TLR4/NF-κB-mediated Formononetin On The Immune Regulation Of Mouse Peritoneal Macrophages And KCs Cells

Formononetin(FMN)is a natural isoflavone compound:and it is the main active ingredient in the total flavonoids of Ammopiptanthus seeds.It is distributed in a variety of plants and traditional authentic medicinal materials.Preliminary studies have shown that FMN has a good effect for promoting the immune system of animals,and it is an important direction for the development of new natural non-toxic veterinary immunopotentiators.At present,extraction and purification,anti-inflammatory,anti-oxidant and other effects are mainly fo cused on the research ofFMN.However,there are relatively few studies on FMN’s regulatory effect on the innate immune system of animals and its mechanism.Therefore,in order to discuss the effect of FMN on the innate immune system of animals and its molecular mechanism further,mice peritoneal macrophages(PM)and liver Kupffer cells(KCs)were used as research objects in the study to explore the active effects of FMN on macrophage proliferation,migration,phagocytosis,and cytokine secretion.Concurrently,the signal transduction effect of TLR4/NF-κB signaling pathway was adopted to discover the molecular mechanism for the viability of mice PM and KCs cells regulated by FMN.In this experment,cell viability test(including cell proliferation in vitro cell adhesion count,cell migration,macrophage phagocytosis,cytokine secretion),and expression of key TLR4/NF-κB proteins were used to explore the immunomodulatory effects and mechanism of FMN on PM and KCs cells.The results of the study show:①There is a obvious effect of promoting the proliferation of PM and KCs cells for FMN.Compared with the blank group,within the dose of 2.5～10μg/mL of FMN,it has obvious proli feration on PM and KCs cells,and when the dose of FMN is 2.5μg/mL:its effects is the most prominent.②There is a obvious effect of promoting the adhesion of PM cells for FMN.Within the dose of 2.5～10μg/mL of FMN，the concentration of PM cells is significantly increased,and the cells adhered the most at the 5.0μg/mL dose of FMN.③There is a obvious effect of promoting the migration of PM cells for FMN.Within the dose of 2.5～10μg/mL of FMN:the cells migration rate increased at the 5.Oμg/mL dose of FNN，it is the highest of the PM cells migration rate,and the largest number of cells passing through the polycarbonate membrane as well.④There is aobvious effect of promoting the phagocytic ability of PM and KCs cells for FMN.Within the dose of 2.5～4μg/mL of FMN,it can promote the phagocytosis of neutral red dye by PM and KCs cells,and the quantity of phagocytosis increases with the increasing of FMN doses.When the dose of FMN reach to 40～g/mL,it is the highest for the phagocytic activity of PM and KCs cells.⑤There are various effects of promoting the secretion of cytokines of PM and KCs cells for FMN.Within the dose of 2.5～10μg/mL of FMN,it is good to promote the secretion of IL-1β and IL-6 by PM cells,and at the 5.0μg/mL dose of FMN,the secretion of IL-1β and IL-6 by PM cells reaches maximum.Concurrently,within the dose of 2.5～40μg/mL of FMN,it is good to promote the secretion of IL-1β and IL-6 by KCs cells,and at the 10μg/mL dose of FMN,the secretion of IL-1β and IL-6 in KCs cells reaches maximum.In addition,Within the dose of 2.5～10μg/mL of FMN,it cat significantly promote the secretion of NO by PM and KCs cells.⑥There is a significant effect of promoting the expression of key proteins in TLR4/NF-κB signaling pathway of mice PM and KCs cells for FMN.The expression levels of TLR4,MyD88,and nuclear factor P65 in PM and KCs cells are increased to varying degrees by FMN within the dose of 2.5～10μg/mL.The above experiments have proved that the fimction of the body’s innate immune system was promoted and strengthened obviously that FMN can significantly promote the proliferation of mice PM and KCs cells,enhance the phagocytic ability of PM and KCs cells and the secretion of cytokines within a certain dose range,thereby greatly enhancing the activity of mice maicrophages.Concurrently,it is also proved that FMN can regulate the viability of mouse PM and KCs cells by TLR4/NF-κB signalind pathway to regulate and enhance the body’s innate immune system.In addition,studies have also proved that FMN possess the two-way regulation of the viability of PM and KCs cells.It is an inhibitory effect that the doses of FMN exceed over a certain range on the viability of PM and KCs cells.