Anti-inflammatory Activity Of Allicin And Its Effect On TLR4/NF-κB/MAPKs Signaling Pathway

Garlic belongs to the genus Allium of the Liliaceae,a biennial herb.Garlic and other related plants such as shallots,chives,and onions are commonly used Chinese herbal medicines.Garlic is an ancient medicinal and edible treasure,which is called "health protection god" abroad.It is rich in many nutrients and has certain nutritional value.As the main biologically active substance in garlic,allicin has many beneficial biological functions for the human body,including anti-inflammatory,anti-oxidant,anti-tumor,anti-bacterial,and so on.To investigate the anti-inflammatory effect of allicin,we used 1 μg/m L lipopolysaccharide to stimulate mouse primary peritoneal macrophages as an in vitro inflammatory model,and dexamethasone as a positive control to explore its anti-inflammatory activity and its effect on TLR4/NF-κB/MAPKs signaling pathway at cellular and molecular levels,so as to provide experimental basis for the development and utilization of allicin in clinical anti-inflammatory effect.The research methods and results of this article are as follows:(1)MTT method was used to detect the cytotoxicity of allicin at different concentrations on mouse peritoneal macrophages and selected 40,80,and 160 μg/m L as low,medium,and high concentrations of allicin.Considering the effects of different concentrations of LPS and dexamethasone on NO secretion and cytotoxicity of mouse peritoneal macrophages.1 μg/m L LPS was used as the stimulating concentration to construct the inflammatory model of mouse peritoneal macrophages in vitro and 100 μg/m L dexamethasone as the positive control in this experiment.(2)Neutral red phagocytosis assay was used to detect the effect of allicin on the phagocytic function of mouse peritoneal macrophages.The results showed that allicin could effectively activate mouse peritoneal macrophages and thus promote their phagocytosis.(3)Griess,ELISA and Western blot methods were used to detect the inflammatory factors and related proteins.The results showed that allicin pretreatment could significantly inhibit the COX-2 enzyme activity,NO,IL-6 and IL-1β secretion of mouse peritoneal macrophages induced by LPS,and significantly inhibit the relative expression of COX-2,i NOS,IL-6 and IL-1β proteins.(4)RT-q PCR method was used to detect the effect of allicin on the m RNA expression of i NOS,COX-2,IL-6,and IL-1β in LPS-induced mouse peritoneal macrophages.The results showed that after 1μg/m L LPS stimulated mouse peritoneal macrophages,allicin pretreatment inhibited the expression of i NOS,COX-2,IL-6 and IL-1β m RNA in different degrees,and the inhibitory effect was more significant with the increase of allicin concentration,showing an obvious dose-effect relationship.(5)Western Blot method was used to detect the effect of allicin on TLR4-mediated NF-κB and MAPKs pathway related proteins in mouse peritoneal macrophages induced by LPS.The results showed that after 1 μg/m L LPS stimulated mouse peritoneal macrophages,the protein expression of TLR4 was significantly up-regulated,while allicin pretreatment could effectively inhibit the protein expression of TLR4 after LPS stimulation.Compared with the blank control group,the phosphorylation levels of P38,JNK,and ERK in mouse peritoneal macrophages increased significantly after LPS stimulation.Compared with the LPS group,allicin pretreatment at 40,80,and 160 μg/m L could inhibit the phosphorylation of P38,JNK,and ERK in different degrees.After 1 μg/m L LPS stimulated mouse peritoneal macrophages,the phosphorylation level of P65 increased significantly.Compared with the LPS group,allicin pretreatment at40,80,and 160 μg/m L could significantly inhibit the phosphorylation of P65.In summary,research shows that allicin can inhibit pro-inflammatory cytokines production by down-regulating TLR4 expression protein and inhibiting the activation of NF-κB and MAPKs in mouse peritoneal macrophages stimulated by LPS.