Immunomodulatory Effects of a Standardized Botanical Mixture Comprising Angelica gigas Roots and Pueraria lobata Flowers Through the TLR2/6 Pathway in RAW 264.7 Macrophages and Cyclophosphamide-Induced Immunosuppression Mice

Abstract

Background: As the population ages, enhancing immune function is crucial to mitigating age-related physiological decline. Since immunostimulant drugs are known to have potential side effects, medicinal plants emerge as promising candidates offering a safer alternative. To leverage the advantages of medicinal plants with fewer side effects and develop a potent immune-enhancing agent, we investigated the efficacy of a novel immunomodulatory candidate derived from the combination of Angelica gigas and Pueraria lobata (CHL). Methods: In vitro, CHL was treated in RAW 264.7 macrophages at various time points, and the experiments conducted in the study were performed using ELISA, Western blot, and RT-qPCR analysis. In vivo, C57BL/6 mice were administrated CHL for 16 days (p.o.) and CTX on the three days (i.p.), and experiments were conducted with ELISA, western blot, RT-qPCR analysis, H&E staining, flow cytometry, gut microbiome, and correlation analysis. Results: In vitro, CHL has upregulated NO and cytokines expression, substantially enhancing the NF-κB and MAPK activation. Furthermore, CHL promoted the TAK1, TRAF6, and MyD88 via TLR2/6 signaling. In vivo, the CHL improved the reduced body weight and immune organs' indices and recovered various cytokines expression, NK cell cytotoxicity activity, and immune cell population. CHL also improved the histological structure and tight junction markers, mucin-2, and TLR2/6 in the intestines of CTX-induced mice. Conclusions: Overall, CHL demonstrated immunostimulatory potential by enhancing immune responses and restoring immune function, suggesting its promise as a safe and effective immune-enhancing agent.

Keywords: immunomodulatory; intestine; macrophages; medicinal herb; toll-like receptor 2/6.

Figure 1

Effects of CHL on immune mediator production and expression in RAW 264.7 macrophages. (A,B) Cells were stimulated with A. gigas (100 μg/mL), P. lobata (100 μg/mL), CHL (50, 100, or 200 μg/mL), or LPS (5 ng/mL) for 24 h. (C) Cells were pretreated with polymyxin B (0.1 μg/mL) and then stimulated with CHL (200 μg/mL) or LPS (5 ng/mL) for 24 h. (D–K) Cells were stimulated with CHL (50, 100, or 200 μg/mL) or LPS (5 ng/mL). LPS was used as a positive control and β-actin was used as an internal control. Data are presented as mean ± SEM of three independent experiments. ** p < 0.01, *** p < 0.001 vs. CON; ### p < 0.001 vs. LPS-treated cells.

Figure 2

Effects of CHL on the TLR2/6 signaling pathway in RAW 264.7 macrophages. (A–I) Cells were stimulated with CHL (50, 100, or 200 μg/mL) or LPS (5 ng/mL) for 15–30 min or 6 h. LPS was used as a positive control and β-actin was used as an internal control. Data are presented as mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CON.

Figure 3

Effects of CHL on body weight, immune organ indices, and cytokine expression in CTX-treated mice. (A) Body weights and indices of (B) spleen and (C) MLN were measured at the end of the animal experiments. Data are presented as mean ± SEM (n = 23–24). (D–H) Cytokine production and (G–M) mRNA expression of IL-12, IFN-γ, TNF-α, IL-4, and IL-6. Data are presented as mean ± SEM (n = 7). # p < 0.05 vs. CON group; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CTX group.

Figure 4

Effects of CHL on natural killer (NK) cell activity and characterization of innate immune cell population in CTX-treated mice. (A) NK cell activity of CHL, cell ratio between splenocytes and YAC-1 = 1:5, 1:10, or 1:20. Population of (B) CD3⁻/NK1.1⁺ NK cells, (C) CD11b⁺/MHC II⁺ dendritic cells, (D) CD11b⁺/Ly6C⁺ monocytes, (E) CD11b⁺/Ly6C⁺/F4/80⁺ macrophages, and (F) CD11b⁺/Ly6G⁺ neutrophils. Data are presented as mean ± SEM (n = 6–7). # p < 0.05 vs. CON group; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CTX group.

Figure 5

Effects of CHL on the characterization of the adaptive immune cell population in CTX-treated mice. Populations of (A) CD3⁺ T cells, (B) CD3⁺/CD4⁺ T helper cells, (C) CD3⁺/CD4⁺/IFN-γ⁺ Th1 cells, (D) CD3⁺/CD4⁺/IL-4⁺ Th2 cells, (E) CD3⁺/CD4⁺/IL-17⁺ Th17 cells, and (F) CD3⁺/CD4⁺/CD25⁺/FoxP3⁺ regulatory T cells. Data are presented as mean ± SEM (n = 6–7). # p < 0.05 vs. CON group; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CTX group.

Figure 6

Effects of CHL on histological changes in the intestine and the expression of tight junction markers and mucin 2 in CTX-treated mice. Histological changes in the (A) small intestine and (B) colon. (C–L) Protein and mRNA expression of tight junction-related markers (ZO-1, occludin, and claudin-1) and MUC2 in the small and large intestines. Data are presented as mean ± SEM (n = 6–7). # p < 0.05 vs. CON group; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CTX group.

Figure 7

Effects of CHL on gut microbiome composition in CTX-treated mice. Analysis of microbial diversity: (A) Principal coordinate analysis (PCoA) plots; (B) Chao1 index; (C) Simpson index between each group. (D–G) The relative ratio of Firmicutes, Deferribacteres, Bacteroidetes, and Proteobacteria. (H,I) GPR41 and GPR43 mRNA expression in the colon. Correlation analysis between gut microbiota and (J) immune response and (K) intestinal immunity. Data are presented as mean ± SEM (n = 6–7). # p < 0.05 vs. CON group; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CTX group.

Figure 8

Effects of CHL on TLR2/6 signaling pathway in CTX-treated mice. (A) TLR2 and (B) TLR6 mRNA expressions of the small intestine. Data are presented as the means ± SEM (n = 7). # p < 0.05 vs. CON group; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CTX group. Correlation analysis between gut microbiota and (C) TLR2/6. Regression analysis between TLR2/6 and (D,E) Firmicutes; (F,G) Deferribacteres; (H,I) Bacteroidetes; and (J,K) Proteobacteria. R means correlation coefficient value and P means p value.

Figure 9

Representative HPLC chromatograms of CHL and standards (nodakenin and tectoridin). HPLC chromatograms of (A) nodakenin standard and CHL at 330 nm and (B) tectoridin standard and CHL at 260 nm.