Ginsenoside Rh2 Alleviates LPS-Induced Inflammatory Responses by Binding to TLR4/MD-2 and Blocking TLR4 Dimerization

Lipopolysaccharide (LPS) triggers a severe systemic inflammatory reaction in mammals, with the dimerization of TLR4/MD-2 upon LPS stimulation serving as the pivotal mechanism in the transmission of inflammatory signals. Ginsenoside Rh2 (G-Rh2), one of the active constituents of red ginseng, exerts potent anti-inflammatory activity. However, whether G-Rh2 can block the TLR4 dimerization to exert anti-inflammatory effects remains unclear. Here, we first investigated the non-cytotoxic concentration of G-Rh2 on RAW 264.7 cells, and detected the releases of pro-inflammatory cytokines in LPS-treated RAW 264.7 cells, and then uncovered the mechanisms involved in the anti-inflammatory activity of G-Rh2 through flow cytometry, fluorescent membrane localization, Western blotting, co-immunoprecipitation (Co-IP), molecular docking and surface plasmon resonance (SPR) analysis in LPS-stimulated macrophages. Our results show that G-Rh2 stimulation markedly inhibited the secretion of LPS-induced interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and nitric oxide (NO). Additionally, G-Rh2 blocked the binding of LPS with the membrane of RAW 264.7 cells through direct interaction with TLR4 and MD-2 proteins, leading to the disruption of the dimerization of TLR4 and MD-2, followed by suppression of the TLR4/NF-κB signaling pathway. Our results suggest that G-Rh2 acts as a new inhibitor of TLR4 dimerization and may serve as a promising therapeutic agent against inflammation.


Introduction
Toll-like receptor 4 (TLR 4 ), a member of the toll-like receptor (TLR) family, is involved in innate immunity and mediates inflammatory responses by recognizing lipopolysaccharide (LPS) [1,2].LPS-induced bimolecular TLR 4 /MD-2 dimerization is the basis of LPS-induced signal transduction.The lipid A component of Escherichia coli LPS features two phosphorylated glucosamines linked by a β (1-6) linkage and acylated by six lipid chains.Among these, five fatty acid chains are deeply embedded within a pocket, while the sixth chain extends to the surface of MD-2, engaging in hydrophobic interactions with the invariant phenylalanine residues of the adjacent TLR 4 *.Subsequently, the two phosphate groups on lipid A attach to the TLR 4 /MD-2 complex through electrostatic interactions with positively charged amino acids in TLR 4 , TLR 4 * and MD-2 [3].The formation of the TLR 4 /MD-2/TLR 4 */MD-2* heterodimer ultimately triggers the dimerization of the cytoplasmic domains, culminating in the recruitment of downstream adaptor proteins and the initiation of intracellular signaling cascades, ultimately leading to an immune response [4].Studies have shown that TLR 4 dimerization is crucial for activating downstream inflammatory signaling pathways, including the NF-κB pathway, which in turn promotes the secretion of TNF-α, IL-6 and IL-1β [5,6].Several studies have demonstrated that a range of substances functioning as TLR 4 /MD-2 inhibitors can produce anti-inflammatory outcomes by blocking the dimerization of TLR 4 with MD-2, such as total tanshinones, isoacteoside and ginsenoside Rb1 [7][8][9].Therefore, blocking TLR 4 dimerization has become a new strategy to inhibit inflammation.
G-Rh 2 , first isolated from red ginseng, is a steroidal saponin belonging to the protopanaxadiol type, and has various potent biological functions, including antitumor, anti-obesity, anti-inflammatory and antioxidant activities, preventing neurodegenerative diseases and so on [10][11][12][13][14].In this study, we aimed to explore the anti-inflammatory effect of G-Rh 2 by constructing an LPS-induced RAW 264.7 cell inflammatory model in vitro.Furthermore, we determined whether G-Rh 2 blocked the TLR 4 /MD-2 dimerization to exert anti-inflammatory effects.

Effect of G-Rh 2 on the Levels of TNF-α, IL-6 and NO
To explore whether G-Rh 2 exerted potential cytotoxicity action on RAW 264.7 cells, cell viability was detected by an MTT assay after incubation for 24 h in the absence or presence of different concentrations of G-Rh 2 .Our findings indicated that G-Rh 2 , at concentrations ranging from 0 to 80 µg/mL, did not notably impact cell viability in a dose-dependent manner (p > 0.05) (Figure 1A).
Because TNF-α, IL-6 and NO are the main markers of inflammation, we analyzed the effects of G-Rh 2 on TNF-α, IL-6 and NO secretion in LPS-induced RAW 264.7 cells in vitro.As shown in Figure 1B-D, TNF-α, IL-6 and NO secreted by RAW 264.7 cells were significantly increased under LPS stimulation (p < 0.01).Compared with the LPS group, the secretion of TNF-α, IL-6 and NO was significantly decreased by different concentrations of G-Rh 2 (p < 0.01).Among these, the inhibitory effects of G-Rh 2 (15 and 30 µg/mL) on TNF-α secretion were better than those of the positive drug (quercetin 10 µg/mL).
downstream inflammatory signaling pathways, including the NF-κB pathway, which in turn promotes the secretion of TNF-α, IL-6 and IL-1β [5,6].Several studies have demonstrated that a range of substances functioning as TLR4/MD-2 inhibitors can produce antiinflammatory outcomes by blocking the dimerization of TLR4 with MD-2, such as total tanshinones, isoacteoside and ginsenoside Rb1 [7][8][9].Therefore, blocking TLR4 dimerization has become a new strategy to inhibit inflammation.
G-Rh2, first isolated from red ginseng, is a steroidal saponin belonging to the protopanaxadiol type, and has various potent biological functions, including antitumor, antiobesity, anti-inflammatory and antioxidant activities, preventing neurodegenerative diseases and so on [10][11][12][13][14].In this study, we aimed to explore the anti-inflammatory effect of G-Rh2 by constructing an LPS-induced RAW 264.7 cell inflammatory model in vitro.Furthermore, we determined whether G-Rh2 blocked the TLR4/MD-2 dimerization to exert anti-inflammatory effects.

Effect of G-Rh2 on the Levels of TNF-α, IL-6 and NO
To explore whether G-Rh2 exerted potential cytotoxicity action on RAW 264.7 cells, cell viability was detected by an MTT assay after incubation for 24 h in the absence or presence of different concentrations of G-Rh2.Our findings indicated that G-Rh2, at concentrations ranging from 0 to 80 µg/mL, did not notably impact cell viability in a dosedependent manner (p > 0.05) (Figure 1A).
Because TNF-α, IL-6 and NO are the main markers of inflammation, we analyzed the effects of G-Rh2 on TNF-α, IL-6 and NO secretion in LPS-induced RAW 264.7 cells in vitro As shown in Figure 1B-D, TNF-α, IL-6 and NO secreted by RAW 264.7 cells were significantly increased under LPS stimulation (p < 0.01).Compared with the LPS group, the secretion of TNF-α, IL-6 and NO was significantly decreased by different concentrations of G-Rh2 (p < 0.01).Among these, the inhibitory effects of G-Rh2 (15 and 30 µg/mL) on TNFα secretion were better than those of the positive drug (quercetin 10 µg/mL).

Effect of G-Rh2 on the Binding of FITC-LPS to the Cell Membrane
To investigate the effect of G-Rh2 on the binding of FITC-LPS to the cell membrane, firstly, we further detected the effect of G-Rh2 on the binding of FITC-LPS to RAW 264.7 cells by flow cytometry.As shown in Figure 2, the fluorescence intensity in the FITC-LPS stimulation group was significantly increased to 12.6% (p < 0.01).And compared with the FITC-LPS group, the fluorescence intensity of G-Rh2 groups were significantly decreased to 5.21%, 5.16% and 2.33%, respectively (p < 0.01), in a concentration-dependent manner.
Secondly, we measured the fluorescence intensity of the cell membrane by CLSM, as shown in Figure 3.The cell membranes fluoresced strongly after FITC-LPS stimulation, whereas the G-Rh2 group fluoresced weakly.

Effect of G-Rh 2 on the Binding of FITC-LPS to the Cell Membrane
To investigate the effect of G-Rh 2 on the binding of FITC-LPS to the cell membrane, firstly, we further detected the effect of G-Rh 2 on the binding of FITC-LPS to RAW 264.7 cells by flow cytometry.As shown in Figure 2, the fluorescence intensity in the FITC-LPS stimulation group was significantly increased to 12.6% (p < 0.01).And compared with the FITC-LPS group, the fluorescence intensity of G-Rh 2 groups were significantly decreased to 5.21%, 5.16% and 2.33%, respectively (p < 0.01), in a concentration-dependent manner.
Secondly, we measured the fluorescence intensity of the cell membrane by CLSM, as shown in Figure 3.The cell membranes fluoresced strongly after FITC-LPS stimulation, whereas the G-Rh 2 group fluoresced weakly.

Effect of G-Rh 2 on the Activation of the TLR 4 /NF-κB Signaling Pathway
To further confirm that G-Rh 2 inhibits the production of pro-inflammatory factors by blocking the TLR 4 signaling pathway, we examined the effect of G-Rh 2 on the expression of key proteins of the TLR 4 signaling pathway.As shown in Figure 4, G-Rh 2 dose-dependently inhibited LPS-induced phosphorylation levels of NF-κB p65 and protein expression of TLR 4 and MD-2 (p < 0.01).

Effect of G-Rh 2 on the Dimerization of TLR 4 and MD-2
Only the dimerization of TLR 4 and MD-2 can trigger TLR 4 intracellular signaling pathways and finally induce NF-κB into the nucleus [15].Therefore, we further investigated the effect of G-Rh 2 on LPS-induced dimerization of TLR 4 and MD-2 by means of Co-IP.As shown in Figure 5, the co-precipitation of TLR 4 /MD-2 was significantly increased in the LPS group (p < 0.01), while G-Rh 2 (15 µg/mL) treatment significantly inhibited LPSinduced TLR 4 /MD-2 complex formation (p < 0.01).The results indicated that G-Rh 2 could prevent the transmission of LPS signals to cell membranes and inhibit the dimerization of TLR 4 and MD-2.

Effect of G-Rh2 on the Activation of the TLR4/NF-κB Signaling Pathway
To further confirm that G-Rh2 inhibits the production of pro-inflammatory factors by blocking the TLR4 signaling pathway, we examined the effect of G-Rh2 on the expression of key proteins of the TLR4 signaling pathway.As shown in Figure 4, G-Rh2 dose-dependently inhibited LPS-induced phosphorylation levels of NF-κB p65 and protein ex- To further confirm that G-Rh2 inhibits the production of pro-inflammatory factor blocking the TLR4 signaling pathway, we examined the effect of G-Rh2 on the express of key proteins of the TLR4 signaling pathway.As shown in Figure 4, G-Rh2 dose pendently inhibited LPS-induced phosphorylation levels of NF-κB p65 and protein pression of TLR4 and MD-2 (p < 0.01).

Effect of G-Rh2 on the Dimerization of TLR4 and MD-2
Only the dimerization of TLR4 and MD-2 can trigger TLR4 intracellular signal pathways and finally induce NF-κB into the nucleus [15].Therefore, we further inve gated the effect of G-Rh2 on LPS-induced dimerization of TLR4 and MD-2 by means of IP.As shown in Figure 5, the co-precipitation of TLR4/MD-2 was significantly increased the LPS group (p < 0.01), while G-Rh2 (15 µg/mL) treatment significantly inhibited L induced TLR4/MD-2 complex formation (p < 0.01).The results indicated that G-Rh2 co prevent the transmission of LPS signals to cell membranes and inhibit the dimerization TLR4 and MD-2.

Effect of G-Rh2 on the Dimerization of TLR4 and MD-2
Only the dimerization of TLR4 and MD-2 can trigger TLR4 intracellular signaling pathways and finally induce NF-κB into the nucleus [15].Therefore, we further investigated the effect of G-Rh2 on LPS-induced dimerization of TLR4 and MD-2 by means of Co-IP.As shown in Figure 5, the co-precipitation of TLR4/MD-2 was significantly increased in the LPS group (p < 0.01), while G-Rh2 (15 µg/mL) treatment significantly inhibited LPSinduced TLR4/MD-2 complex formation (p < 0.01).The results indicated that G-Rh2 could prevent the transmission of LPS signals to cell membranes and inhibit the dimerization of TLR4 and MD-2.

Discussion
G-Rh2, a protopanaxadiol saponin from ginseng, has been reported to exhibit antiinflammatory and anticancer effects [16][17][18].This provides strong support for us to further explore its anti-inflammatory mechanism.Therefore, the aim of this study was to verify the anti-inflammatory activity of G-Rh2, and to explore the underlying mechanisms in a cell inflammation model.Our results revealed that G-Rh2 could significantly decrease the secretion of TNF-α, IL-6 and NO in LPS-induced RAW 264.7 cells and indicated G-Rh2 exerted quite evident anti-inflammatory effects.Both TNF-α and IL-1β are reported to be NF-κB target genes, and the expression of these two target genes is significantly increased in LPS-stimulated macrophages [19].Quercetin inhibits the NF-κB, Akt and JNK signaling pathway, thereby reducing the expression of TNF-α and IL-1β in LPS-stimulated RAW 264.7 cells [20].We speculate that G-Rh2 could block the binding of LPS with the membrane of RAW 264.7 cells through direct interaction with TLR4 and MD-2 proteins, followed by suppression of the TLR4/MD-2 mediated downstream NF-κB signaling pathway, so as to better reduce the expression of TNF-α.
LPS, a macromolecular glycolipid, is unable to cross cell membranes by itself but must bind to membrane receptors to exert its biological effects [21,22].To further explore Next, we evaluated the interaction between G-Rh 2 and TLR 4 /MD-2 through SPR experiments; as shown in Figure 6B, G-Rh 2 binds to TLR 4 /MD-2 protein in a dose-dependent manner and presents a "fast up, fast down" binding pattern.G-Rh 2 and TLR 4 /MD-2 have a specific binding force and an affinity of KD = 71.20 µM.

Discussion
G-Rh 2 , a protopanaxadiol saponin from ginseng, has been reported to exhibit antiinflammatory and anticancer effects [16][17][18].This provides strong support for us to further explore its anti-inflammatory mechanism.Therefore, the aim of this study was to verify the anti-inflammatory activity of G-Rh 2 , and to explore the underlying mechanisms in a cell inflammation model.Our results revealed that G-Rh 2 could significantly decrease the secretion of TNF-α, IL-6 and NO in LPS-induced RAW 264.7 cells and indicated G-Rh 2 exerted quite evident anti-inflammatory effects.Both TNF-α and IL-1β are reported to be NF-κB target genes, and the expression of these two target genes is significantly increased in LPS-stimulated macrophages [19].Quercetin inhibits the NF-κB, Akt and JNK signaling pathway, thereby reducing the expression of TNF-α and IL-1β in LPS-stimulated RAW 264.7 cells [20].We speculate that G-Rh 2 could block the binding of LPS with the membrane of RAW 264.7 cells through direct interaction with TLR 4 and MD-2 proteins, followed by suppression of the TLR 4 /MD-2 mediated downstream NF-κB signaling pathway, so as to better reduce the expression of TNF-α.
LPS, a macromolecular glycolipid, is unable to cross cell membranes by itself but must bind to membrane receptors to exert its biological effects [21,22].To further explore the anti-inflammatory mechanism of G-Rh 2 in LPS-induced RAW 264.7 cells, we next detected the effect of G-Rh 2 on the binding of LPS to RAW 264.7 cells by flow cytometry, and further determined the effect of G-Rh 2 on the binding of LPS to cell membranes with laser confocal technology.Flow cytometry showed that G-Rh 2 could significantly inhibit the fluorescence intensity of FITC-LPS binding to the cells, indicating that G-Rh 2 could significantly inhibit the binding of LPS to RAW 264.7 cells.Laser confocal analysis showed that G-Rh 2 could significantly inhibit the fluorescence intensity of FITC-LPS binding to cell membranes.These results suggest that G-Rh 2 may inhibit the binding of LPS to cell membranes to alleviate LPS-induced cell inflammation.
TLR 4 is responsible for the recognition of LPS and then induces the activation of the NF-κB signaling pathway [23].Therefore, we then explored the effects of G-Rh 2 on the TLR 4 /NF-κB signaling pathway.Regarding the results, G-Rh 2 significantly inhibited the protein expression of TLR 4 and MD-2 and phosphorylation of NF-κB p65 in LPS-induced RAW 264.7 cells.These results indicated that G-Rh 2 may block the combination of TLR 4 and MD-2, and then inhibit TLR 4 /NF-κB signaling pathway activation.To further explore whether G-Rh 2 interfered with the combination of TLR 4 and MD-2, we explored whether G-Rh 2 influences the LPS-induced dimerization of TLR 4 and MD-2 by Co-IP.The findings demonstrated that the co-precipitation of TLR 4 /MD-2 exhibited a marked increase in the LPS group.Conversely, the administration of G-Rh 2 markedly impeded the formation of the LPS-induced TLR 4 /MD-2 complex.
According to the previous report, LPS-induced TLR 4 /MD-2 dimerization is the key to inflammation signaling transduction [24].A previous study has reported that anthocyanins mostly fit into the hydrophobic pocket of MD-2 and bind to TLR 4 , which results in the inhibition of NF-κB activity to attenuate lipopolysaccharide-induced inflammation [25].TTAK-242 inhibits the activation of the TLR 4 signaling pathway by binding to the Cys747 amino acid site of TLR 4 's intracellular TIR domain [26,27].However, how G-Rh 2 affects the dimerization of TLR 4 /MD-2 is not clear.The molecular docking study predicted that G-Rh 2 would bind to the hydrophobic pocket of MD-2, which led to G-Rh 2 occupying the position of LPS in the hydrophobic pocket of MD-2, resulting in the inability of TLR 4 to dimerize.It has been demonstrated that amino acid residues 119-132 of MD-2 play a key role in the recognition and binding of LPS, while amino acids 46~50, 79~83 and 90~105 are key binding sites of MD-2 and TLR 4 [3].G-Rh 2 interacts with MD-2 amino acids PHE119 and PHE121 that bind to important sites of LPS, blocking the binding of LPS to MD-2.In addition, G-Rh 2 can block the binding of TLR 4 to MD-2 ILE46 and VAL48 amino acids, blocking the formation of a complex between TLR 4 and MD-2.
It was found that G-Rh 2 bound to TLR 4 /MD-2 dose-dependently in SPR studies, and the binding interaction occurred in a "fast up and fast down" manner, indicating that G-Rh 2 specifically bound to TLR 4 /MD-2 with a strong affinity, KD = 71.20 µM.Shin et al. showed that the KD value for the binding of LPS to MD-2 was 2.33 µM.Although G-Rh 2 has a lower affinity than LPS, the results of Western blot and Co-IP showed that G-Rh 2 significantly inhibited the formation of the TLR 4 /MD-2 complex and suppressed the downstream signal transduction.Combined with the results of molecular docking, G-Rh 2 is mated into the hydrophobic pocket of TLR 4 /MD-2.We conjectured that the binding affinity between G-Rh 2 and MD-2 hydrophobic pockets may be higher than that of LPS, which leads to fewer LPS lipid chains falling into hydrophobic pockets; TLR 4 then cannot form a dimerization structure, and then its downstream signal transduction is inhibited.Regarding the crystal structure of LPS/MD-2/TLR 4 , the affinity between LPS and TLR 4 /MD-2 mainly includes the interaction between five lipid chains of LPS and residues of the hydrophobic pocket of MD-2, and the hydrophobic interaction between another lipid chain and the conserved phenylalanine of TLR 4 * [3].More important is that the two phosphate groups on lipid A bind to the TLR 4 /MD-2 complex through interactions with positively charged amino acids in TLR 4 , TLR 4 * and MD-2.A previous study has demonstrated that these two phosphate groups within the lipid A moiety exert a profound influence on the endotoxic properties of LPS.The removal of either phosphate group results in a 100-fold reduction in endotoxic activity, with the remaining monophosphoryl LPS exhibiting relatively weak stimulatory properties with regard to the innate immune response [28].This suggests that the affinity of LPS may depend mainly on two phosphate groups, while the affinity of LPS binding to MD-2 may be low.Therefore, LPS may have a lower affinity than G-Rh 2 when only the capacity for binding to the hydrophobic pockets of MD-2 is compared.
Recently, lipid A derivatives like lipid IVa and eritoran, which possess four fatty acid chains, have been demonstrated to selectively interact with the hydrophobic cavity of MD-2.This interaction serves to disrupt TLR 4 dimerization, with them acting as antagonists of the TLR 4 /MD-2 complex [29].This also supports our hypothesis that when the number of lipid chains of LPS falling into MD-2 pockets is reduced, this becomes an inhibitor of TLR 4 signaling transduction.
These results suggest that G-Rh 2 is targeted by preempting LPS and TLR 4 /MD-2, and that G-Rh 2 targeting TLR 4 /MD-2 blocks the binding of LPS to TLR 4 /MD-2 and the dimerization of TLR 4 /MD-2, which will inevitably attenuate the LPS-induced TIR wobble and the recruitment of downstream adapters.This will ultimately inhibit the activation of the NF-κB signaling pathway.

Cell Culture and Viability Assay
RAW 264.7 cells were obtained from the Kunming Cell Bank of Chinese Academy of Sciences and cultured in DMEM, which was supplemented with 10% FBS, 100 U/mL penicillin and 100 U/mL streptomycin at 37 • C in a humidified incubator with 5% CO 2 [30].The MTT assay was used for measurement of cell viability, as described by Yan et al. [31].Cells were distributed in 96-well plates at a density of 5 × 10 5 cells/mL and incubated for 12 h.G-Rh 2 was prepared at 6 concentrations of G-Rh 2 (0, 5, 10, 20, 40, 80 µg/mL) to be added to the well (nine wells were repeated for each dose group) for 24 h.Next, an MTT assay was performed in accordance with the original method.The absorbance of each well was measured at 490 nm using a microplate reader (Multiskan GO Thermo Scientiific, Waltham, MA, USA).

Detection of Pro-Inflammatory Factors
The dosage of G-Rh 2 was determined by the MTT assay and a previous report [32].In order to assay the production of TNF-α, IL-6 and NO, the supernatant of RAW 264.7 cells was collected after they were co-treated with G-Rh 2 (7.5, 15 and 30 µg/mL) or quercetin (10 µg/mL) in conjunction with LPS (1 µg/mL) for a period of 12 h [33].TNF-α and IL-6 levels were quantified using ELISA kits, following the manufacturer's instructions.NO production was determined using a Griess reagent nitrite measurement kit.

Flow Cytometric Analysis of FITC-LPS Binding
The RAW 264.7 cells (1 × 10 6 cells/mL) were seeded into a 6-well plate for 6 h, then incubated with FITC-LPS (5 µg/mL) with or without G-Rh 2 (7.5, 15, 30 µg/mL) for the same time.Finally, cells in each well were collected and centrifuged at 1000 rpm for 3 min.The final cells were suspended with PBS, and the bound FITC-LPS was examined by flow cytometry (Beckman, Brea, CA, USA) according to reference [34].

Determination of Membrane Localization of FITC-LPS
The localization of FITC-LPS on the cell membrane was determined with reference to Hua et al. [35].In short, RAW 264.7 cells (1 × 10 4 cells/mL) were seeded in 6-well plates with cover slides placed in advance for 6 h, then incubated with FITC-LPS (5 µg/mL) with or without G-Rh 2 (15 µg/mL) for the same time.Subsequently, the supernatant was removed and the cells were rinsed twice with PBS and stained with 0.2 µM DID.In the end, the cells were fixed in 4% PFA, and the localization of FITC-LPS on the cell membrane was observed with a confocal laser scanning microscope (CLSM) (ZEISS LSM900, excitation 488 nm and 507 nm, Oberkochen, Germany).

Western Blot Assay
A Western blot assay was carried out as previously described [31].Briefly speaking, RAW 264.7 cells (6 × 10 5 cells/mL) were seeded in 6-well plates for 12 h.Then, the cells were incubated with G-Rh 2 and LPS for the same time as in the previous experiment.The total proteins were lysed, collected, determined, separated and transferred to PVDF membranes as previously described [31].Next, the membranes were blocked with 5% nonfat milk and incubated with primary antibodies (TLR 4 , MD-2, NF-κB p65, p-NF-κB p65) and secondary antibodies.Finally, the membranes were detected by enhanced chemiluminescence.The protein levels were normalized against the included β-actin standards and subsequently analyzed using ImageJ software (Version 1.54j) (available at https://imagej.net/,accessed on 24 February 2023).

Co-Immunoprecipitation (Co-IP)
The investigation of TLR 4 /MD-2 complex formation was carried out as previously described [36].Briefly, RAW 264.7 cells (6 × 10 5 cells/mL) were seeded into a 6-well plate for 6 h, then incubated with G-Rh 2 (15 µg/mL) in the presence or absence of LPS (1 µg/mL) for 12 h.Subsequently, the cells were lysed with buffer containing protease and phosohatase inhibitors.Cell extract was incubated with adequate amounts of anti-MD-2 antibody at 4 • C overnight, and then precipitated with protein A/G magnetic beads under the same conditions.After boiling, the released protein was detected by immunoblotting using anti-TLR 4 antibody.The experimental procedure was consistent with the Western blot assay as before.

Molecular Docking Study
Molecular docking of G-Rh 2 with TLR 4 /MD-2 was conducted with reference to Yao et al. [37] and performed in Autodock Vina software (version 1.1.2).Firstly, the threedimensional structures of the compound G-Rh 2 were downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov,accessed on 9 November 2022).Next, the crystal structures of TLR 4 /MD-2 (PDB ID: 2Z65) were downloaded from the PDB database (https://www.rcsb.org/,accessed on 9 November 2022).Then, we imported them into AutoDockTools software (version 1.5.6) to remove water molecules, add nonpolar hydrogens and calculate the Gasteiger charges of the structures, and saved them as PDBQT files.
Lastly, the docking procedure was undertaken for the purpose of analyzing the results using both PyMOL (version 2.5) and AutoDock Vina software.

SPR Analysis
A Biacore T200 Biomolecular Interaction Analysis system (Company, Shanghai, China) and Series Sensor Chip CM5 (10246576) were used to determine the binding affinity of G-Rh 2 to recombinant human TLR 4 /MD-2.SPR analysis was carried out as previously described [38].Briefly, the TLR 4 /MD-2 protein (in acetate acid buffer pH 5.0) was loaded onto the sensors, and then an EDC and NHS mixture was added for activation of the chip.Different concentrations of G-Rh 2 (200, 100, 50, 25, 12.5, 6.25, 3.125, 1.562 and 0 µM) were prepared with a running buffer (PBS, 0.2% Tween-20, 5% DMSO, pH 7.4).The sensor and sample plate were positioned within the instrument.Following the manufacturer's guidelines, interactions were assessed at a flow rate of 50 µL/min, comprising an association phase of 60 s and a dissociation phase of 60 s.The collected data were analyzed using BIAcore T200 Evaluation software (version 3.2.1).Binding kinetic parameters, including KD values, were determined through global fitting of the kinetic data obtained from different concentrations of Blumeatin, employing a 1:1 Langmuir binding model.

Statistical Analysis
Statistical analysis was conducted using SPSS 17.0 (SPSS Inc., Chicago, IL, USA).The results are reported as mean ± standard deviation (SD) and were evaluated through one-way analysis of variance (ANOVA).p < 0.05 was deemed indicative of statistical significance.

Figure 2 .Figure 2 . 13 Figure 3 .
Figure 2. Effect of G-Rh2 on FITC-LPS binding to RAW 264.7 cells.LPS was incubated with RAW 264.7 cells alone or together with G-Rh2 for 6 h.Cells were collected and the mean fluorescence values of cells in each group were detected by flow cytometry.(A) The bound FITC-LPS with RAW 264.7 was examined by flow cytometry.(B) The fluorescence intensity was analyzed.The results are shown as means ± SD (n = 3) of at least three independent experiments (**, p < 0.01, compared with the mean fluorescence values of cells in the control group.##, compared with the mean fluorescence values of cells in the LPS group, p < 0.01).

Figure 3 .
Figure 3. G-Rh 2 blocks the localization of FITC-LPS on the cell membrane.Morphologically, the effect of G-Rh 2 on the localization of FITC-LPS on the cell membrane was observed by CLSM.Protein on the cell membrane was observed as red fluorescence circles on the DID line; FITC-LPS with green fluorescence was bound to the membrane, and was observed as green circles on the cell membrane.

Figure 4 .
Figure 4. Effects of G-Rh2 on the expression of TLR4 signaling pathway proteins.(A-D) LPS wa incubated with RAW 264.7 cells alone or together with G-Rh2 for 12 h.The effects of G-Rh2 on M 2, TLR4 and P-NF-κB p65 were analyzed by Western blotting.The results are shown as means ± (n = 3) of at least three independent experiments (**, p < 0.01, compared with the control group.compared with the LPS group, p < 0.01).

Figure 5 .
Figure 5. G-Rh2 inhibits the formation of the TLR4/MD-2 complex.A Co-IP assay detected the e fect of G-Rh2 on the complex formation of TLR4 and MD-2.The results are shown as means ± SD = 3) of at least three independent experiments (**, p < 0.01, compared with the control group.## compared with the LPS group, p < 0.01).

Figure 4 .Figure 4 .
Figure 4. Effects of G-Rh 2 on the expression of TLR 4 signaling pathway proteins.(A-D) LPS was incubated with RAW 264.7 cells alone or together with G-Rh 2 for 12 h.The effects of G-Rh 2 on MD-2, TLR 4 and P-NF-κB p65 were analyzed by Western blotting.The results are shown as means ± SD (n = 3) of at least three independent experiments (**, p < 0.01, compared with the control group.##, compared with the LPS group, p < 0.01).

Figure 5 .
Figure 5. G-Rh2 inhibits the formation of the TLR4/MD-2 complex.A Co-IP assay detected the effect of G-Rh2 on the complex formation of TLR4 and MD-2.The results are shown as means ± SD (n = 3) of at least three independent experiments (**, p < 0.01, compared with the control group.##, compared with the LPS group, p < 0.01).

Figure 5 .
Figure 5. G-Rh 2 inhibits the formation of the TLR 4 /MD-2 complex.A Co-IP assay detected the effect of G-Rh 2 on the complex formation of TLR 4 and MD-2.The results are shown as means ± SD (n = 3) of at least three independent experiments (**, p < 0.01, compared with the control group.##, compared with the LPS group, p < 0.01).

2. 5 .
G-Rh 2 Binds to TLR 4 /MD-2, Blocking the Formation of LPS-TLR 4 /MD-2 Complexes To investigate the mechanism of G-Rh 2 inhibiting the dimerization of TLR 4 and MD-2, we performed molecular simulations of complexes formed by G-Rh 2 and TLR 4 /MD-2 using molecular docking software (version 1.1.2).As shown in Figure 6A, G-Rh 2 is mated into the hydrophobic pocket of TLR 4 /MD-2, which binds to TLR 4 /MD-2 and may presentNext, we evaluated the interaction between G-Rh2 and TLR4/MD-2 through SPR experiments; as shown in Figure6B, G-Rh2 binds to TLR4/MD-2 protein in a dose-dependent manner and presents a "fast up, fast down" binding pattern.G-Rh2 and TLR4/MD-2 have a specific binding force and an affinity of KD = 71.20 µM.