Effects Of Total Flavonoids From Allium Mongolicum Regel On Anti-inflammation In LPS Stressed Mouse Peritoneal Macrophages

The present study aims to examine the protection and anti-inflammatory activity of total flavonoids originated from Allium Mongolicum regel on lipopolysaccharide(LPS)stressed mouse peritoneal macrophages(MPM).Therefore,LPS stressed MPM were used as the stress model to study the effects of different concentrations of total flavonoids originated from Allium Mongolicum regel on inflammatory mediators,cytokines,relative genes and NF-κB pathway,to provide scientific basis for the further development and utilization of flavonoids of Allium Mongolicum regel.This study consists of 4 experiments:Experiment 1 Selection of optimum additive concentration of total flavonoids.CCK-8 method was applied to select concentrations that facilitate MPM cell viability.Results showed that,total flavonoids of Allium Mongolicum regel stimulated cell viability when the concentration was 12.5~200μg/m L compared to the control group.When the concentration was 200μg/m L,the cell viability tended to decline,therefore in the following experiments the additive concentration of total flavonoids were 25~100μg/m L.Experiment 2 Effects of total flavonoids of Allium Mongolicum regel on inflammatory factors in culture medium of LPS stressed MPM.Treatments include control,LPS stressed group(1μg/m L LPS),pretreatment of total flavonoids + LPS group(pretreatment of 25,50 or 100μg/m L of total flavonoids for 1h before addition of 1μg/m L LPS).Cells were cultured with different treatments for 24 h and then Griess method was used to determine cellular NO content;and contents of TNF-α,IL-6,IL-1β and IL-10 in culture medium were examined by enzyme linked immunosorbent assay(ELISA).Results showed that,total flavonoids of Allium Mongolicum regel decreased contents of NO,TNF-α,IL-6 and IL-1β in LPS stressed MPM(p<0.01)and enhanced IL-10 content significantly(p<0.01).Experiment 3 Effects of total flavonoids of Allium Mongolicum regel on genes encoding inflammatory factors in LPS stressed MPM.Cells were cultured with different treatments for 4-12 h.The m RNA expression of TNF-α,IL-6,IL-1β,IL-10,i NOS and COX-2 was determined using RT-PCR.Results showed that,compared to LPS stressed group,total flavonoids of Allium Mongolicum regel significantly reduced expression of i NOS,TNF-α,IL-6,IL-1β and COX-2,and increased expression of IL-10 in MPM.Experiment 4 Effect of total flavonoids of Allium Mongolicum regel on NF-κB pathway.Cells were cultured with different treatments for 4-12 h and gene expression of Myd88 and NF-κB was examined.Results showed that,compared to LPS stressed group,total flavonoids of Allium Mongolicum regel significantly reduced expression of Myd88 and NF-κB when treated for 4h(p<0.01);however,with the increase of treatment time,m RNA abundance of Myd88 and NF-κB decreased but showed no difference with LPS stressed group(p>0.05).Overall,proper concentrations of total flavonoids from Allium Mongolicum regel could inhibit contents of cytokines and relative gene expressions in LPS stressed MPM directly,and the possible mechanism was to suppress m RNA expression of pro-inflammatory cytokines by inhibition of NF-κB pathway.