Panax ginseng abuse exhibits a pro‐inflammatory effect by activating the NF‐κB pathway

Abstract P. ginseng (Panax ginseng C. A. Meyer) is a well‐known traditional medicine that has been used for thousands of years to treat diseases. However, “ginseng abuse syndrome” (GAS) often occurs due to an inappropriate use such as high‐dose or long‐term usage of ginseng; information about what causes GAS and how GAS occurs is still lacking. In this study, the critical components that potentially caused GAS were screened through a step‐by‐step separation strategy, the pro‐inflammatory effects of different extracts on messenger RNA (mRNA) or protein expression levels were evaluated in RAW 264.7 macrophages through quantitative real‐time polymerase chain reaction (qRT‐PCR) or Western blot, respectively. It was found that high‐molecular water‐soluble substances (HWSS) significantly increased the expression of cytokines (cyclooxygenase‐2 (COX‐2), inducible nitric oxide synthase (iNOS), and interleukin 6 (IL‐6)) and cyclooxygenase 2 (COX‐2) protein; gel filtration chromatography fraction 1 (GFC‐F1) further purified from HWSS showed prominent pro‐inflammatory effects by increasing the transcription of cytokines (COX‐2, iNOS, tumor necrosis factor alpha (TNF‐α), and interleukin 1β (IL‐1β)) as well as the expression of COX‐2 and iNOS protein. Moreover, GFC‐F1 activated nuclear factor‐kappa B (NF‐кB) (p65 and inhibitor of nuclear factor‐kappa B alpha (IκB‐α)) and the p38/MAPK (mitogen‐activated protein kinase) signaling pathways. On the other hand, the inhibitor of the NF‐κB pathway (pyrrolidine dithiocarbamate (PDTC)) reduced GFC‐F1‐induced nitric oxide (NO) production, while the inhibitors of the MAPK pathways did not. Taken together, GFC‐F1 is the potential composition that caused GAS through the production of inflammatory cytokines by activating the NF‐кB pathway.

To date, the pharmacological functions of ginseng are attributed to abundant pharmacologically active compounds, such as ginsenosides, polysaccharides, peptides, polyacetylene alcohols, fatty acids, and proteins (Cho et al., 2014;Kim et al., 2017;Ru et al., 2015). Many studies have been focused on the mechanisms for the pharmacological role of such active compounds. For example, ginsenosides exerting antitumor effect are attributed to inhibit tumor invasion or metastasis and induce tumor cell apoptosis; their anti-inflammatory effect takes place through inhibition of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced COX-2 expression (Vazquez & Aguera Ortiz, 2002) or the production of pro-inflammatory cytokines by regulating the transcription factor NF-κB and activator protein-1 (AP-1) (Jee et al., 2014); on the other hand, polysaccharides also play indispensable roles in the medicinal value of ginseng, such as enhancing immunity (Yu et al., 2014), regulating blood glucose levels (Ratan et al., 2021), exerting antioxidant effects , and causing antitumor effects (Jee et al., 2014), possibly by activating transcription factors (NF-κB and AP-1) along with their upstream signal transduction enzymes such as extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) (Byeon et al., 2012). Although ginseng abuse or misuse always occurs in daily life, the underlying reason for producing GAS is almost ignored.
Inflammation is an innate immune response marked by capillary dilatation, leukocytic infiltration, redness, heat, and pain, and it can eliminate toxic stimuli and damaged tissues (Kim et al., 2017). As the primary immune cells for innate defense against infections (Kang & Min, 2012), macrophages play a crucial role in the inflammatory response. First, macrophages recognize harmful stimuli through toll-like receptors (TLRs) and leucine-rich repetitive sequence receptors (nucleotide oligomerization domain (NOD)-like receptors (NLRs)) (Kim et al., 2017). Subsequently, these receptors trigger a series of signaling cascades, such as NF-кB and MAPK pathways; NF-кB is a crucial transcription factor connected to immune and acute inflammatory responses. The MAPK pathway, which activates downstream transcription factors that cause the transcription of pro-inflammatory genes and the secretion of pro-inflammatory cytokines (such as TNFα, IL-1β, and IL-6), is intimately tied to the activation of NF-кB pathway (Shin et al., 2018). The transcriptional expression of inducible nitric oxide synthase (iNOS) will promote the persistent production of NO. COX-2 is also an inducible enzyme and associated to the pathogenesis of inflammatory-related diseases (Bai et al., 2019). However, the uncontrolled production of pro-inflammatory mediators will evoke an inflammatory response (Azike et al., 2015;Deng et al., 2020). The continuous inflammatory reaction will lead to the overactivation of the innate immune system, in turn causing harmful pathological consequences to the host (García-Lafuente et al., 2009).
Due to excessive consumption of P. ginseng inducing inflammation-like symptoms, whether or not the causal mechanism for GAS involves the occurring process of inflammation and how to make such GAS are still unclear. This study aims to explore the bioactive components that caused GAS and reveal the underlying mechanism for producing GAS. In this study, we extracted different fractions from P. ginseng roots through an activity-directed separation strategy and investigated their possible effects in vitro. Results showed that it is the water-soluble fraction, GFC-F1 (gel filtration chromatography fraction 1), from high-molecular water-soluble substances (HWSS) of P. ginseng that significantly induce the levels of pro-inflammatory cytokines by activating NF-κB, not the MAPK signaling pathway.

| Material and treatments
Fresh six-year-old P. ginseng roots were collected from the ginseng planting research base in Jilin province. First, the pro-inflammatory components were screened from ginseng roots using a stepwise screening method (Yan et al., 2014). As shown in Figure 1a, 1 kg of ginseng roots was rinsed, dried, and added with 2 L of ultrapure water for homogenization. Next, the ginseng homogenate was centrifuged, and the resulting supernatant and precipitate were used to prepare water-soluble substances (WSS) and ethyl acetate extracts (EAE), respectively. Then, WSS was separated into high-molecular water-soluble substances (HWSS) and low-molecular compounds (LWSS) through dialysis with dialysis bags (8-14 kDa cut-off) at 4°C for 3-5 days. The HWSS was further purified by gel filtration chromatography (GFC). After being filtered with a 0.  Cell viability was performed to determine the growth inhibitory effects of ginseng extracts on RAW 264.7 cells at different concentrations with a cell counting kit-8 (CCK-8) testing kit (Zoman). In brief, the cells were cultured in a 96-well plate at a density of 1 × 10 4 cells/well. After 24-h preincubation, the cells were treated with different ginseng extracts for 18 h, respectively. Subsequently, 10 μl of CCK-8 solution was added to each well, and the cells were further incubated for 2 h. Finally, the absorbance was determined at 450 nm using a microplate reader (Tecan M200 PRO). All experiments were performed with three replicates. 72°C for 10 s. The β-actin gene was used as an internal standard.
The gene expression levels relative to the control were analyzed using the 2 −△△Ct method. The primers of each gene for qRT-PCR are shown in Table 1.

| Nitric oxide (NO) assay
After 18-h cell incubation with samples of different concentrations, the nitrite (sodium nitrite (NaNO 2 )) content was quantified using a Total Nitric Oxide Assay Kit (Beyotime, Shanghai, China). Briefly, 50 μl of culture supernatants was mixed with 50 μl of Griess reagents І and II. The absorbance at 540 nm was measured. Nitric oxide (NO) concentrations were calculated with NaNO 2 as the standard curve.
In the inhibitor experiment, the cells were pretreated with inhibitors for 2 h before the sample treatments.

| Western blot analysis
After 12-h cell incubation with samples of different concentrations, the cells were washed with prechilled phosphate-buffered saline (PBS) three times and lysed with lysis buffer. After centrifugation (12,000 g, 4°C, 5 min), the supernatant was collected for Western blot analysis. The proteins were quantified with a bicinchoninic acid assay (BCA) kit.

| Statistical analysis
All results were analyzed with SPSS Statistics v.19 and shown as the mean ± standard error (SE). The statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Duncan's multiple range tests for comparison among different groups. The differences at p < .05 were considered to be statistically significant. The signaling pathway map was created with BioRe nder.com.

| Effects of LWSS and HWSS on cell viability and pro-inflammatory activity
The On the other hand, no significant difference in such genes' mRNA levels was found between the control and the LWSS-treated groups.
In addition, the COX-2 protein content was also markedly induced by HWSS, similar to the treatment of LPS, while the COX-2 protein content in the LWSS-treated group was similar to the control (Figure 3e-f).

| Effects of GFC fractions on cell viability and pro-inflammatory activity
The HWSS with pro-inflammatory activity was further separated into two fractions (GFC-F1 and GFC-F2) by GFC (Figure 1b)  are responsible for GFC-F1-induced macrophage activation, immunoblot analysis was utilized to assess the phosphorylation state of crucial enzymes in NF-κB and MAPK pathways. The transcription factor NF-κB, consisting of the subunits p65 and p50, is involved in the transcriptional regulation of pro-inflammatory kinases. NF-κB activation requires the phosphorylation and degradation of its endogenous inhibitor (IκBα). Compared to the control, the protein levels of p-p65 (the phosphorylation state of p65) were clearly increased, and the p-IκBα (the phosphorylation state of IκBα (inhibitor of nuclear factor-kappa B alpha)) protein levels were slightly increased by LPS or GFC-F1 treatment; however, the p65 or IκBα protein levels were similar among treatments (Figure 6a). In addition to the NF-κB transcription factor, MAP kinases (p38, JNK, and ERK) are involved in the production of cytokines and NO. It was found that the p-p38

| GFC-F1 activated NFκ B and MAPK
protein level was higher in LPS or GFC-F1-treated group than in the control; except for the p-p38 protein level, the protein levels of other enzymes were similar among different treatments (Figure 6b).
To provide further evidence of GFC-F1-activated NF-κB and MAPK pathways, RAW 264.7 macrophages were pretreated with inhibitors of NF-κB or MAPKs, and their effects on NO production in GFC-F1-treated RAW 264.7 cells were assessed. As illustrated in Figure 7, NO production was significantly increased in GFC-F1treated RAW 264.7 cells compared to the control group; PDTC (pyrrolidine dithiocarbamate) (NF-κB inhibitor) can efficiently inhibit NO production in GFC-F1-stimulated RAW 264.7 macrophages.

| DISCUSS ION
Panas ginseng is the most well-known traditional herbal medicine used as a therapeutic supplement or functional food to treat various diseases for a long time (Um et al., 2020). Previous studies have believed that the pharmacological effects of ginseng are derived from its various active components, mainly including ginsenosides, polysaccharides, peptides, etc. (Kim et al., 2017), and most researches mainly focus on the functions of ginsenosides and polysaccharides.
Of them, ginsenosides, the major bioactive constituents of P. ginseng, show different therapeutic effects, including anti-inflammatory, anticancer, anti-oxidative, and antidiabetic effects (Kim, 2018). While ginseng polysaccharides, the most abundant components of ginseng, have antitumor, anti-inflammatory, antioxidative, and immunomodulatory activities (Guo et al., 2021). Although P. ginseng is beneficial to human health, excessive intake of P. ginseng can still cause some side effects, such as "ginseng abuse syndrome" (GAS), which is a generally accepted adverse event related to P. ginseng (Siegel, 1979). A previous study has also reported that overconsumption of Korean red ginseng, the steamed root of P. ginseng, could induce "shanghuo" .
There are similarities in symptoms between GAS and "shanghuo," which are related to inflammation. It was reported that the watersoluble substances in "heating fruits" may be the key components to induce inflammation. For example, the water-soluble extracts of litchi, longan, and dried longan significantly increased the production of prostaglandin E2 (PGE 2 ) and the expression of COX-2 protein in RAW 264.7 macrophages (Huang & Wu, 2002). Moreover, water-soluble F I G U R E 3 Effects of LWSS and HWSS on RAW 264.7 macrophages. Cells were treated with various concentrations of LWSS and HWSS (100, 200, 500, and 1000 μg/ml) for 18 h. LPS (1 μg/ml) as a positive control. (a) The viability was measured by a CCK assay kit. Cells were treated with 500 μg/ml of LWSS/HWSS for 18 h, and the mRNA levels of COX-2 (B), iNOS (C), and IL-6 (D) were measured by qRT-PCR. (e,f) The COX-2 protein level was measured by Western blot analysis. Data are presented as the mean ± SE (n = 3), and different lowercase letters on the bar indicate significantly different values (p < .05). CCK, cell counting kit; COX-2, cyclooxygenase-2; HWSS, highmolecular water-soluble substances; IL-6, interleukin-6; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; LWSS, low-molecular water-soluble substances; mRNA, messenger RNA; qRT-PCR, quantitative real-time polymerase chain reaction.
extract of satsumas mandarin promoted pro-inflammatory mediators' production, showing an apparent pro-inflammatory effect (Yan et al., 2014). However, they found no obvious effects of EAE on PGE 2 production (Huang & Wu, 2002;Yan et al., 2014). In this study, we also found that WSS significantly increased the expression levels of proinflammatory cytokines (Figure 2b-d). What is more, it was HWSS that remarkedly enhanced the transcription level of pro-inflammatory mediators (Figure 3b-d) rather than EAE and LWSS. Although EAE and LWSS had minimal effects on pro-inflammatory mediators, it might be attributed to an emergency reaction of that fast response to eliminate adverse reaction of external stimulus, even slight stimuli by producing cytokines (Um et al., 2020). In addition, we further purified two fractions (GFC-F1 and GFC-F2) from HWSS and found that GFC-F1 induced a significant inflammation response (Figure 4b-f). Therefore, we could suggest that the high-molecular water-soluble substance of P. ginseng, GFC-F1, plays a significant role in the pro-inflammatory effect in the case of excessive ginseng consumption. Although the molecular weight of GFC-F1 is 11-60 kDa with four primary components (Figure 1c), and some studies suggested that polysaccharides or proteins in the high-molecular watersoluble substances are the key components that cause inflammation (Li et al., 2010;Wang et al., 2016;Yan et al., 2014). For instance, the aqueous extract containing polysaccharides of North American ginseng has an immunostimulatory effect and can increase the production of NO, TNFα, and IL-6 (Azike et al., 2015). Azike et al. (2015) also found that acidic polysaccharide fractions with molecular weight ≥ 100 kDa enhanced the production of NO and TNFα. There was also a study showing that acidic polysaccharide of Korean red ginseng activated macrophages through NF-κB and AP-1 as well as the enzymes of 255 signaling pathway, including ERK and JNK, thereby resulting in the activation of transcription factors (Byeon et al., 2012). Although a novel protein of American ginseng can dose-dependently enhance NO production in murine peritoneal macrophages (Qi et al., 2016), there is currently no research to substantiate the pro-inflammatory effect of proteins in P. ginseng. Therefore, the exact constituent of GFC-F1 causing the occurrence of GAS needs further study in the future.
Inflammation, an immune response that protects our body against various stimuli, is closely associated with the release of proinflammatory mediators such as iNOS, COX-2, and pro-inflammatory cytokines such as interleukins (ILs) and TNFα (Bak et al., 2012).
The expression of pro-inflammatory cytokines is regulated by the NF-κB and MAPK pathways at both transcriptional and posttranscriptional levels (Bak et al., 2012;Choi et al., 2008). It is reported that thaumatin-like protein isolated from Litchi chinensis has pro-inflammatory activity and can enhance the expression of inflammation-related genes (such as COX-2, iNOS, TNFα, and p65; its pro-inflammatory activity might be regulated by the NF-κB pathway (Chen et al., 2020).
Furthermore, P. ginseng containing various pharmacological components has been well known as an immune modulator (Son et al., 2020), enhancing macrophage activity and producing many cytokines and inflammatory mediators, such as NO (Kang & Min, 2012 pathway (Friedl et al., 2001). In addition, the water extract of wildsimulated ginseng increased the production of pro-inflammatory mediators (such as NO, iNOS, COX-2, IL-1β, IL-6, and TNFα) and activated macrophages through MAPK, NF-κB, and PI3K/AKT signaling pathways (Um et al., 2020). Herein, the present study found that WSS, HWSS isolated from WSS, and GFC-F1 purified from HWSS enhanced the expression levels of pro-inflammatory genes, such as COX-2, iNOS, TNFα, and IL-1β and NO production. Moreover, GFC-F1 activated key factors in both NF-κB and MAPK pathways in RAW 264.7 macrophages ( Figure 6). However, NF-κB inhibitor efficiently inhibited NO production in GFC-F1-stimulated RAW 264.7 macrophages, while MAPK inhibitors increased NO production rather than reducing NO production ( Figure 7). Hence, we could conclude that the pro-inflammatory activity of GFC-F1 takes place through the regulation of the NF-κB signaling pathway (including the phosphorylation of p65 and IκBα) rather than the MAPK pathway ( Figure 8).

| CON CLUS IONS
Panas ginseng can activate macrophages to enhance the immune system. However, excessive consumption of ginseng may cause overactivation of the immune system forming GAS. The present results suggested that GFC-F1 (the molecular weight range of 11-60 kDa) purified from the high-molecular water-soluble substance of ginseng is the crucial component that caused GAS. Moreover, GFC-F1 activates the NF-κB pathway to trigger the immune system leading to inflammatory responses by inducing the phosphorylation of p65 and IκBα. These results provide a theoretical basis for improving ginseng quality and guidelines for the scientific consumption of ginseng.
However, the specific component that may cause GAS still needs further study.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.