Effect And Mechanism Of Mongolian Medicine Danggong-3 Extract On LPS-induced Inflammatory Response In RAW264.7 Cells

Objective:To Observe the effect and mechanism of Mongolian medicine Danggong-3extract(DG-3)on lipopolysaccharide(LPS)induced RAW264.7 cell inflammatory response.Methods: 1.Cell Culture of Mouse Mononuclear Macrophage Line RAW264.7.2.Establishment of LPS induced inflammation model of RAW264.7 cells,and detection of tumor necrosis factor by ELISA kit α(TNF-α)、 Interleukin-1β(IL-1β)inflammatory factor content to determine the success of modeling.3.The activity of RAW264.7 cells was detected by CCK8 method.RAW264.7 cells were pretreated with DG-3 extract at different concentrations(50,100,200,400,800,1600,3200 μg/ml)for 24 hours to determine the safe dose range of DG-3.4.RAW264.7 cells were randomly divided into blank control group,LPS group(1μg / ml LPS stimulated cells in vitro)and DG-3 treatment group(LPS stimulation + two concentration groups with better effect).Blank control group: cells without any treatment;LPS group: cells were stimulated with 1μg/ml LPS;LPS+DG-3 group: 100,200μg/ml DG-3 were added respectively,and the supernatant was taken after 20 hours culture,and the levels of inflammatory factors TNF-α,IL-6 were detected by enzyme-linked immunosorbent assay(ELISA).5.The m RNA levels of JAK2,STAT3,TNF-α,IL-6 in RAW264.7cells were polymerase chain reaction by RT-PCR.6.The content of JAK2 and STAT3 protein in RAW264.7 cells was determined by western-blot.Results : 1.The results of CCK8 showed that the safe range of DG-3 was100-200μg/ml.The concentrations of DG-3 were 100μg/ml and 200μg/ml,respectively.2.Elisa results showed that DG-3 could significantly inhibit the expression of TNF-α and IL-6 in RAW264.7 cells induced by LPS.Compared with the blank control group,the expression of TNF-α,IL-6 in LPS Group was significantly increased(P<0.05),and the expression of TNF-α,IL-6 in DG-3100μg/ml and 200μg/ml groups were significantly decreased compared with the LPS Group(P<0.01),the inhibitory effect was most obvious when the concentration of DG-3 was 200 μg/ml.3.RT-PCR results showed that DG-3 could significantly decrease the expression of JAK2,STAT3,TNF-α,IL-6 m RNA.Compared with the blank control group,the expression of JAK2,STAT3,TNF-α and IL-6 m RNA in LPS Group increased(P<0.05),the m RNA expressions of JAK2,STAT3,TNF-α and IL-6 in DG-3 100 and 200μg/ml groups were significantly decreased(P<0.01).4.Western Blot results showed that DG-3 could significantly decrease the expression of JAK2 and STAT3 protein.Compared with the blank control group,the expression of JAK2 and STAT3 protein in LPS Group increased(P<0.05),while the expression of JAK2 and STAT3 protein in DG-3 100μg/ml and 200μg/ml groups decreased significantly(P<0.01).Conclusion:1.DG-3 has anti-inflammatory effect and has no toxicity to RAW264.7cells.2.DG-3 could inhibit the expression of inflammatory factors TNF-α and IL-6 in RAW264.7 cells induced by LPS.3.Anti-inflammatory mechanism of DG-3 is related to the inhibition of the expression of JAK2,STAT3,TNF-α,IL-6,m RNA and the expression of JAK2,STAT3 protein.