| Objective: To investigate the effect of naringin(NAR)on the polarization state of macrophages stimulated by high glucose and its related mechanism.Methods: Naringin was dissolved in dimethyl sulfoxide(DMSO)to prepare a parent solution.The mice macrophage Raw264.7 logarithmic phase are used in preliminary experiment.The CCK 8 method is adopted to detect naringin effects on cell proliferation activity,set up the inoculum glucose concentration gradient of 1 g/L,2 g/L,4 g/L,5 g/L,6 g/L,the preparation of different concentration of glucose DMEM medium,and by tags to sugar concentration(a,b,c,d,e).The cells were cultured in an incubator for 24 h.The activity of inducible nitric oxide synthase(i NOS)in cells was detected by the NOS typing test box,and the sugar concentrations in the medium of low and high glucose were screened according to the active-concentration curve.The experimental groups were as follows: control group: normal control(NG),osmotic pressure control(NG+M),high glucose control(HG).Drug treatment group: high glucose + naringin(HG+NAR),high glucose + fasudil(HG+F),high glucose +C3 transferase(HG+C3),and drug intervention group: fasudil and C3 transferase were set as RhoA/ROCK pathway blockers as positive control.The macrophage RAW264.7 of mice in each group was cultured for 24 h after intervention according to the group conditions,and the supernatant was collected for ELISA detection.The detection indexes were as follows:interleukin 10(IL-10),interleukin 6(IL-6),tumor necrosis factor-α(TNF-α).After cell lysis,the proteins were extracted and analyzed by Western blot.β-actin was selected as the internal reference running out electrophoresis bands,which were respectively the following protein molecules: RhoA/ROCK pathway associated proteins(Rho A,ROCK1,ROCK2,MYPT1,p-MYPT1),i NOS,and arginin-1(Arg-1).In order to investigate the phenotype of RAW264.7,antibodies to CD80 and i NOS were used to label M1,and antibodies to Arg-1 and CD206 were used to label M2.RAW264.7 was sorted by flow cytometry to detect the number and proportion of M1 and M2 of macrophages in each group(M1/M2).Results: Compared with the NG group,the experimental results of the NG+M group were the same without statistical significance(P>0.05),indicating that the osmotic pressure of the solution had no effect on the experimental results,and the results of the other groups were different from the normal control group,with statistical significance,which could be used for subsequent statistical analysis.Compared with NG group,high glucose stimulated RAW264.7 to activate RhoA/ROCK pathway,and the expression of related protein molecules(Rho A,ROCK1,ROCK2,MYPT1,p-MYPT1),pro-inflammatory mediator i NOS protein were increased,and the expression of anti-inflammatory mediator Arg-1 was decreased(P<0.05).The secretion of inflammatory cytokines interleukin-6 and TNF-α was increased,and the secretion of anti-inflammatory cytokines interleukin-10 was decreased(P<0.05).The results of Flow cytometry showed that the number of M1-type macrophages was increased,and the M1/M2 ratio was increased(P<0.05).Compared with HG group,the expression of RhoA/ROCK pathway protein molecules,i NOS protein and the secretion of IL-6 and TNF-α were inhibited,while the expression of Arg-1 protein and the secretion of IL-10 were increased(P<0.05).In contrast to the high glucose control group,the number of M2 type was increased and M1/M2 was decreased(P<0.05).Pairwise comparisons of HG +NAR,HG+F and HG +C3 showed no statistical difference(P>0.05),which further confirmed the conclusion of this experiment.Conclusion: Naringin may promote the differentiation of macrophages stimulated by high glucose to M2 type by down-regulating RhoA/ROCK signaling pathway. |
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