Anti-Inflammatory Effects and Immunomodulatory Efficacy of Unitein (Fermented Glycine max, Panax ginseng, and Chenpi Mixture) in RAW 264.7 Macrophages and Mice Splenocytes along with Component Analysis

Abstract

Inflammation and the immune system are intricately linked, with the immune system serving as a vital defense mechanism in the human body. Consequently, there is great emphasis placed on the regulation of both the body’s inflammatory response and the immune system. This study investigated the anti-inflammatory properties and immunomodulatory effects of Unitein (fermented Glycine max, Panax ginseng, and chenpi mixture) in both RAW 264.7 macrophages and mice splenocytes while also comprehensively analyzing its components. To this end, the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay was employed to evaluate the cytotoxicity of the samples to RAW 264.7 cells. The Griess assay was utilized to determine nitrite concentrations, while the enzyme-linked immunosorbent assay (ELISA) was employed to assess the levels of inflammation-related cytokines in RAW 264.7 cells and isolated mouse splenocytes. Additionally, quantitative polymerase chain reaction was employed to quantify the mRNA expression of inflammation-related genes in the cells, while the lactate dehydrogenase assay was used to analyze natural killer (NK) cell activity. Our results revealed that Unitein exhibited no toxicity to RAW 264.7 cells at concentrations below 1.5 mg/mL. However, Unitein effectively suppressed the release of proinflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and interferon (IFN)-γ, as well as enzymes COX-2 and iNOS from RAW 264.7 cells, while concurrently promoting the release of anti-inflammatory cytokines IκB-α, IL-10, and IL-4. Furthermore, Unitein inhibited the increase in TNF-α, IL-1β, IFN-γ, IL-12, nuclear factor (NF)-κB p50, NF-κB p65, and iNOS induced by lipopolysaccharides (LPSs) in isolated mouse splenocytes. Notably, Unitein also exhibited the potential to stimulate NK cell activity. Metabolite analysis indicated that Unitein contained more active compounds with anti-inflammatory and immunity-enhancing properties than did unfermented Unitein. This study highlights the potential therapeutic role of Unitein in mitigating inflammation and enhancing immune responses, thereby deepening our understanding of its biological activities and underlying mechanisms. These findings underscore the significance of Unitein as a potential candidate for the development of novel anti-inflammatory and immunomodulatory interventions.