Structural analogues in herbal medicine ginseng hit a shared target to achieve cumulative bioactivity

Abstract

By a pilot trial on investigating immunomodulatory activity and target of ginsenosides, the major bioactive components of ginseng, here we report that structural analogues in herbal medicines hit a shared target to achieve cumulative bioactivity. A ginsenoside analogues combination with definite immunomodulatory activity in vivo was designed by integrating pharmacodynamics, serum pharmacochemistry and pharmacokinetics approaches. The cumulative bioactivity of the ginsenoside analogues was validated on LPS/ATP-induced RAW264.7 macrophages. The potentially shared target NLRP3 involved in this immunomodulatory activity was predicted by systems pharmacology. The steady binding affinity between each ginsenoside and NLRP3 was defined by molecular docking and bio-layer interferometry assay. The activation of NLRP3 inflammasomes in LPS/ATP-induced RAW264.7 was significantly suppressed by the combination, but not by any individual, and the overexpression of NLRP3 counteracted the immunomodulatory activity of the combination. All these results demonstrate that the ginsenoside analogues jointly hit NLRP3 to achieve cumulative immunomodulatory activity.

Fig. 1. Pharmacochemical, pharmacokinetic and pharmacodynamic analyses determined the analogues’ combination as ginsenoside Rb₁ (1247.32 ng/mL, 1.12 µM), Rd (5510.30 ng/mL, 5.82 µM), Rg₃ (16.25 ng/mL, 20.72 nM) and F₂ (3.07 ng/mL, 3.91 nM).

a Body weight (n = 10), b spleen index (n = 5), c thymus index (n = 5), d IL-2 (n = 10), e IL-6 (n = 10), f IFN-γ (n = 10), g IgG (n = 10), h IgM (n = 10) and i splenocyte proliferative (n = 3). j HPLC-TQ-MS/MS chromatograms (negative ion mode) of ginsenoside Rb₁ and its metabolites in serum. k Proposed in vivo metabolic pathway of ginsenoside Rb₁. l–o Concentration–time curves of ginsenoside Rb₁ and its metabolites (Rd, Rg₃ and F₂) after the oral administration of ginsenoside Rb₁ (160 mg/kg) on the 7th day (n = 3). p–t Dynamic pharmacodynamic evaluation after the oral administration of ginsenoside Rb₁ (160 mg/kg) on the 7th day (n = 8). Data are expressed as mean ± SD. Compared with Con, ^#P < 0.05, ^##P < 0.0¹; compared with Mod, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001; ns represents no significant difference. Con: control group; Mod: model group; L: Rb₁-treated group with low dose (40 mg/kg); M: Rb₁-treated group with middle dose (80 mg/kg); H: Rb₁-treated group with high dose (160 mg/kg).

Fig. 2. Ginsenoside analogues’ combination achieved stronger immunomodulatory activity in vitro than individual ginsenosides alone at the same dosages.

a Cell proliferation (n = 6), b, c, g cell cycle, d, h cell migration, e, i cell invasion, f, j cell pyroptosis, k qRT-PCR assay for mRNA expressions and l ELISA for levels of pro-inflammatory (iNOS, IL-1β and TNF-α) and anti-inflammatory (IL-4 and IL-10) cytokines (b–l, n = 3). Data are expressed as mean ± SD. Compared with Mod, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001; compared with Com, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001.

Fig. 3. Systems pharmacology predicted NLRP3 as a key shared target in mediating the immunomodulatory activity of ginsenoside analogues.

a PPI network analysis, b KEGG analysis, c compounds-targets-pathways network and d centrality algorithm analysis of the network.

Fig. 4. Binding affinity of the ginsenoside analogues to NLRP3 was evaluated by molecular docking and bio-layer interferometry.

Molecular docking predicted the binding sites of ginsenoside analogues: a, e Rb₁, b, f Rd, c, g Rg₃, d, h F₂ to NLRP3. The protein structure is demonstrated in ribbon format while ligand is represented in ball and stick format. Note: Red areas mean oxygen atom, grey areas mean carbon atom, blue areas mean nitrogen atom, white areas mean hydrogen atom and green areas mean other. The green dotted lines represent hydrogen bonds. Bio-layer interferometry validated the binding affinity of the ginsenoside analogues Rb₁, Rd, Rg₃, F₂ to NLRP3. Typical kinetic characterization of NLRP3 to various concentrations of analogues by using BLI assay (n = 5). i Biosensors were exposed to various concentrations (marked 1–5) of Rb₁ solution (1.44, 7.21, 36.1, 180.3, 901.7 μM) for association (600 s) and dissociation (600 s). j Biosensors were exposed to Rd solution (9.06, 18.1, 36.3, 72.6, 145.2 μM) for association (300 s) and dissociation (300 s). k Biosensors were exposed to Rg₃ solution (7.18, 14.4, 28.7, 57.4, 114.8 μM) for association (240 s) and dissociation (240 s). l Biosensors were exposed to F₂ solution (14.2, 28.3, 56.6, 113.2, 226.4 μM) for association (180 s) and dissociation (180 s). Association and dissociation steps are divided by the dotted line. Panels (m–p) represent Rb₁, Rd, Rg₃ and F₂ in the association and dissociation steps in order to fit results globally with a 1:1 binding model and get the best values of k_on, k_dis and K_D.

Fig. 5. The combination, but not individuals, significantly inhibits NLRP3 inflammasome activation in LPS/ATP-stimulated RAW264.7 macrophages.

Immunostaining assay for activated NLRP3 (a) and western blot assay for determining protein expressions: b NLRP3, c NEK7, d ASC, e cleaved caspase-1/caspase-1, f cleaved IL-1β /IL-1β, g IL-18 in cell lysates. GAPDH was used as the loading control. All data are expressed as mean ± SD (n = 3). Compared with Mod, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001; compared with Com, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001.

Fig. 6. Overexpression of NLRP3 counteracted the immunomodulatory activity of the combination in LPS/ATP-stimulated RAW264.7 macrophages.

a Cell proliferation (n = 6), b, c, g cell cycle (n = 6), d, h cell migration, e, i cell pyroptosis, f, j cell apoptosis, k immunostaining assay for activated NLRP3. l, mWestern blot assay for determining protein expressions of NLRP3, NEK7, ASC, caspase-1, cleaved caspase-1, IL-1β, cleaved IL-1β and IL-18 in cells lysates (GAPDH was measured as the loading control). n ELISA for the levels of pro-inflammatory (iNOS, TNF-α and IL-1β) and anti-inflammatory (IL-4 and IL-10) cytokines in cell supernatants (d–n, n = 3). All data are expressed as mean ± SD. ^*P < 0.05, ^**P < 0.01; ns represents no significant difference.