Study on the immunomodulatory effect of saponins from Panax ginseng and Panax ginseng on RAW 264.7 cells in vitro
Macrophages involved in non-specific immunity of the body are the first line of defense against pathogens. Activated macrophages can release various inflammatory factors and cytokines (such as NO and TNF – α) to participate in the body’s immune response and regulate the immune response. Research has shown that saponins in traditional Chinese medicine can activate macrophages, enhance cell phagocytic ability, promote the secretion of related active molecules, and upregulate the expression of related proteins. Radix Pseudostellaria fibroblast roots saponins (RPFRS) are one of the bioactive components isolated from the ginseng roots of Pseudostellaria heterophylla, which have the effects of enhancing the body’s immunity, antioxidation, and so on. Nowadays, the main root of Panax ginseng is used as medicine, while the root silk is discarded, causing great waste. Research has found that the average content of total saponins in the root silk of Panax ginseng is 1.14 times that of the root tuber. The preliminary research of the research group has shown that the extract of Panax ginseng and Panax ginseng, mainly composed of saponins, has an immunoprotective effect on immunosuppressed mice and can enhance their immune function. At present, there are few reports on the immune effects of RPFRS alone, and mouse mononuclear macrophages (RAW 264.7 cells) are often used as a model for in vitro immune activity studies. Therefore, this experiment targeted RAW 264.7 cells and studied the immunomodulatory effects and possible mechanisms of RPFRS in vitro by detecting immunological indicators such as cell proliferation, phagocytic activity, related cytokines, and mRNA expression. The aim of the study is to enrich the immunological content of RPFRS and provide theoretical basis for the development and utilization of Panax ginseng.

 

The effect of RPFRS on the proliferation of RAW 264.7 cells. Saponins have certain hemolytic and toxic properties, and have lower toxic doses compared to other plant extracts, so attention should be paid to dosage when using them. To determine whether RPFRS has toxic effects on RAW 264.7 cells and screen for the optimal concentration of RPFRS for experiments, the MTT method was used to determine the effect of different concentrations of RPFRS on the proliferation of RAW 264.7 cells. The results of this experiment showed that RPFRS could significantly promote the proliferation of RAW 264.7 cells in the concentration range of 12.5-800 μ g/mL (P<0.01), and showed a dose-response relationship in the concentration range of 12.5-100 μ g/mL. Therefore, concentrations of 12.5-200 μ g/mL were selected for subsequent experiments.

The effect of RPFRS on the phagocytic activity of RAW 264.7 cells. Phagocytosis is one of the main functions of macrophages. In non-specific immunity, macrophages can kill or digest pathogens within cells, dead cells of the body, and other large particle antigens through phagocytosis. In specific immunity, they can play a role in immune regulation and antigen presentation. There are research reports that both Platycodon grandiflorus saponins D and Magnolia grandiflorus saponins can enhance the phagocytic ability of mouse macrophages. Consistent with previous studies, the results of this experiment indicate that RPFRS can increase the OD value of RAW 264.7 cell phagocytosis of neutral red in a dose-dependent manner at concentrations of 12.5-200 μ g/mL (P<0.01), indicating that RPFRS can enhance the phagocytic activity of RAW 264.7 cells and enhance their immune function.

The effect of RPFRS on the secretion of NO and iNOS mRNA expression in RAW 264.7 cells. Macrophages can eliminate pathogens by triggering immune inflammatory responses and assist the body in regulating specific immune responses, while NO synthesized by nitric oxide synthase (iNOS) is closely related to its inflammatory immune response function. Yang et al. found that the protein hydrolysate of Panax ginseng can promote RAW 264 cells to secrete NO and activate macrophages with TNF – α, enhancing their immune activity. The results of this experiment showed that compared with the blank control group, RPFRS could significantly increase the content of NO in the supernatant of RAW 264.7 cells (P<0.05), and at the same time, the expression of iNOS in RAW 264.7 cells was also significantly increased (P<0.01), both of which showed a dose-dependent increase. This result is consistent with the findings of Li et al. that Astragaloside IV can increase the expression of NO and iNOS mRNA in the supernatant of RAW 264.7 cells.
The effect of RPFRS on cytokine secretion and mRNA expression in RAW 264.7 cells. Cytokines such as IL-6, IL-10, IL-1 β, and TNF – α mainly play a role in local and systemic inflammatory responses. IL-6 can induce B lymphocyte precursors to become antibody secreting cells. IL-10, as an anti-inflammatory factor, can inhibit macrophage activity and inflammatory cytokines such as IL-6 and TNF – α. IL-1 β can promote the activation and degranulation of macrophages and neutrophils, as well as the expression of other inflammatory factors. TNF α is the main pro-inflammatory cytokine produced by macrophages in response to various stimuli, which can promote the secretion of cytokines such as IL-6 and IL-8. Sun et al. found that soybean saponin Ab can increase the levels of TNF – α and IL-1 β in the supernatant of RAW 264.7 cells. Li et al. found that Astragaloside IV can increase the levels and mRNA expression of IL-6, IL-1 β, and TNF – α in the supernatant of RAW 264.7 cells. The results of this study showed that compared with the blank control group, RPFRS could dose dependently increase the levels of IL-6, IL-10, IL-1 β, and TNF – α in the supernatant of RAW 264.7 cells. RPFRS also increased the mRNA expression levels of IL-6, IL-10, IL-1 β, and TNF – α in RAW 264.7 cells to varying degrees, indicating that RPFRS can regulate their immune function by stimulating the secretion of cytokines.
The effect of RPFRS on the expression of NF – κ B pathway related proteins in RAW 264.7 cells. NF – κ B, as a nuclear transcription factor, can activate the expression of various genes related to early defense responses, thereby regulating physiological processes such as cell apoptosis, immune response, inflammation, and tissue remodeling. The activation of the NF – κ B signaling pathway is stimulated by various signaling molecules. TLR2 and TLR4, as members of the pattern recognition receptor family, are highly expressed on the surfaces of macrophages, B cells, and DC cells, and can monitor and recognize disease-related molecular patterns to initiate signal transduction in the signaling pathway. The latter can also stimulate MyD88 to trigger a signaling cascade and induce NF – κ B activation. MyD88 and TRIF bind to TLR binding proteins in the cytoplasm, activate the MAPK pathway under the action of ubiquitin, promote I κ B – α phosphorylation to activate the NF – κ B pathway, and regulate downstream gene expression. He et al. found that after intervention with different doses of total saponins from Platycodon grandiflorum, the levels of IL-6, IL-1 β, and TNF – α in the synovial tissue of the ankle joint of collagen induced arthritis rats decreased, while the levels of TLR2, TLR4, MyD88 and other proteins decreased. Shin et al. found that heat-treated ginsenosides Rg1 and Rg3 can promote the secretion of IL-6 and TNF – α in RAW264.7 cells, as well as the phosphorylation of MAPK and the degradation of I κ B – α to activate the NF – κ B signaling pathway. The results of this experiment showed that under the action of RPFRS, the mRNA expression of TLR4 in RAW 264.7 cells decreased, while the mRNA expression of MyD88 and NF – κ B increased; Consistent with this, the results of WB showed a decrease in the expression of TLR4, I κ B α, and cytoplasmic NF – κ B proteins, while the expression of TLR2, MyD88, TRIF, and nuclear NF – κ B proteins increased, indicating the translocation of NF – κ B from the cytoplasm to the nucleus. After activation of the NF – κ B pathway, it can reverse the transcription induction mediated by pro-inflammatory cytokines, which corresponds to the results of cytokine content and mRNA expression mentioned above. This indicates that RPFRS can activate RAW 264.7 cells through the TLR2 and TLR4 mediated TRIF/MyD88-NF – κ B transduction pathway.
RPFRS can promote the proliferation of RAW 246.7 cells, enhance phagocytic activity, increase the content and mRNA expression of related cytokines, downregulate the mRNA expression of TLR4, upregulate the expression of TLR2, MyD88, TRIF, and nuclear NF – κ B protein, downregulate the expression of TLR4, I κ B – α, and cytoplasmic NF – κ B protein, and show a certain dose effect. RPFRS can exert immune protection by inhibiting the stimulation of TLR4 by LPS, and may also activate RAW 264.7 cells through the TLR2/4-MyD88/TRIF-NF – κ B signaling pathway, participating in the immune response process and exerting immune regulatory effects. Further research is needed to investigate the relationship between RPFRS and other signaling pathways involved in immune regulation of RAW 264.7 cells, as well as their components and immune activity.