| Objective:To study the effect of scutellarin on oxidative damage of mouse monocyte /macrophage RAW264.7 induced by ox-LDL,and to determine the effect of scutellarin on the expression of p38 MAPK and oxidative stress related factors NRF2,HO-1 and KEAP-1 in RAW264.7 cells induced by ox-LDL.To explore the potential molecular mechanism of scutellarin regulating p38 MAPK/NRF2/HO-1 signaling pathway.Methods:1.Mouse monocyte/macrophage RAW264.7 cells were cultured in vitro to replicate ox-LDL-induced injury model;the effects of different ox-LDL on the proliferation of RAW264.7 cells at different time points were detected by CCK-8 method.2.The effect of different concentrations of scutellarin on the proliferation of RAW264.7 cells induced by ox-LDL was detected by CCK-8 method.3.The effects of scutellarin on the contents of ROS,SOD,CAT and GSH in ox-LDL-induced RAW264.7 cells were detected.4.The effects of scutellarin on the expression of NRF2,HO-1,Keap1 and p38 MAPK mRNA in ox-LDL-induced RAW264.7 cells were detected by RT-qPCR.5.The effect of scutellarin on the expression of NRF2,HO-1,KEAP-1 and P-p38 MAPK protein in RAW264.7 cells induced by ox-LDL was detected by immunofluorescence.6.The effects of scutellarin on the expression of NRF2,HO-1,KEAP-1,p38 MAPK and P-p38 MAPK in ox-LDL-induced RAW264.7 cells were detected by Western blot.Results:1.Compared with the normal control group,the proliferation of RAW264.7 cells was significantly promoted after treatment with different concentrations of ox-LDL for 24 h and 48 h,and 75 mg/L ox-LDL had a significant effect on cell proliferation after 24 h.Therefore,75 mg/L ox-LDL was used to induce the proliferation of RAW2647 cells for 24 h.2.The results of CCK-8 assay showed that compared with the normal control group,the cell proliferation in the model group was significantly higher(P<0.01).Compared with the model group,the cell proliferation was decreased after 24 h of scutellarin treatment(P<0.05 or P<0.01).The cell proliferation of vitamin C group and 10μmol/L simvastatin group was significantly decreased(P<0.01).20 μmol/L p38 MAPK inhibitor SB203580 group and 20 μmol/L SB203580+100 μmol/L scutellarin group also inhibited cell proliferation(P<0.05 or P<0.01).Compared with p38 MAPK inhibitor SB203580 group,the proliferation of SB203580+scutellarin group was relatively weakened,but there was no statistical difference(P>0.05).3.The effects of scutellarin on the contents of ROS,SOD,CAT and GSH in ox-LDL-induced RAW264.7 cells were detected by kit.The results showed that compared with the normal control group,the ROS content in the model group was significantly increased after ox-LDL-induced injury(P<0.01),and the contents of SOD,CAT and GSH were significantly decreased(P<0.01).Compared with the model group,the content of ROS in each concentration of scutellarin group,vitamin C group,p38 MAPK inhibitor SB203580 group and SB203580+scutellarin group decreased to varying degrees,and the levels of SOD,CAT and GSH increased(P<0.05 or P<0.01).In addition,the content of ROS in simvastatin group was also significantly decreased(P<0.01).Compared with p38 MAPK inhibitor SB203580 group,the content of ROS in SB203580+scutellarin group was decreased,and the contents of SOD,CAT and GSH were increased,but there was no significant difference(P>0.05).4.The results of RT-qPCR showed that compared with the normal control group,the expression of NRF2 mRNA in the model group decreased(P<0.05),and the expression of KEAP-1 and p38 MAPK mRNA increased(P<0.01).Compared with the model group,the expression of NRF2 and HO-1 mRNA in 12.5 μmol/L,25μmol/L,50 μmol/L,100 μmol/L scutellarin group,10 μmol/L simvastatin group and p38 MAPK inhibitor SB203580 group increased,and the expression of KEAP-1 and p38 MAPK mRNA decreased(P<0.05 or P<0.01).5.Immunofluorescence results showed that compared with the normal control group,the fluorescence expression of NRF2 in the model group was decreased(P<0.05),and the fluorescence expression of KEAP-1 and P-p38 MAPK was increased(P<0.01).Compared with the model group,the fluorescence expression of NRF2 and HO-1 in12.5 μmol/L,25 μmol/L,50 μmol/L,100 μmol/L scutellarin group,10 μmol/L simvastatin group and p38 MAPK inhibitor SB203580 group was enhanced,and the fluorescence expression of KEAP-1 and P-p38 MAPK was decreased(P<0.05 or P<0.01).6.Western blot results showed that compared with the normal control group,the expression of NRF2 protein in the model group decreased(P<0.05),and the fluorescence expression of KEAP-1 and P-p38 MAPK increased(P<0.01).Compared with the model group,the protein expressions of NRF2 and HO-1 in 12.5,25,50,100μmol/L scutellarin group,10 μmol/L simvastatin group and p38 MAPK inhibitor SB203580 group were significantly increased,and the protein expressions of KEAP-1and P-p38 MAPK were decreased(P<0.05 or P<0.01).There was no significant change in p38 MAPK protein level in each group(P>0.05).Conclusion(s):1.Scutellarin can significantly inhibit the abnormal proliferation of mouse monocyte/macrophage RAW264.7 induced by ox-LDL,reduce ROS content and increase the content of SOD,CAT and GSH in cells,and reduce oxidative damage.2.The protective effect of scutellarin on ox-LDL-induced oxidative stress in mouse monocyte/macrophage RAW264.7 cells is related to the regulation of p38MAPK/NRF2/HO-1 signaling pathway. |
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