Kadsura japonica fruits exert immunostimulatory and anti-obesity activity in RAW264.7 and 3T3-L1 cells

ABSTRACT

Under the COVID-19 pandemic, interest in immune enhancement and anti-obesity is increasing. Thus, in this study, we investigated whether Kadsura japonica fruits (KJF) exhibits immunostimulatory activity and anti-obesity activity. KJF increased the production of immunostimulatory factors and phagocytosis in RAW264.7 cells. Inhibition of TLR2 and TLR4 blocked KJF-mediated production of immunostimulatory factors in RAW264.7 cells. In addition, the inhibition of MAPK and PI3 K/AKT signaling pathway reduced KJF-mediated production of immunostimulatory factors, and the activation of MAPK and PI3 K/AKT signaling pathway by KJF suppressed the inhibition of TLR2/4. KJF attenuated the lipid accumulation and the protein expression such as CEBPα, PPARγ, perilipin-1, adiponectin, and FABP4 related to the lipid accumulation in 3T3-L1 cells. In addition, KJF inhibited excessive proliferation of 3T3-L1 cells and protein expressions such as β-catenin and cyclin D1 related to cell growth. These findings indicate that KJF may have immunostimulatory activity and anti-obesity activity.

KEYWORDS:

• Anti-obesity activity
• immunostimulatory activity
• Kadsura japonica
• macrophages
• adipocytes

1. Introduction

Plants of the genus Kadsura belonging to the Schisandraceae family have been economically and medicinally important resources (Liu et al., 2014). The fruits, leaves, stems, and roots of the plants of the genus Kadsura have been used as folk medicines with different medicinal applications because they have different pharmacological efficacy (Liu et al., 2014). The fruit of a plant of the genus Kadsura, similar to Schisandra chinensis, is used for the treatment of chronic cough and dyspnea, nocturnal emission and spermatorrhea, chronic diarrhea, palpitations, and insomnia (Liu et al., 2014). Their leaves have been used to treat traumatic bleeding and to relieve swelling and pain (Liu et al., 2014). Among plants of the genus Kadsura, Kadsura japonica (KJ) has been used to treat customs, duodenal ulcer, acute gastroenteritis, menstrual pain, postpartum colic, and joint pain in China (Liu et al., 2014). In Japan, the fruits of KJ (KJF), called “Nan-gomishi” have been used as an anti-tussive and tonic (Ookawa et al., 1995). In Korea, KJF has a unique taste like Schisandra chinensis fruits, so it is used as a favorite food such as tea. In addition, KJF is regarded as a valuable medicinal resource because of its good activity in anti-aging and relieving hangover (Kim et al., 2010). Recently, it has been reported that KJ has anti-hepatitis activity and GABA A receptor modulatory activity (Kuo et al., 2005; Zaugg et al., 2011). However, there are no other studies on the pharmacological activity of KJ. In addition, KJ is mainly used as a substitute for Schisandra chinensis although KJ is recognized as a valuable medicinal resource. Therefore, various pharmacological and functional studies of KJ are required to develop a promising natural agent by utilizing KJ.

In this study, as interest in immunity and obesity is increasing amid the prevalence of COVID-19, we evaluated the immunostimulatory and anti-obesity activity of KJ. Furthermore, the mechanism of action of how KJ exhibits the immunostimulatory and anti-obesity activity was investigated.

2. Materials and methods

2.1. Chemical reagents

Kinase inhibitors (ERK1/2 inhibitor PD98059, p38 inhibitor SB203580 and JNK inhibitor SP600125), TLRs inhibitors (TLR2 inhibitor C29 and TLR4 inhibitor TAK-242), 3-(4,5-Dimethylthiazol-2-yl)−2,5-Diphenyltetrazolium Bromide (MTT), dexamethasone, 3-Isobutyl-1-methylxanthine (IBMX), insulin and Oil Red O staining solution were purchased from Sigma Aldrich (St. Louis, MO, USA). The primary and secondary antibodies were purchased from Cell Signaling (Bervely, MA, USA).

2.2. Sample preparation

Kadsura japonica was provided after botanical identification from Forest Medicinal Resources Research Center, National Institute of Forest Science, Yongju, Korea. To prepare the sample, 20 g of Kadsura japonica fruits, leaves, and branches were immersed in 400ml distilled water and then extracted at 40 ^oC for 24 h. After 24 h, the extracts were centrifuged at 12,000 rpm for 10 min at 4 ^oC to obtain a clear supernatant. Then, the recovered supernatant was lyophilized. Freeze-dried Kadsura japonica fruits (KJF), leaves (KJL), and branches (KJB) were stored at −80 ^oC until use. KJF, KJL, and KJB were dissolved in sterile distilled water, and aliquoted in small portions, and stored at −80 ^oC until use.

2.3. Cell culture of RAW264.7 cells and 3T3-L1 cells

RAW264.7 cells and 3T3-L1 cells were purchased from American Type Culture Collection (Manassas, VA, USA). RAW264.7 cells were cultured in Dulbecco's 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 at 37 ^oC under a humidified atmosphere of 5% CO₂. 3T3-L1 cells were cultured in DMEM/F-12 1:1 Modified medium containing 10% bovine calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 ^oC under a humidified atmosphere of 5% CO₂.

2.4. MTT assay for measuring the cell viability of RAW264.7 cells

RAW264.7 cells were plated in a 96-well plate until the cells were more than 80% full. Then, KJF was treated at 37 ^oC under a humidified atmosphere of 5% CO₂ for 24 h. After 24 h, 50 μl of MTT solution (1 mg/ml) was added and then the cells were incubated at 37 ^oC under a humidified atmosphere of 5% CO₂ for 4 h. Then, the cell culture medium was removed, and 100 μl of DMSO was added for eluting the resulting crystals. Then, the absorbance of the eluted resulting crystals was measured at 570 nm using a UV/Visible spectrophotometer (Human Cop., Xma-3000PC, Seoul, Korea).

2.5. Cell proliferation assay for measuring the growth of 3T3-L1 cells

After the treatment was completed, the growth of 3T3-L1 cells was measured using NucleoCounter NC-250 (Chemometec, Allerod, Denmark) according to the manufacturer’s protocol.

2.6. Griess assay for measuring NO secreted by RAW264.7 cells

RAW264.7 cells were plated in a 12-well plate for 24 h. After 24 h, the cells were treated with KJF at 37 ^oC under a humidified atmosphere of 5% CO₂ for the additional 24 h. Then, the cell culture media and Griess reagent (Sigma Aldrich) were mixed at a ratio of 1:1 at room temperature for 15 min, and the absorbance was measured at 540 nm using UV/Visible spectrophotometer (Human Cop., Xma-3000PC, Seoul, Korea).

2.7. Neutral red uptake assay for measuring the phagocytic activity of RAW264.7 cells

RAW264.7 cells were plated in a 12-well plate for 24 h. After 24 h, the cells were treated with KJF at 37 ^oC under a humidified atmosphere of 5% CO₂ for the additional 24 h.

Then, the cell culture media was removed, and the cells were stained with a 0.01% Neutral red solution in a humidified atmosphere of 5% CO₂ for 2 h. After staining, the cells were washed 3 times with 1×PBS, and the stained Neutral red was eluted with a cell lysis buffer (ethanol: acetic acid = 1: 1). The absorbance of the eluted Neutral red was measured at 540 nm using a UV/Visible spectrophotometer (Human Cop., Xma-3000PC, Seoul, Korea).

2.8. Oil Red O staining for measuring lipid accumulation in 3T3-L1 cells

Two days after 3T3-L1 cells were 100% full (designated as day 0), 3T3-L1 cells were cultured with DMI media (DMEM/F-12 containing 10% FBS, 1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 10 μg/ml insulin) for 48 h. Then, on day 2, 3T3-L1 cells were cultured in insulin media (DMEM/F-12 containing 10% FBS and 10 μg/ml insulin) for 48 h. After that, the medium (DMEM/F-12 containing 10% FBS) was then changed every 2 days (days 4 and 6). The treated cells were recovered on day 8. For Oil Red O staining, 3T3-L1 cells were gently washed three times with cold 1 Χ PBS, and then fixed with 10% formalin for 1 h at room temperature. After fixation, 3T3-L1 cells were washed three times with DH₂O, and then 3T3-L1 cells were left with 60% isopropanol for 5 min at room temperature. After 5 min, 3T3-L1 cells were completely dried and stained with Oil Red O solution (60% isopropanol and 40% water) for 20 min at room temperature. After washing with DH₂O five times, stained lipid droplets of 3T3-L1 cells were visualized and photographed with a light microscope (Olympus, Tokyo, Japan), and then dissolved in 100% isopropanol and quantified by measuring the absorbance at 500 nm with a microplate reader (Human Cop., Xma-3000PC, Seoul, Korea).

2.9. Reverse transcription polymerase chain reaction (RT–PCR) for measuring mRNA level in RAW264.7 cells

The isolation of mRNA from RAW264.7 cells and the synthesis of cDNA using the isolated mRNA were carried out using an RNeasy Mini Kit (Valencia, CA, USA) and a Verso cDNA kit (Thermo Scientific, Pittsburgh, PA, USA), respectively. The amplification of the target gene was performed using a PCR Master Mix Kit (Promega, Madison, WI, USA) and the primers. The sequences of the primers used in this study were as follows: iNOS: forward 5’-ttgtgcatcgacctaggctggaa-3’ and reverse 5’-gacctttcgcattagcatggaagc-3’, IL-1β: forward 5’-ggcaggcagtatcactcatt-3’ and reverse 5’-cccaaggccacaggtattt-3’, IL-6: forward 5′-gaggataccactcccaacagacc-3′ and reverse 5′-aagtgcatcatcgttgttcataca-3′, TNF-α: forward 5′-tggaactggcagaagaggca-3′ and reverse 5′-tgctcctccacttggtggtt-3′, GAPDH: forward 5’-ggactgtggtcatgagcccttcca-3’ and reverse 5’-actcacggcaaattcaacggcac-3’. The PCR results were visualized using agarose gel electrophoresis. The density of mRNA bands was calculated using the software UN-SCAN-IT gel version 5.1 (Silk Scientific Inc. Orem, UT, USA).

2.10. Western blot analysis for measuring protein level in RAW264.7 cells and 3T3-L1 cells

The samples for SDS-PAGE were prepared by extracting proteins from RAW264.7 cells and 3T3-L1 cells with RIPA buffer (Boston Bio Products, Ashland, MA, USA) containing protease inhibitor (Sigma-Aldrich) and phosphatase inhibitor (Sigma-Aldrich), and the protein amounts were quantified by BCA protein assay (Thermo Fisher Scientific, Waltham, MA USA). After sample preparation, SDS-PAGE was performed for protein separation, and then proteins separated on the gel were transferred to the PVDF membrane. After the transfer, the PVDF membrane was blocked with 5% nonfat milk in Tris-buffered saline containing 0.05% Tween 20 (TBS-T) at room temperature for 1 h, and then the PVDF membranes were treated with the primary antibodies in 5% BSA in TBS-T at 4 ^oC for overnight. After the primary antibody treatment was completed, the PVDF membranes were treated with the secondary antibodies in 5% nonfat milk in TBS-T at room temperature for 1 h. 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). The density of Western blot bands was calculated using the software UN-SCAN-IT gel version 5.1 (Silk Scientific Inc. Orem, UT, USA).

2.11. Statistical analysis

All the data are shown as mean ± SD (standard deviation). Statistical significance was determined by Student’s t-test. Differences with *P <0.05 and ^#P <0.05 were considered statistically significant.

3. Results

3.1. KJF activates mouse macrophages, RAW264.7 cells

First, we compared the production of the immunostimulatory factors such as NO, iNOS, IL-1β, IL-6, and TNF-α secreted by macrophages in RAW264.7 cells treated with KJF, KJL, or KJB to determine whether KJ induces macrophage activation. As shown in Figure 1(A), the production levels of NO, iNOS, IL-1β, and IL-6 were highest in RAW264.7 cells treated with KJF, but the production of the level of TNF-α was similar in RAW264.7 cells treated with KJF, KJL, or KJB. Therefore, we selected KJF for further study. KJF dose-dependently increased the production of NO, iNOS, IL-1β, IL-6, and TNF-α, and phagocytotic activity in RAW264.7 cells (Figure 1(B and C)). However, KJF was not cytotoxic to RAW264.7 cells (Figure 1D). Moreover, the induction of TNF-α production was similar in RAW264.7 cells treated with KJF or SCF, but the level of NO, iNOS, and IL-1β production was higher in RAW264.7 cells treated with KJF compared to SCF (Figure 1E).

Figure 1. Effect of KJF on macrophage activation in RAW264.7 cells. (A) RAW264.7 cells were treated with KJF, KJL and KJB for 24 h. NO level and mRNA level were measured by Griess assay and RT-PCR, respectively. (B) RAW264.7 cells were treated with KJF for 24 h. NO level and mRNA level were measured by Griess assay and RT-PCR, respectively. (C) RAW264.7 cells were treated with KJF for 24 h. Phagocytic activity was measured by a neutral red assay. (D) RAW264.7 cells were treated with KJF for 24 h. Cell viability was measured by MTT assay. (E) RAW264.7 cells were treated with KJF and SCF for 24 h. NO level and mRNA level were measured by Griess assay and RT-PCR, respectively. *P < 0.05 compared to the cells without the treatment.

3.2. KJF increases the production of immunostimulatory factors through TLR2/4 stimulation in RAW264.7 cells

To investigate toll-like receptor 2 (TLR2) and toll-like receptor 4 (TLR4) are contributing to KJF-mediated increase in production of immunostimulatory factors in macrophages, RAW264.7 cells were pre-treated with C29 (TLR2 inhibitor) or TAK-242 (TLR4 inhibitor) for 2 h and then co-treated with KJF for 24 h. As shown in Figure 2(A and B), inhibition of TLR2 by C29 only decreased the level of NO produced by KJF, and inhibition of TLR4 by TAK-242 attenuated KJF-mediated induction of NO, iNOS, IL-1β, and TNF-α in RAW264.7 cells. However, the production of IL-6 by KJF had no change in the inhibition of TLR2 and TLR4 in RAW264.7 cells.

Figure 2. Effect of TLR2 and TLR4 on KJF-mediated production of immunostimulatory factors in RAW264.7 cells. RAW264.7 cells were pretreated with C29 (TLR2 inhibitor, 100 μM) or TAK-242 (TLR4 inhibitor, 5 μM) for 2 h and co-treated with KJF (100 μg/ml) for 24 h. NO level (A) and mRNA level (B) were measured by Griess assay and RT-PCR, respectively. *P < 0.05 compared to the cells without the treatment. ^#P < 0.05 compared to the cells with KJF alone.

3.3. KJF increases the production of immunostimulatory factors through MAPKs and PI3 K signaling-dependent TLR2/4 stimulation in RAW264.7 cells

To investigate mitogen-activated protein kinases (MAPKs) and phosphoinositide 3-kinase (PI3 K) signaling contributes to KJF-mediated increase in production of immunostimulatory factors in macrophages, RAW264.7 cells were pre-treated with PD98059 (Extracellular signal-regulated protein kinase 1/2 (ERK1/2) inhibitor), SB203580 (p38 inhibitor), SP600125 (c-Jun-N-terminal kinases (JNK) inhibitor) or LY294002 (PI3 K inhibitor) for 2 h, and then co-treated with KJF for 24 h. As shown in Figure 3A, ERK1/2 inhibition by PD98059 reduced the increases in IL-1β, IL-6, and TNF-α by KJF. The inhibition of p38 by SB203580 blocked the production of iNOS, IL-1β, IL-6, and TNF-α by KJF. The inhibition of JNK by SP600125 attenuated the production of NO, iNOS, IL-1β, and IL-6 by KJF. The inhibition of PI3 K by LY294002 decreased the production of NO, iNOS, and IL-6 by KJF. To find out whether KJF activates MAPKs and PI3 K signaling, RAW264.7 cells were treated with KJF by time, and phosphorylation level of ERK1/2, p38, JNK, and PI3 K was investigated by Western blot analysis. As shown in Figure 3B, phosphorylation of ERK1/2 was increased at 3 h after KJF treatment, and phosphorylation of p38, JNK, and PI3 K was increased at 5 min after KJF treatment. Finally, we analyzed whether TLR2 and TLR4 contribute to KJF-induced activation of MAPKs and PI3 K signaling. As shown in Figure 3C, ERK1/2 phosphorylation by KJF was blocked by TAK-242-mediated TLR4 inhibition. The phosphorylation of p38, JNK, and PI3 K by KJF was attenuated by inhibition of C29-mediated TLR2 and TAK-242-mediated TLR4 inhibition.

Figure 3. Effect of MAPK signaling pathway on KJF-mediated production of immunostimulatory factors in RAW264.7 cells. (A) RAW264.7 cells were pretreated with PD98059 (ERK1/2 inhibitor, 40 μM), SB203580 (p38 inhibitor, 40 μM) or SP600125 (JNK inhibitor, 40 μM) for 2 h and then co-treated with KJF (100 μg/ml) for 24 h. NO level and mRNA level were measured by Griess assay and RT-PCR, respectively. (C) RAW264.7 cells were treated with KJF (100 μg/ml) for the indicated times. The protein levels were determined by Western blot analysis. Actin was used as a loading control. (C) RAW264.7 cells were pretreated with C29 (TLR2 inhibitor, 100 μM) or TAK-242 (TLR4 inhibitor, 5 μM) for 2 h and co-treated with KJF (100 μg/ml) for 5 min or 180 min. Protein levels were measured by Western blot analysis. Actin was used as a loading control. *P < 0.05 compared to the cells without the treatment. ^#P < 0.05 compared to the cells with KJF alone.

3.4. KJF inhibits lipid accumulation through downregulating adipogenesis-related proteins in 3T3-L1 cells

To investigate the anti-obesity activity of KJ, we compared the inhibitory effect of KJF, KJL, and KJB against lipid accumulation in 3T3-L1 cells. As shown in Figure 4A, inhibitory activity against lipid accumulation by KJ parts was highest in the order of KJF, KJB, and KJL. Thus, we selected KJF for further study. KJF dose-dependently blocked DMI/insulin-mediated lipid accumulation (Figure 4B) and down-regulated adipogenesis-related proteins such as CCAAT/enhancer-binding protein alpha (CEBPα), peroxisome proliferator-activated receptor-gamma (PPARγ), perilipin-1, fatty acid-binding protein 4 (FABP4), and adiponectin (Figure 4C). In addition, we observed that inhibitory activity against lipid accumulation was higher in KJF treatment than SCF treatment (Figure 4D).

Figure 4. Inhibitory effect of KJF against lipid accumulation in 3T3-L1 cells. (A) 3T3-L1 cells treated with KJF, KJL, and KJB in presence of DMI/insulin. Lipid accumulation was determined by measuring Oil Red O staining. (B) 3T3-L1 cells treated with KJF in presence of DMI/insulin. Lipid accumulation was determined by measuring Oil Red O staining. (C) 3T3-L1 cells treated with KJF in presence of DMI/insulin. Protein levels were measured by Western blot analysis. Actin was used as a loading control. (D) 3T3-L1 cells treated with KJF and SCF in presence of DMI/insulin. Lipid accumulation was determined by measuring Oil Red O staining. *P < 0.05 compared to the cells without KJF treatment.

3.5. KJF inhibits the growth of 3T3-L1 cells in the adipogenesis process

We investigate the effect of KJF on the proliferation of 3T3-L1 cells in the absence or presence of DMI/insulin treatment. As shown in Figure 5A, KJF did not affect the proliferation of 3T3-L1 cells in absence of DMI/insulin treatment, but KJF inhibited the proliferation of 3T3-L1 cells treated with DMI/insulin. In presence of DMI/insulin treatment, KJF dose-dependently suppressed the proliferation of 3T3-L1 cells (Figure 5B) and down-regulated the expression of cell growth-related proteins such as β-catenin and cyclin D1 (Figure 5C).

Figure 5. Inhibitory effect of KJF against the growth of 3T3-L1 cells. (A) 3T3-L1 cells were treated with KJF in the absence or presence of DMI/insulin. Cell growth was determined by NucleoCounter NC-250. (B) 3T3-L1 cells were treated with KJF in presence of DMI/insulin. Cell growth was determined by NucleoCounter NC-250. (C) 3T3-L1 cells treated with KJF in presence of DMI/insulin. Protein levels were measured by Western blot analysis. Actin was used as a loading control.

3.6. KJF inhibits the lipid accumulation and the growth of 3T3-L1 cells in the early and late phases of adipogenesis

To investigate whether KJF affects the late phase of adipogenesis in 3T3-L1 cells, KJF was treated at D0-D8, D2-D8, D4-D8, or D6-D8 during the adipogenesis process, and then lipid accumulation and cell growth were measured. As shown in Figure 6(A and B), KJF treatment during D0-D8 significantly inhibited DMI/insulin-mediated lipid accumulation and proliferation of 3T3-L1 cells. In addition, the lipid accumulation and the proliferation of 3T3-L1 cells were inhibited during KJF treatment of D2-D8, D4-D8, and D6-D8 although inhibitory activity against the lipid accumulation and the proliferation of 3T3-L1 cells decreased as the treatment time of KJF was shortened.

Figure 6. Effect of HML on early phase and late phase in adipogenesis of 3T3-L1 cells. All 3T3-L1 cells were incubated with DMI from D0 to D2. Then, on D2, 3T3-L1 cells were treated with 10 μg/ml of insulin from D2 to D4. After that, the medium (DMEM/F-12 containing 10% FBS) was then changed every 2 days (D4 and D6). KJF (200 μg/ml) was treated concurrently with DMI (KJF D0), treated concurrently with insulin (KJF D2), treated when the medium was changed every 2 days (KJF D4 and KJF D6). The treated cells were recovered on D8. (A) Lipid accumulation was determined by measuring Oil Red O staining. (B) Cell growth was determined by NucleoCounter NC-250. *P < 0.05 compared to the cells without KJF treatment.

4. Discussion

The human body maintains homeostasis by fighting external pathogens through an innate and adaptive immune response. It is known that the innate immune response, which is the first line of defense against foreign pathogens, plays an important role in the initial recognition of the invasion of foreign pathogens and initiating a continuous immune response (Mogensen, 2009). On the other hand, the adaptive immune responses play role in eliminating foreign pathogens in the late phase of infection (Mogensen, 2009). In fact, in recent years, strengthening the innate and adaptive immune system using immunostimulants has been proven to be an effective way to increase resistance to diseases (Zhao et al., 2014). It is known that the innate immune response is mainly mediated by phagocytic cells and antigen-presenting cells such as granulocytes, macrophages, and dendritic cells (Iwasaki & Medzhitov, 2004).

Among the innate immune cells, macrophages are known to maintain body homeostasis by removing invading external pathogens and internal wastes through phagocytosis and repairing damaged tissues (Hirayama et al., 2018). In addition, macrophages are known to contribute to strengthening the adaptive immune system because macrophages could present antigens to T-cells and immunostimulatory factors such as NO, iNOS, IL-1β, IL-6, and TNF-α secreted by activated macrophages contribute to the activation of T-cells and B-cells, which are adaptive immune cells (Hirayama et al., 2018). Therefore, the development of natural products that stimulate macrophages has been required. In this study, we found that KJF treatment further increased the secretion of immunostimulatory factors in RAW264.7 cells compared to KJL and KJB. In addition, KJF increased macrophage phagocytosis. These results show that KJF is a potential natural product that can strengthen the innate immune system through the stimulation of macrophages.

SCF, a well-known traditional Chinese medicine, has been widely used in traditional medicine and functional food for a long time (Zhao et al., 2014). Recently, it has been reported that SCF enhances the immune system through TLR4-mediated activation of macrophages. KJF belonging to the Schisandraceae family is mainly used as a substitute for SCF. Interestingly, it was found that the secretion of immunostimulatory factors of macrophages was higher in KJF treatment than in SCF treatment. These results show that KJF could be used as a main raw material rather than a substitute for SCF in the development of functional agents related to immune enhancement.

Macrophages remove foreign pathogens through an inflammatory response when foreign pathogens invade the body (Hirayama et al., 2018). The inflammatory response by macrophages to remove foreign pathogens initiates when pattern recognition receptors (PRRs) present in the cell membrane of macrophages recognize pathogen-associated molecular patterns (PAMPs) of foreign pathogens (Hirayama et al., 2018). Thus, it has been reported that activation of PRRs can enhance not only innate immunity but also adaptive immunity (Akira et al., 2006; Kawai & Akira, 2009). Among the PRRs, TLRs, which play a central role in the response to foreign pathogens, are known to mainly recognize pathogenic bacteria or viruses (Kawai & Akira, 2011; Schenten & Medzhitov, 2011). In addition, TLR agonists have been used as potential vaccine adjuvants (Kumar et al., 2019). Recently, plant-derived products have attracted attention as potential materials for the development of new vaccine adjuvants because they have no toxicity and side effects (Granell et al., 2010). In this study, it was observed that KJF-mediated increase in the secretion of immunostimulatory factors such as NO, iNOS, IL-1β, and TNF-α was mainly dependent on TLR4 in RAW264.7 cells. These findings show that KJF promotes the secretion of immunostimulatory factors in macrophages. Lipopolysaccharide (LPS), which has strong immunostimulatory activity through TLR4 stimulation, has been studied as a vaccine adjuvant, but its use has been limited due to the toxicity of LPS. (Toussi & Massari, 2014). Thus, it is considered that KJF without cytotoxicity can be used as a vaccine adjuvant that stimulates TLR4.

The secretion of immunostimulatory factors in macrophages results from the activation of the signaling pathways such as MAPKs and PI3 K mediated by TLRs activation (Peroval et al., 2013). In this study, we found that the inhibition of MAPKs and PI3 K signaling pathways blocks KJF-mediated increase in the secretion of immunostimulatory factors of RAW264.7 cells. In addition, KJF increased the phosphorylation of MAPK components (ERK1/2, p38, and JNK) and PI3 K, and the inhibition of TLR2/4 blocked KJF-mediated phosphorylation of MAPK components (ERK1/2, p38, and JNK) and PI3 K. These results show that KJF promotes the secretion of immunostimulatory factors by activating MAPKs and PI3 K signaling pathways through TLR2/4 stimulation.

Obesity, characterized by excessive body weight and abnormal body fat accumulation, is considered a serious disease worldwide because it leads to various metabolic diseases such as hyperlipidemia, type 2 diabetes, cardiovascular disease, and cancer (Chang et al., 2017; Han & Lean, 2016; Keller & Lemberg, 2003; Pi-Sunyer, 2009). In addition, it is reported that obesity weakens the immune system of the human body and increases the infection rate and mortality from viral infectious diseases such as COVID-19 (Andrade et al., 2021). Thus, we also investigated the anti-obesity activity of KJ. In this study, the inhibitory activity of KJF against lipid accumulation in adipocytes was higher than that of KJL and KJB. In addition, KJF exhibited higher inhibitory activity against lipid accumulation than SCF, which was previously reported to have anti-obesity activity (Park et al., 2012). These results show that KJF could be used as a main raw material rather than a substitute for SCF in the development of anti-obesity agents.

It is known that the lipid accumulation in differentiated adipocytes is caused by various factors such as CEBPα, PPARγ, perilipin-1, FABP4, and adiponectin. Indeed, it was reported that when PPARγ was ectopically expressed in fibroblasts, adipogenesis was induced in the fibroblasts, and other factors did not induce adipogenesis in the absence of PPARγ (Farmer, 2006; Rosen & MacDougald, 2006; Tontonoz et al., 1994). CEBPα is an essential transcriptional factor related to adipogenesis in adipocytes (Rosen et al., 2002). It has been reported that a deficiency of CEBPα in fibroblasts reduces adipogenesis (Wu et al., 1999). Perilipin bound to lipid droplets in adipocytes is known to be involved in lipolysis (Greenberg et al., 1991). Phosphorylation of perilipin promotes lipolysis by facilitating the access of hormone-sensitive lipase to lipid droplets (Greenberg et al., 1991). Indeed, it was confirmed that overexpression of perilipin in adipocytes reduced lipolysis (Souza et al., 2002), and the decomposition of basal lipids was promoted in mice deficient in perilipin, resulting in a decrease in body weight of the mice (Tansey et al., 2001). Adiponectin and FABP4 have been reported to be responsible for the formation of mature adipocytes (Moseti, 2016). In this study, we found that KJF reduced the level of adipogenesis-related proteins such as CEBPα, PPARγ, perilipin-1, FABP4, and adiponectin. These results show that KJF could inhibit lipid accumulation in differentiated adipocytes by downregulating the level of adipogenesis-related proteins.

It is known that excessive proliferation of adipocytes is closely related to the increase in adipose tissue observed in obesity (Knittle et al., 1979). Therefore, inhibition of the proliferation of adipocytes is considered a potential target for suppressing obesity. Indeed, it has been reported (-)-Epigallocatechin-3-gallate inhibits the lipid accumulation in adipocytes by suppressing the growth of adipocytes (Chan et al., 2011). In this study, we observed that KJF inhibits DMI/insulin-mediated excessive proliferation of 3T3-L1 cells through downregulation of β-catenin and cyclin D1 protein. β-catenin is known to inhibit the differentiation of preadipocytes into mature adipocytes by downregulating PPARγ and CEBPα (Rosen & MacDougald, 2006). However, it has been reported that β-catenin is overexpressed in mature adipocytes rather than in preadipocytes, which is involved in lipid accumulation in mature adipocytes (Chen et al., 2020). In addition, it was reported that inhibition of β-catenin in mature adipocytes attenuated obesity induced by a high-fat diet and reduced the mass of adipose tissue by inhibiting adipocyte proliferation (Chen et al., 2020). Thus, inhibition of adipocyte proliferation by downregulating β-catenin and cyclin D1 is considered to contribute to the inhibitory activity of KJF against lipid accumulation in mature adipocytes.

5. Conclusion

In conclusion, KJF induced the activation of macrophages through TLR4-mediated activation of MAPK signaling pathways and inhibited the lipid accumulation in adipocytes by downregulating lipid accumulation- and proliferation-related protein expression, which indicates that KJF may have immunostimulatory activity and anti-obesity activity. it is believed that KJF can be developed as an agent related to immune enhancement and anti-obesity in the future. However, animal-based preclinical studies related to KJF’s immune enhancement and anti-obesity are required since this study was cell-based.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2018R1A6A1A03024862).