| Objective:To study the effect of Corilagin on the proliferation and foaming of macrophages RAW264.7 cells with lipopolysaccharide(LPS)-induced damage.Determination the levels of mRNA and protein expression of CD40,CD40L,tumor necrosis factor receptor-associated factor-1(TRAF-1),interleukin-6(IL-6),inducible nitric oxide synthase(iNOS)and y-interferon(IFN-y).This study provides a theoretical basis for regulation of Corilagin on the CD40-CD40L signaling pathway.Methods:1.In vitro culture of mouse monocytes/macrophages RAW264.7,CCK-8 method for determination of different concentrations(0.4μg/mL,0.6μg/mL,0.8μg/mL,1.0μg/mL,1.2μg/mL)and different time(12h,24h,48h)of LPS has different effects on the viability of RAW264.7 cells,determine the optimal LPS induction concentration and time.2.Experimental groups:normal control group,LPS injury model group,positive control group(simvastatin 10μmol/L),Corilagin different dose groups(6.25 μmol/L,12.5μmol/L,25μmol/L,50μmol/L,100μmol/L),DRI-C21045(CD40-CD40L signaling pathway inhibitor)group and DRI-C21045+corilagin 100μmol/L group.3.The CCK-8 method was used to determine the effects of different doses of Corilagin(6.25μmol/L,12.5μmol/L,25μmol/L,50μmol/L,100μmol/L)on the proliferation of RAW264.7 cells with LPS-induced injury,and draw a dose-response curve to get IC50.4.The red O method and cholesterol kit were used to determine he effects of different doses of Corilagin(6.25μmol/L,12.5μmol/L,25μmol/L,50μmol/L,100μmol/L)on the foaming of RAW264.7 cells with LPS-induced injury.5.Real-time quantitative polymerase chain reaction(RT-qPCR)detection mRNA expression of CD40,CD40L,IL-6,iNOS and IFN-γ in RAW264.7.6.Western blot detection of CD40,CD40L,IL-6,iNOS and IFN-γ protein expression in RAW264.7.7.Immunohistochemical methods were used to detect the protein expression of CD40,CD40L and TRAF-1 in RAW264.7.Results:1.The effect of 1.0μg/mL of LPS for 24h has the most obvious proliferation effect on RAW264.7 cells,1.0μg/mL of LPS is the optimal induction concentration,and 24h is the optimal induction time.2.Corilagin has an inhibitory effect on the proliferation of RAW264.7 cells injured by LPS,and its IC50 is 41.25μmol/L.3.Corilagin can inhibit the foaming of RAW264.7 cells injured by LPS.4.RT-qPCR results showed that compared with the normal control group,the mRNA expressions of CD40,CD40L,TRAF-1,IL-6 and IFN-γ in model group were increased,while the mRNA expression of iNOS was decreased.Compared with the model group,the mRNA expression of CD40,CD40,TRAF-1,IL-6 and IFN-y were decreased in RAW264.7 cells of positive control group,Corilagin 25,50,100μmol/L groups,while the mRNA expression of iNOS increased.And the Corilagin 6.25μmol/L and 12.5μmol/L groups compare with model group showed no significant difference(P>0.05).5.Western blot results showed that compared with the normal control group,the protein expressions of CD40,CD40L,TRAF-1,IL-6 and IFN-γ in model group were increased,while the protein expression of iNOS was decreased.Compared with the model group,the protein expression of CD40,CD40,TRAF-1,IL-6 and IFN-γ were decreased in RAW264.7 cells of positive control group,Corilagin 25,50,100μmol/L groups,while the protein expression of iNOS increased.And the Corilagin 6.25μmol/L and 12.5μmol/L groups compare with model group showed no significant difference(P>0.05).6.The results of immunofluorescence showed that compared with the normal control group,the expression of CD40,CD40L and TRAF-1 in the model group was increased.Compared with model group,the expression of CD40,CD40L and TRAF-1 in RAW264.7 cells were decreased in positive control group and Corilagin 100μmol/L group.Conclusion:1.Corilagin can significantly inhibit the abnormal proliferation and foaming of RAW264.7 cells injured by LPS.2.Corilagin can down-regulates mRNA and protein expression of CD40,CD40L,TRAF-1,IL-6,IFN-γ,and up-regulates mRNA and protein expression of iNOS in RAW264.7 cells with LPS-induced injury.3.Corilagin inhibited the proliferation and foaming of RAW264.7 cells induced by LPS is closely related to the regulation of the CD40-CD40L signaling pathway. |
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