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Journal of Ginseng Research logoLink to Journal of Ginseng Research
. 2025 Feb 10;49(3):282–293. doi: 10.1016/j.jgr.2025.02.001

Ginsenoside Rg2, a principal effective ingredient of Panax ginseng, attenuates DSS-induced ulcerative colitis through NF-κB/NLRP3 pathway

Ji Zhang a,1, Jing Xie a,1, Zhiqiang Niu b, Long You a, Yanan Liu b, Rui Guo b, Guigui Yang a, Ziliang He a, Ting Shen b, Honggang Wang c,⁎⁎, Qi Yan d,⁎⁎⁎, Weicheng Hu b,⁎
PMCID: PMC12125587  PMID: 40453354

Abstract

Background

Ginsenoside Rg2 (G-Rg2), a major active compound of Panax ginseng, exhibits a wide range of pharmacological properties, including anticancer, antioxidant and neuroprotective effects. However, the mechanisms by which G-Rg2 mitigates ulcerative colitis (UC) have not been clearly elucidated.

Aims

In the present study, we aimed to elucidate the underlying mechanisms by which G-Rg2 mitigated UC.

Methods

In this study, we investigated the efficacy of G-Rg2 in ameliorating dextran sulfate sodium (DSS)-induced UC and its potential mechanisms using a DSS-induced UC mouse model and Lipopolysaccharides (LPS)/nigericin (Nig)-induced NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome activation on immortalized bone marrow-derived macrophages (iBMDMs).

Results

Oral administration of G-Rg2 at doses of 10 and 20 mg/kg significantly mitigated weight loss, normalized food and water intake, and improved colon histopathology in DSS-induced UC mice. G-Rg2 also restored mRNA expression levels of occludin, claudin-3, zona occluden (ZO)-1 and mucin 2, thereby enhancing intestinal barrier integrity. G-Rg2 significantly suppressed the nuclear translocation of p65, the subunit of nuclear factor kappa-B (NF-κB), as well as downregulated NLRP3, cleaved IL-1β and caspase1 p20 expression induced by LPS/Nig in iBMDMs.

Conclusion

G-Rg2 effectively reduced colon inflammation in DSS-induced UC mice and diminishes inflammatory responses under LPS/Nig conditions by regulating NF-κB/NLRP3 pathway, thereby inhibiting NLRP3 inflammasome activation, which may serve as a potent therapeutic agent for UC.

Keywords: Ginsenoside Rg2, Ulcerative colitis, NLRP3 inflammasome, NF-κB

Graphical abstract

Image 1

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Highlights

  • •

    Ginsenoside Rg2 (GRg2) is a tetracyclic triterpenoid saponin derived from Panax ginseng.

  • •

    GRg2 mitigates symptoms in mice with DSS-induced colitis.

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    GRg2 suppresses the activation of NLRP3 inflammasomes in DSS mice.

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    GRg2 inhibits inflammasome activation in iBMDMs.

1. Introduction

Ulcerative colitis (UC) is a chronic inflammatory condition of the colon's mucosal layer, characterized by symptoms such as abdominal pain, cramping, diarrhea, urgency to defecate, and weight loss, which significantly affect quality of life [1]. The pathogenesis of UC primarily involves damage to the intestinal barrier, dysbiosis of the gut microbiota, and hyperactivation of immune cells [[2], [3], [4]]. Currently, glucocorticoids are primarily used to treat UC due to their anti-inflammatory and immunosuppressive effects. However, there is growing evidence that prolonged use of glucocorticosteroids can lead to various side effects, including metabolic disorders, increased digestive burden, neuroinflammation, and diabetes [5,6]. Consequently, it is essential to investigate natural products to improve treatment options for UC patients.

The NLRP3 inflammasome is a multi-protein complex comprised of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and caspase-1. It is activated by pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), including Nig, adenine triphosphate (ATP), and monosodium urate crystals (MSU) [7,8]. As a critical component of the innate immune system, excessive activation of the NLRP3 inflammasome is associated with various inflammatory diseases in humans, such as neurodegenerative diseases, acute lung injury (ALI), sepsis, atherosclerosis, and peritonitis [9,10]. Emerging evidence supports that modulation of NLRP3 inflammasome activation is a promising strategy for alleviating UC. For example, L-Fucose has been shown to reduce inflammation in rat colons and inhibit macrophage M1 polarization by diminishing NF-κB/NLRP3 inflammasome pathway activity [11]. Furthermore, shaoyao decoction has restored intestinal barrier integrity and suppressed pro-inflammatory cytokines by inhibiting the MKP1/NF-κB pathways in vivo [12]. Recent findings also highlight those small molecules and natural products, such as MCC950 and oridonin, can mitigate NLRP3-associated diseases by specifically targeting NLRP3 in vivo [13,14]. Meanwhile, excessive pro-inflammatory cytokines resulting from disrupted intestinal barrier function have been addressed by interventions like G-Rg3, which protects the intestinal barrier and reduces intestinal dysbiosis, as found by Liu et al. [15]. These studies suggest that a variety of potential therapeutics could alleviate colonic inflammation and intestinal barrier dysfunction by inhibiting NLRP3 inflammasome activation.

Ginseng is an herb renowned for its anti-inflammatory, antioxidant, and immune-boosting properties [16]. G-Rg2, a tetracyclic triterpenoid saponin derived from ginseng, exhibits a broad spectrum of pharmacological effects. Previous research also demonstrated that G-Rg2 can lower blood lipids, inhibit platelet aggregation, and mitigate cardiovascular diseases such as atherosclerosis [17]. Additionally, G-Rg2 has shown anti-cancer properties and has been reported to protect breast cancer cells [18]. However, the anti-inflammatory effects of G-Rg2 on UC as well as its potential mechanisms remain poorly defined. To address this gap, our study aimed to investigate the protective effects of G-Rg2 against UC and explore the potential mechanism of LPS/Nig-induced inflammasome activation in iBMDMs.

2. Materials and methods

2.1. Materials and reagents

G-Rg2 (Purity ≥98 %) was obtained by Professor Li Fu as a gift from the Natural Products Research Center of Chengdu Institute of Biology, Chinese Academy of Sciences. Dextran Sulfate Sodium Salt (60316EH25) was acquired from Yeasen, Shanghai, China. 5-ASA (A12982) and Nigericin (N102401) were sourced from Aladdin, Shanghai, China. TRizol reagent (15596026) was obtained from Ambion (Austin, TX, USA). Phosphate-buffered saline (PBS) (SH30256.01), Dulbecco's Modified Eagle's Medium (DMEM) (AH29882652), and penicillin-streptomycin antibiotics (SH40003.01) were purchased from Cytiva (Marlborough, MA, USA). Thiazolyl Blue Tetrazolium Bromide (MTT), LPS (L2880) and fetal bovine serum (FBS) (F8318) were procured from Sigma-Aldrich, (St. Louis, MO, USA). ECL reagent (CW0049M) was purchased from CWBio (Jiangsu, China). The NovoStart® SYBR qPCR SuperMix Plus kit (E096-01A) was sourced from Novoprotein (Shanghai, China). The LDH cytotoxicity assay kit (C0017) and BCA protein quantification kit were obtained from Beyotime Insititute of Biotechnology (Nantong, China). Mouse IL-1β ELISA kit (VAL601) and mouse TNF-α ELISA kit (VAL609) were acquired from Novus Bio (Centennial, CO, USA). Cleaved-caspase1 (#89332), cleaved-IL-1β (#63124), NLRP3 (#15101), pro-IL-1β (#12242), pro-caspase1 (#24232), NF-κB p65 (#D14E12), phosphorylated IκBα (#2859), IκBα (#9242S), ERK (#4695), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (#4370), JNK (#4370), phospho-SAPK/JNK (Thr183/Tyr185) (#4671), p38 MAPK (#9212), phospho-p38 MAPK (Thr180/Tyr182) (#4511), and ASC (#67824) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). GAPDH (ET1601-4) was acquired from HuaBio (Hangzhou, China). Anti-Rabbit IgG (HRP) (ab32513), anti-Mouse IgG (HRP) (ab6789), GSDMD (ab21980), and Cleaved-GSDMD (ab255603) were purchased from Abcam (Cambridge, UK). Cell culture dish (430166), 96-well plate (3599), and 6-well plate (3516) were obtained from Corning Incorporation (Oneonta, NY, USA).

2.2. Cell culture and treatment

iBMDMs were provided by Prof. Feng Shao from NIBS (Beijing China). The cells were cultured in DMEM medium supplemented with 10 % FBS and 1 % penicillin-streptomycin antibiotics at 37 °C and 5 % CO2. For the western blot assay and RT-qPCR experiments, cells were seeded at a density of 1 × 106 cells/well in a 6-well plate. 12 h later, the cells were pre-treated with G-Rg2 for 30 min, followed by treatment with 1 μg/mL LPS for 3 h, and subsequently, the cells were stimulated with 10 μM Nig to activate the inflammasome.

2.3. Cell viability analysis

iBMDMs were seeded at a density of 1 × 104 cells/well in a 96-well plate and incubated for 12 h. The cells were then treated with different concentration of G-Rg2 (0, 5, 10, 20, 30, 40, 50 μM) for 24 h. After discarding the supernatant, the cells were exposure to 100 μL of MTT solution (0.5 mg/mL) and incubated for an additional 4 h. Subsequently, 100 μL of MTT stopping buffer were added to each well to stop the reaction. The optical density at 550 nm was measured using multimode microplate reader (Tecan Group Ltd., Männedorf, Switzerland).

2.4. LDH release, ROS accumulation, and mitochondrial membrane potential assay

The LDH cytotoxicity assay kit was used to measure the release of LDH from iBMDMs culture supernatants following treatment with LPS/Nig. A reactive oxygen species (ROS) detection kit and a mitochondrial membrane potential assay kit with JC-1 were used to measure the level of ROS and mitochondrial membrane potential in iBMDMs after treatment as described above. All commercial test kits were used according to the manufacturer's instructions.

2.5. Scanning electron microscope (SEM)

iBMDMs were seeded at a density of 1 × 106 cells/well in a 12-well plate and incubated for 12 h. Cells were subsequently treated with G-Rg2 for 30 min followed by 1 μg/mL LPS for 3 h, and then 10 μM Nig. After treatment, the cells were washed with PBS and fixed with 2.5 % glutaraldehyde at 4 °C for 2 h. Post fixation, the cells were washed with PBS and sequentially dehydrated using increasing ethanol concentrations (20 %, 50 %, 70 %, 90 %, 100 %). They were then dried in a freeze-drying oven, mounted on conductive adhesive, and gold-coated in preparation for scanning electron microscopy (SEM) analysis.

2.6. Immunostaining and fluorescence microscopy

After washing with PBS, iBMDMs were fixed with 4 % paraformaldehyde at 4 °C for 15 min. Cells were then permeabilized with 0.5 % Triton X-100 for 10 min and blocked with 3 % BSA for 1 h. Primary antibodies were incubated for 12 h at 4 °C, followed by secondary antibodies for 2 h at room temperature. The nuclei were stained with DAPI solution. The cover glass was cleaned with PBS and permanently sealed with nail polish. Finally, the samples were observed under a fluorescence microscope (Scope AI, ZEISS).

2.7. Animals and treatment

The mice were acquired from SPF Biotechnology (Beijing, China). Forty mice were randomly assigned into five groups (n = 8 per group): (1) control, (2) DSS model, (3) treated with G-Rg2 (10 mg/kg) and DSS, (4) treated with G-Rg2 (20 mg/kg) and DSS, and (5) treated with 5-ASA (50 mg/kg) and DSS. To model UC, all groups except the control were administered 3 % DSS in their drinking water for 7 days. Throughout this period, the DSS group did not receive any G-Rg2 or 5-ASA treatments. Both G-Rg2 and 5-ASA were suspended in 0.5 % CMC-Na (Sigma-Aldrich, St. Louis, MO, USA) and delivered via gavage. Changes in body weight, food and water intake were recorded daily. At the study's conclusion, the mice were euthanized and administered Zoletil®50 (Virbac, Carros, France). Blood was collected via orbital bleeding, and the colon length was measured. The spleen was weighed, and samples of serum, colon and spleen tissue were collected for analysis. Disease Activity Index (DAI) scores were calculated as specified in the provided in Table S1. All animal experiments were approved by Yangzhou University-Institutional Animal Care and Use Committee at Nov 27, 2023 (No.202312006).

2.8. Hematoxylin and Eosin (H&E) staining

The colon was fixed in 4 % paraformaldehyde, and then embedded by paraffin. The cut sections were stained with H&E for histopathology. Images were taken using a microscope (Nikon Eclipse E100, Sendai, Japan).

2.9. Reverse transcriptase-polymerase chain reaction analysis

RNA from cells and tissues was extracted using Trizol, and cDNA was synthesized using a reverse transcription kit (Takara, Otsu, Japan). The sequences of the primers used for qRT-PCR are detailed in Table S2. These primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The reaction system was configured according to the NovoStart® SYBR qPCR SuperMix Plus kit instructions and carried out using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Results were calculated using the comparative cycle threshold method (2-ΔΔCT).

2.10. Western blot analysis

The total proteins were extracted using Radio immunoprecipitation assay (RIPA) lysis buffer supplemented with the cocktail of protease and phosphatase inhibitors. Nuclear proteins were extracted using a commercial kit (Beyotime, Nantong, China) following the manufacturer's instructions. After treatment, cells were collected, lysed, vortexed, and centrifuged. The resulting precipitate was resuspended and lysed in nuclear protein extraction reagent to obtain the nuclear proteins. Protein concentrations were quantitated through BCA protein assay. The western blot was performed as described previously [19].

2.11. Enzyme-linked immunosorbent assay (ELISA)

According to the manufacturer's protocol, mouse IL-1β and TNF-α ELISA kits were used to measure and quantify the cytokines in supernatants derived from cell cultures and serum.

2.12. Data analysis

Graphs and statistical analyses were conducted using GraphPad Prism 9.5 software (GraphPad Software Inc., San Diego, CA, USA). The results are presented as the mean ± standard deviation. The data were replicated at least three times and analyzed using ANOVA, with p < 0.05 considered statistically significant.

3. Results

3.1. Effects of G-Rg2 on cell viability and pyroptosis in iBMDMs

As shown in Fig. 1A, G-Rg2 did not exhibit any cytotoxic effects on iBMDMs when treated with at a concentration of 50 μM for 24 h. Therefore, subsequent studies employed G-Rg2 concentrations of 5, 10, and 20 μM. LPS and Nig were used to induce iBMDMs in an in vitro experimental model to investigate the role and mechanisms of G-Rg2. As indicated in Fig. 1B, LDH release was significantly increased in iBMDMs induced by LPS and Nig compared to the control group. However, treatment with G-Rg2 significantly inhibited LDH release in a concentration-dependent manner. To confirm this effect, SEM was later performed to observe the microstructural morphology of the iBMDMs. Compared to the control group, cells treated with LPS and Nig showed swelling and the formation of pores on the cell membrane; however, these symptoms were restored after treatment with G-Rg2, as illustrated in Fig. 1C. The inhibitory effects of G-Rg2 on LDH release and pore formation in the cell membrane suggest that G-Rg2 may inhibit pyroptosis in iBMDMs. To confirm this, protein levels of GSDMD and cleaved-GSDMD were analyzed by western blot analysis (Fig. 1D). Compared to the control group, GSDMD and cleaved-GSDMD levels were increased when cells were induced by LPS and Nig. However, there was a significant reduction in cleaved-GSDMD levels under the treatment of 20 μM G-Rg2.

Fig. 1.

Fig. 1

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Effects of G-Rg2 on cell viability, LDH release and pyroptosis in iBMDMs. (A) The effect of G-Rg2 on the cell viability of iBMDMs by MTT assay. (B) G-Rg2 reduces the release of LDH. (C) Detailed morphology of iBMDM cells photographed by SEM, magnification: × 20000, scale bar: 5 μm. (D) The expressions of GSDMD and Cleaved-GSDMD in iBMDMs.

3.2. G-Rg2 inhibited LPS and Nig induced inflammation through the NF-κB/NLRP3 pathway

Western blot analysis showed that treatment with LPS and Nig significantly increased the expression of NLRP3 in lysates, as well as cleaved Caspase-1 and IL-1β in cell culture supernatants, confirming NLRP3 inflammasome activation. Notably, pre-treatment with G-Rg2 (5, 10, and 20 μM) for 30 min resulted in a concentration-dependent reversal of cleaved Caspase-1 and IL-1β levels. Although G-Rg2 did not affect the expression of pro-caspase 1, pro-IL-1β and ASC, 20 μM G-Rg2 markedly decreased NLRP3 expression (Fig. 2A–D). ASC oligomerization was detected by western blotting (Fig. 2E), and the result demonstrated that levels of ACS monomer, dimer and oligomers were remarkably increased by LPS and Nig induction. However, the treatment of G-Rg2 led to a reduction in these levels compared to the model group. These results suggest that G-Rg2 inhibited the ASC oligomerization, thereby suppressing NLRP3 inflammasome activation. Furthermore, immunofluorescence staining of ASC further supported the anti-inflammatory effects of G-Rg2 and its role in modulating the NLRP3 inflammasome in iBMDMs (Fig. 2F).

Fig. 2.

Fig. 2

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Effects of G-Rg2 on NLRP3 inflammasome activation in iBMDMs. (A–D) The expressions of NLRP3, pro-caspase 1, pro-IL-1β and ASC in iBMDM cells, and the p20 and p17 in iBMDMs culture supernatant by Western blot. (E) The oligomerization of ASC in iBMDM cells. (F) The speckles of ASC in iBMDM cells were tasted by immunofluorescence. Scale bar: 5 μm. Values are presented as the mean ± SEM (n = 3). #p < 0.05 vs control group. ∗p < 0.05 vs LPS and Nig group.

In addition, ELISA results indicated that LPS and Nig treatment enhanced the expression of pro-inflammatory cytokines IL-1β and TNF-α in supernatants, which G-Rg2 effectively reversed (Fig. 3A and B). Further analyses using RT-qPCR to examine changes in NLRP3-related gene expression showed that pre-treatment with G-Rg2 (5, 10, and 20 μM) before LPS exposure significantly suppressed IL-1β and NLRP3 mRNA levels, with the most substantial inhibition at 10 μM (Fig. 3C and D). The expression of phosphorylated IκBα and NF-κB p65 was higher in LPS-induced iBMDMs compared to the control. However, G-Rg2 treatment led to a marked reduction in these protein levels at early time stage of LPS induction (Fig. 3E and F). Immunofluorescent staining and western blot analysis further demonstrated an increase in NF-κB p65 following LPS treatment, including its nuclear translocation, which was significantly diminished with 20 μM G-Rg2 (Fig. 3G).

Fig. 3.

Fig. 3

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Effect of G-Rg2 on the NF-κB/NLRP3 pathway and ROS accumulation in iBMDMs. TNF-α (A) and IL-1β (B) secretion was determined by ELISA in iBMDMs culture supernatant. Values are presented as the mean ± SEM (n = 3). #p < 0.05 vs control group. ∗p < 0.05 vs LPS and Nig group. The effect of G-Rg2 on NLRP3 (C) and pro-IL-1β (D) gene expression was determined by RT-qPCR. Values are presented as the mean ± SEM (n = 5). #p < 0.05 vs control group. ∗p < 0.05 vs LPS group. (E, F) The expressions of NF-κB signaling pathway related proteins were detected by Western blot. (G) The localization of NF-κB p65 in iBMDMs was tasted by immunofluorescence. Scale bar: 5 μm. (H) The expression and phosphorylation levels of MAPKs in iBMDMs. (I) The ROS accumulation in iBMDMs was assayed by fluorescent probes. Scale bar: 10 μm.

In addition, the effects of G-Rg2 on mitogen activated protein kinases activation were detected by western blotting analysis. As shown in Fig. 3H, G-Rg2 significantly downregulated the levels of phosphorylated kinases, including p-ERK, p-JNK, and p-p38. Similarly, ROS production in LPS and Nig-activated iBMDMs was also markedly reduced by G-Rg2 (Fig. 3I). These results demonstrated the modulatory effect of G-Rg2 on the priming signaling of the NLRP3 inflammasome. In contrast, G-Rg2 failed to reverse mitochondrial dysfunction (Fig. 4). Overall, these findings suggest that G-Rg2 attenuates inflammation in iBMDMs by inhibiting the NF-κB/NLRP3 pathway in vitro.

Fig. 4.

Fig. 4

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Mitochondrial membrane potential of iBMDMs. The effect of G-Rg2 on mitochondrial function in iBMDMs were presented as Mitochondrial membrane potential assay with JC-1, and was detected by fluorescence microscopy (A), scale bar: 10 μm and flow cytometry (B), JC-1 red/green ratio are presented as the mean ± SEM (n = 3). ∗p < 0.05 vs control group.

3.3. G-Rg2 attenuated DSS induced UC symptoms

To evaluate the anti-inflammatory properties of G-Rg2 in UC, a model was induced using 3 % DSS for a duration of 7 days. Daily monitoring encompassed parameters such as body weight, fecal consistency, and overall well-being of the mice (Fig. 5). After 7 days, a significant decrease in body weight was observed in the DSS-treated mice. During the feeding period, food and water intake were notably reduced in the DSS group compared to the control. Conversely, the G-Rg2 treated groups exhibited an upward trend (Fig. 5B–E). Significant improvements in colon length were noted between the G-Rg2 and 5-ASA groups; the colon length in the control group averaged 8.5 cm, whereas it was significantly shorter in the DSS group, at approximately 5.9 cm. Mice treated with G-Rg2 and 5-ASA displayed marked improvements in colon length (Fig. 5F and G). Furthermore, spleen weight and spleen index in the G-Rg2 groups were significantly reduced compared to the DSS group (Fig. 5H and I). In conclusion, these findings indicate that G-Rg2 has a potent anti-inflammatory effect on DSS-induced UC in mice.

Fig. 5.

Fig. 5

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G-Rg2 attenuated DSS induced UC symptoms. (A) UC modelling and treatment scheme. (B) Daily body weight. (C) Food consumption. (D) Water intake. (E) DAI. (F) Representative images of colon. (G) Length of mice colon. (H) Representative images of spleen. (I) Spleen index of mice. Values are presented as the mean ± SEM (n = 8). #p < 0.05 vs Control group. ∗p < 0.05 vs DSS group.

3.4. G-Rg2 decreased pro-inflammatory cytokines

ELISA and RT-qPCR analyses demonstrated that in the DSS group, the levels of IL-1β in serum and TNF-α and IL-6 in the colon were significantly elevated. However, treatment with G-Rg2 (10 and 20 mg/kg) and 5-ASA effectively reduced the expression of these cytokines. Further investigation into the protective effects of G-Rg2 against DSS-induced damage revealed notable improvements (Fig. 6A–C). Compared to the control group, H&E staining showed that DSS treatment led to substantial inflammatory cell infiltration and severe disruption of crypt structure; these histological changes were markedly alleviated in G-Rg2-treated mice. Additionally, while the DSS group exhibited a significant reduction in goblet cells, treatment with G-Rg2 and 5-ASA significantly increased goblet cell counts (Fig. 6D). Overall, these findings suggest that G-Rg2 effectively mitigated DSS-induced damage and inflammation in the colon.

Fig. 6.

Fig. 6

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G-Rg2 decreased pro-inflammatory cytokines. IL-1β (A) secretion was determined by ELISA in mouse serum. TNF-α (B) and IL-6 (C) gene expression in mouse colon was determined by RT-qPCR. (D) Histological examination of mouse colon tissue was assessed by hematoxylin and eosin (H&E) staining. Values are presented as the mean ± SEM (n = 6). #p < 0.05 vs Control group. ∗p < 0.05 vs DSS group.

3.5. G-Rg2 protects the intestinal barrier

Colon levels of tight junction (TJ) proteins, such as occludin, claudin-3, ZO-1, and mucin 2, are commonly used clinical indicators to assess the severity of intestinal barrier impairment in UC. In this study, RT-qPCR was employed to quantify the expression levels of these proteins in the colon, revealing a significant reduction in the DSS group. However, administration of G-Rg2 led to a dose-dependent increase in the mRNA levels of these TJ proteins (Fig. 7A–D). Furthermore, protein levels of occludin and claudin-1 in the colon were detected by immunohistochemistry. As shown in Fig. 7E, DSS treatment decreased the expression of TJ related proteins; however, G-Rg2 treatment significantly increased their levels compared to the model group. These results confirmed the protective effects of G-Rg2 on the mouse intestinal barrier from DSS induced damage.

Fig. 7.

Fig. 7

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G-Rg2 protects the intestinal barrier. The effects of G-Rg2 on ZO-1 (A), occludin (B), claudin-3 (C) and mucin 2 (D) gene expression in mouse colon was determined by RT-qPCR. Values are presented as the mean ± SEM (n = 6). #p < 0.05 vs Control group. ∗p < 0.05 vs DSS group. The protein expression level of occludin and claudin-1 in mouse colon were detected by immunohistochemical analysis (E). Scale bar: 100 μm.

3.6. G-Rg2 inhibits NLRP3 inflammasome activation in UC mice

We investigated whether G-Rg2 exerted an anti-inflammatory effect on DSS-induced UC by suppressing the NF-κB/NLRP3 signaling pathway. Western blot analysis revealed that DSS treatment significantly increased the levels of NLRP3, p20, and p17. However, oral administration of G-Rg2 (10 mg/kg and 20 mg/kg) effectively inhibited these expressions (Fig. 8A–D). Consistent with in vitro findings, serum levels of IL-1β were lower in the G-Rg2 treatment groups compared to the DSS-induced group, indicating that G-Rg2 effectively blocked DSS-induced activation of the NLRP3 inflammasome. In the cellular experiments described earlier, it was confirmed that the NF-κB signaling pathway plays a critical role in inhibiting NLRP3 inflammasome activation by G-Rg2. Furthermore, the regulatory effect of G-Rg2 on NF-κB expression in the DSS-induced UC model was also confirmed. Western blot analysis showed a significant increase in IκBα phosphorylation following DSS treatment. Oral administration of G-Rg2 inhibited IκBα phosphorylation and reduced NF-κB p65 levels in the colon (Fig. 8E). Collectively, these findings suggest that G-Rg2 attenuates NLRP3 inflammasome activation by inhibiting the NF-κB/NLRP3 signaling pathway (Fig. 8F).

Fig. 8.

Fig. 8

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G-Rg2 inhibits NLRP3 inflammasome activation in UC mice. (A–D) The expressions of p17, p20, NLRP3, pro-caspase 1, pro-IL-1β and ASC, and (E) the expressions of NF-κB signaling pathway related proteins in mouse colon were detected by western blot. (F) The molecular mechanism of G-Rg2 against UC: G-Rg2 inhibits nuclear translation of NF-κB, suppresses NLRP3 inflammasome activation, and reduces inflammation, thereby reducing UC. Values are presented as the mean ± SEM (n = 3). #p < 0.05 vs Control group. ∗p < 0.05 vs DSS group.

4. Discussion

UC is recognized as a long-term autoimmune-mediated condition [1,20]. Studies, including those by He et al. [21], have shown that G-Rg2 can ameliorate hepatic fibrosis in high-fat diet-induced mice by modulating the AKT/mTOR signaling pathway at a dosage of 10 mg/kg. Based on these findings, we selected 10 and 20 mg/kg doses of G-Rg2 for treating UC. The UC model was reliably induced using 3 % DSS [22]. In our in vivo studies, we confirmed that administering 3 % DSS water to mice for a continuous seven days resulted in decreased levels of weight, food, and water intake and was accompanied by significant inflammatory cell invasion, destruction of crypt structures, and loss of goblet cells. Consistent with previous findings, G-Rg2 treatment reduced serum levels of inflammatory markers and markedly improved pathological abnormalities [23], suggesting that G-Rg2 may be effective in both preventing and treating UC.

During UC development, DSS stimulates macrophages to release inflammatory factors such as IL-1β, TNF-α, and IL-6, which exacerbate UC progression [24]. Our results indicated that G-Rg2 reduced the levels of IL-1β in serum as well as TNF-α and IL-6 in the colon. The intestinal barrier, comprising epithelial cells, a mucus layer, immune barriers, and a microbial community, plays a crucial role in preventing harmful substances from entering the body. Disruption of this barrier is central to UC pathogenesis [2,4,25]. Elevated plasma levels of LPS, a major component of Gram-negative bacterial cell membranes, have been observed in patients with compromised intestinal barriers [[26], [27], [28]]. LPS can activate the immune system through PAMPs and is a known mechanism for NLRP3 inflammasome activation during its priming phase [7,29]. Our study suggested that DSS may promote the degradation of intestinal barrier proteins in UC mice, leading to NLRP3 inflammasome activation via LPS penetration through the compromised barrier. G-Rg2 appeared to enhance the expression of ZO-1, occludin, and claudin-3 proteins. Furthermore, inhibition of IL-1β and TNF-α has shown beneficial effects on DSS-induced impairments of tight junctions and the intestinal barrier [30,31]. Compounds like paeoniflorin and naringin have also been demonstrated to prevent intestinal barrier disruption and reduce LPS, IL-1β and TNF-α levels, thereby alleviating intestinal inflammation [28,[32], [33], [34]]. Evidence suggests that lysosomal damage is a key regulator of inflammation in UC, with lysosomal proteases implicated in DSS-induced NLRP3 inflammasome activation [35]. The activation of the NLRP3 inflammasome involves two steps: first, NF-κB pathway regulation in response to pathogen-associated molecules which promotes the transcription and translation of NLRP3, pro-IL-1β and IL-18, followed by NLRP3 inflammasome assembly in response to activation signals [7,29,36]. NF-κB, a nuclear transcription factor, plays crucial roles in inflammation, proliferation and apoptosis [37]. Given the role of the NLRP3 inflammasome in UC pathogenesis, inhibiting its activation has been targeted as a therapeutic strategy. Increasing evidence supports those various natural products, such as L-Fucose and betulin, can alleviate DSS-induced UC by inhibiting NF-κB activation and reducing NLRP3 inflammasome activity [11,38]. Western blot analysis confirmed that the DSS group showed significant increases in the expression of p17, p20, NLRP3, NF-κB and p-IκBα in the colon, while G-Rg2 treatment groups exhibited decreases, underscoring the potential anti-inflammatory effects of G-Rg2 through the NF-κB/NLRP3 pathway.

Through the regulation of the NF-κB signaling pathway, LPS activates NLRP3, pro-IL-1β and pro-IL-18 genes, indirectly facilitating the assembly and activation of the NLRP3 inflammasome, which triggers an inflammatory response [29,39]. Among these triggers, iBMDMs can be activated by Nig, ATP, ROS, MSU and other stimulis [40,41]. LDH release is considered an indicator of membrane rupture [42]. Our experiments demonstrated that treatment with G-Rg2 significantly reduced LDH release under LPS and Nig stimulation. Substantial evidence indicates that LPS can induce the production of inflammatory genes by enhancing the activity of the transcription factor NF-κB [43,44]. The results showed an increase in NLRP3 and pro-IL-1β levels following LPS induction in iBMDMs. However, G-Rg2 markedly inhibited the production of these markers. Additionally, upon LPS stimulation, NF-κB p65 dissociates from IκB-NF-κB complexes, becomes activated, and translocates to the nucleus. Treatment with G-Rg2 has the potential to inhibit this process. IL-1β production in iBMDMs, induced by LPS and Nig, serves as an indicator of NLRP3 inflammasome activation [45]. Our results indicated that treatment with LPS and Nig increased the protein expression levels of NLRP3, p20, p17, IL-1β and TNF-α. Consistent with in vivo results, treatment with G-Rg2 reduced NLRP3 protein expression and inhibited the release of p20, p17, and TNF-α in iBMDMs. This study provides insight into the effects of G-Rg2 on NF-κB and NLRP3 inflammasome activation, suggesting that G-Rg2 modulates NF-κB protein activation, thereby influencing the NLRP3 pathway and mitigating the inflammatory response, which can alleviate UC. These findings initially demonstrated that G-Rg2 effectively alleviated inflammatory symptoms in DSS-induced UC mice through suppressing the NF-κB/NLRP3 pathway. In summary, our research demonstrates that G-Rg2 attenuates DSS-induced UC through both in vitro and in vivo experiments, highlighting it as a potential anti-inflammatory therapeutic candidate for DSS-induced UC.

5. Conclusion

As a major active compound of Panax ginseng, G-Rg2 exhibits a wide range of pharmacological properties. In the present study, the underlying mechanisms of G-Rg2 mitigated UC have been investigated based on a DSS-induced UC mouse model and LPS/Nig-induced NLRP3 inflammasome activation iBMDMs. The results showed that G-Rg2 alleviated the progression of colon lesions in DSS-induced UC in mice. Additionally, G-Rg2 treatment reversed the spleen index in UC mice, downregulated the mRNA expression levels of the inflammatory cytokines IL-1β, TNF-α, and IL-6, and upregulated the expression of intestinal tight junction proteins ZO-1, occludin, claudin-3, and mucin 2. Moreover, G-Rg2 reduced the protein levels of NLRP3, IL-1β, Caspase-1, IκBα, p-IκBα, and NF-κB p65. In vitro, G-Rg2 decreased LDH release and the levels of NLRP3, Caspase-1, and IL-1β. Furthermore, G-Rg2 inhibited ROS accumulation and ASC oligomerization in LPS/Nig-induced iBMDMs. Taken together, these results indicated that G-Rg2 mitigated UC by inhibiting the activation of the NLRP3 inflammasome and the release of inflammatory cytokines, modulating the NF-κB/NLRP3 pathways. This study elucidated the mechanisms by which G-Rg2 alleviated inflammatory symptoms in DSS-induced UC mice through the suppression of the NF-κB/NLRP3 pathway, for the first time, highlighting its potential as an anti-inflammatory agent and a promising candidate for the development of anti-inflammatory pharmaceuticals and functional foods.

CRediT authorship contribution statement

Ji Zhang: Methodology, Investigation, Funding acquisition. Jing Xie: Investigation, Writing – original draft, Software. Zhiqiang Niu: Methodology, Investigation. Long You: Methodology, Investigation. Yanan Liu: Methodology, Investigation. Rui Guo: Methodology, Investigation. Guigui Yang: Investigation, Software. Ziliang He: Methodology, Writing – review & editing. Ting Shen: Conceptualization. Honggang Wang: Supervision, Validation. Qi Yan: Methodology, Writing – review & editing. Weicheng Hu: Conceptualization, Investigation, Writing – original draft.

Declaration of competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

This work was financially supported by the National Natural Science Foundation of China (31600281) and the Natural Science Foundation of Jiangsu Higher Education Institutions of China (23KJA550001).

Footnotes

Appendix A

Supplementary data to this article can be found online at doi:mmcdoino

Contributor Information

Honggang Wang, Email: jgzwhg@njmu.edu.cn.

Qi Yan, Email: 18051061221@yzu.edu.cn.

Weicheng Hu, Email: huweicheng@yzu.edu.cn, hu_weicheng@163.com.

Appendix A. Supplementary data

The following is/are the supplementary data to this article:

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