| Objective(s):To investigate the effect of scutellarin on the proliferation and foam cell formation of RAW264.7 cells induced by oxidized low density lipoprotein(ox-LDL).To detect the effect of scutellarin on the m RNA and protein expression of CD40,CD40 L,TRAF6,TAK1,IKKα/β,IKB-α and NF-κB p65,and to explore the regulation of scutellarin on CD40-CD40L/TRAF6 signaling pathway in macrophages.Methods:1.Mouse mononuclear macrophage RAW264.7 was cultured in vitro to replicate ox-LDL-induced injury model.2.Experimental groups : normal control group,ox-LDL injury model group,different concentrations(6.25,12.5,25,50,100 μmol/L)of scutellarin group,Simvastatin group(10 μmol/L),DRI-C21045 group(CD40-CD40 L pathway inhibitor,17.1μmol/L),DRI-C21045+scutellarin group(17.1μmol/L DRI-C21045+100μmol/L scutellarin).3.The effect of scutellarin on the proliferation of RAW264.7 cells induced by ox-LDL was detected by CCK-8 method.4.The effect of scutellarin on the foaming of ox-LDL-induced RAW264.7 cells was detected by cell cholesterol ester content assay kit and oil red O staining.5.The m RNA expression of CD40,CD40 L,TRAF6,TAK1,IKKα,IKB-α and NF-κB p65 in RAW264.7 cells was detected by RT-q PCR.6.The protein expression of CD40,CD40 L,TRAF6 and TAK1 in RAW264.7 cells was detected by immunofluorescence.7.The protein expressions of CD40,CD40 L,TRAF6,TAK1,IKKα/β,IKB-α,NF-κB p65 and p-NF-κB p65 in RAW264.7 cells were detected by Western blot.Results:1.The results of cell proliferation assay showed that compared with the normal control group,the proliferation of RAW264.7 cells in the model group induced by 75 mg/L ox-LDL was significantly increased(P<0.01),and the ox-LDL-induced injury model was successfully replicated;Compared with the model group,after scutellarin intervention,there was no significant difference in cell proliferation between the 6.25 μmol/L and 12.5 μmol/L scutellarin groups and the model group(P>0.05),while the proliferation of cells in the 25,50 and100 μmol/L scutellarin groups decreased,including 25 μmol/L scutellarin group(P <0.05),50 μmol/L scutellarin group(P<0.01),100 μmol/L scutellarin group(P <0.01).The cell proliferation of Simvastatin group,DRI-C21045 group and DRI-C21045+scutellarin group was also significantly reduced,and the difference was statistically significant(P<0.01);Compared with the DRI-C21045 group,the cell proliferation of the DRI-C21045+scutellarin group decreased slightly,but the difference was not statistically significant(P > 0.05).2.The results of intracellular cholesterol ester content showed that compared with the normal control group,the intracellular cholesterol ester content in the model group was significantly increased(P<0.01),and the ox-LDL-induced injury model was successfully replicated;Compared with the model group,after the intervention of scutellarin,there was no significant difference in the content of intracellular cholesterol ester between the scutellarin 6.25,12.5 and 25 μmol/L groups and the model group(P>0.05).The content of intracellular cholesterol ester in the scutellarin 50 μmol/L group and the scutellarin 100 μmol/L group was significantly decreased(P<0.01).The intracellular cholesterol ester content of Simvastatin group,DRI-C21045 group and DRI-C21045+scutellarin group was significantly decreased(P<0.01);Compared with DRI-C21045 group,the content of intracellular cholesterol ester in DRI-C21045+scutellarin group decreased slightly,but the difference was not statistically significant(P> 0.05).3.The results of oil red O staining showed that compared with the normal control group,the red staining area of RAW264.7 cells in the model group was significantly increased,the staining degree was deepened(P<0.01),and the ox-LDL-induced injury model was successfully replicated;Compared with the model group,after scutellarin intervention,there was no significant change in the red staining area and the degree of red staining in the scutellarin 6.25 and 12.5μmol/L groups(P>0.05),while the red staining area and the degree of staining were reduced in the scutellarin 25,50,100 μmol/L groups(P<0.01).And the red staining area and the degree of staining were significantly reduced in the Simvastatin group,DRI-C21045 group and DRI-C21045+scutellarin group(P<0.01);Compared with the DRI-C21045 group,there was no significant change in the degree of red staining in the DRI-C21045+scutellarin group(P>0.05).4.RT-q PCR results showed that compared with the normal control group,the expression of CD40,CD40 L,TRAF6,TAK1,IKKα and NF-κB p65 m RNA in the model group was significantly increased(P<0.01),while the expression of IKB-α m RNA was decreased(P<0.01);Compared with the model group,the expression of CD40,CD40 L,TRAF6,TAK1,IKKα and NF-κB p65 m RNA in the 25,50,100 μmol/L scutellarin group,Simvastatin group and DRI-C21045 group decreased(P<0.01,P<0.05),while the expression of IKB-α m RNA increased(P<0.01,P<0.05).5.Immunofluorescence results showed that compared with the normal control group,the expression of CD40,CD40 L,TRAF6 and TAK1 protein in the model group was significantly increased(P<0.01);Compared with the model group,the expression of CD40,CD40 L,TRAF6 and TAK1 protein in the 25,50,100μmol/L scutellarin group,Simvastatin group and DRI-C21045 group decreased to varying degrees(P<0.01,P<0.05).6.Western blot results showed that compared with the normal control group,the expression of CD40,CD40 L,TRAF6,TAK1,IKKα/β and p-NF-κB p65 protein in the model group increased(P<0.01),and the expression of IKB-α protein decreased(P<0.01),while the expression of NF-κB p65 protein did not change significantly(P> 0.05).Compared with the model group,the expression of CD40,CD40 L,TRAF6,TAK1,IKKα/β and p-NF-κB p65 protein in the 25,50,100 μmol/L scutellarin group,Simvastatin group and DRI-C21045 group decreased to varying degrees(P<0.01,P<0.05),IKB-α protein expression increased(P<0.01,P<0.05),while NF-κB p65 protein expression did not change significantly(P> 0.05).Conclusion(s):1.Scutellarin can significantly inhibit the abnormal proliferation and foaming of macrophage RAW264.7 induced by ox-LDL.2.Scutellarin significantly down-regulated the m RNA expression levels of CD40,CD40 L,TRAF6,TAK1,IKKα and NF-κB p65 in ox-LDL-induced RAW264.7cells,and up-regulated the m RNA expression level of IKB-α.3.Scutellarin significantly down-regulated the expression levels of CD40,CD40 L,TRAF6,TAK1,IKKα/β and p-NF-κB p65 proteins in ox-LDL-induced RAW264.7 cells,and up-regulated the expression level of IKB-α protein.4.The inhibitory effect of scutellarin on the abnormal proliferation and foaming degree of RAW264.7 cells induced by ox-LDL is closely related to the regulation of CD40-CD40L/TRAF6 pathway. |
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