treated with LPS and CB at 37°C incubator. After 24 h, cell culture supernatant was collected for assay. Data are representative of at least three independent experiments in duplicate. Data are expressed as means±SD. *<0.05, **<0.01 versus LPS alone based on Student’s t-test.

Fig. 3. Effect of CB on gene expression of pro-inflammatory cytokine in RAW 264.7 cells. Cells were treated with LPS and CB at 37°C incubator. After 4 h, cell was harvested and total RNA was isolated. The gene expression was assayed with RT-PCR method and GAPDH was used for a control.

Effect of CB on the activation of NF-κB

NF-κB is a major pathway of the inflammatory response in cells and is activated by a variety of stimuli such as LPS and TNF.6) Components of NF-κB are IκB, p65 (RelA), and p50 (RelB). If NF-κB is activated, IκB is degraded and NF-κB subunits such as p65 and p50 are phosphorylated. The p65 and p50 proteins are translocated into the nucleus and transmit various inflammatory signals.8) Therefore, the reduction of phosphorylated NF-κB is associated with the control of inflammation. NF-κB expression levels were measured in the cell lysates. Phospho-NF-κB p65 protein was inhibited by CB in a dose-dependent manner in LPS-induced RAW 264.7 macrophages (Fig. 4).

Fig. 4. Effect of CB on the activation of NF-κB p65 protein in RAW 264.7 cells. Cells were treated with LPS and CB and then cell lysate was collected by chilled cell lysis buffer and used for measurement of phosphorylated NF-κB p65 protein by ELISA method. Data are representative of at least three independent experiments in duplicate. Data are expressed as means±SD. *<0.05, **<0.01 versus LPS alone based on Student’s t-test.
Discussion

Inflammation is an important process in damaged cells and tissues and is modulated by inflammatory mediators such as NO, TNF-α, IL-1β, and IL-6.1,2) NO is associated with innate immunity and inflammatory responses. Additionally, NO is involved in maintaining homeostasis in the body and eliminating pathogenic microorganisms.12-14) TNF-α is the initial mediator of the inflammatory response to pathogens such as bacteria, fungi, and other infectious microbes and produces cytokines such as IL-1β and IL-6 in inflammatory cells. TNF-α is involved in tissue injury and metabolic disorders and increases vascular permeability.15) Additionally, IL-1β and IL-6 are significant mediators in the inflammatory response. IL-1β is induced by COX-2 and iNOS and increases the expression of endothelial cell adhesion molecules.16) IL-6 plays a role in host defense and in the control of T and B lymphocyte functionality.17) Therefore, the control of inflammatory mediators is the most important factor in the inhibition of inflammation. NO production decreased in activated RAW 264.7 cells after CB treatment (Fig. 1). We measured the production of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 in LPS-stimulated macrophages in the absence or presence of CB, and the data showed that CB suppressed cytokine release by macrophages. These results are consistent with that of our previous study.11) Moreover, the mRNA expression levels was suppressed in LPS-stimulated macrophages treated with CB in a dose-dependent manner (Figs. 2 and 3). These results showed that CB reduced the production of inflammatory mediators.

NF-κB was initially observed in B lymphocytes as a κ chain enhancer protein. The novel proteins, a transcription factor, were elucidated using the electrophoretic mobility shift assay (EMSA).18) NF-κB is a protein complex (IκB, p65, and p50), and it is present in the majority of cell types. NF-κB is involved in numerous cellular processes such as inflammation, immunity, wound healing, and tissue repair.1,7) Stimulated NF-κB translocates from the cytosol to the nucleus in inflammatory cells. In this case, IκB is degraded and NF-κB is phosphorylated in the cytosol. Alternatively, p65 and p50 migrate into the nucleus and express signals8) involved in the secretion of inflammatory mediators such as NO and cytokines. Our study showed that CB inhibited the phosphorylation of NF-κB, suggesting that CB suppresses inflammatory signals in LPS-induced RAW 264.7 cells (Fig. 4).

Conclusion

In conclusion, this study elucidated the inhibitory effect of CB on inflammation. Our results showed that CB exhibits anti-inflammatory effects via the suppression of NF-κB activation. Hence, CB is a potential agent for the control of inflammation. Further studies are required to confirm the detailed mechanism and specific target of a variety of inflammatory cells. Further investigation should focus on pre-clinical studies including the pharmacodynamic evaluation of the CB extract.

Acknowledgment

This paper was supported by the Fund of the Sahmyook University in 2019

Conflict of Interest

All authors declare that they have no conflict of interest

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