| Objective:The purpose of this study is to explore the influence of the suppressor of cytokine signal1 ing 1( SOCS1) to the proliferation of vascular smooth muscle cells(VSMCs) which induced by Ang Ⅱ and its possible mechanism.Methods:1) Taking the the human umbilical arterial smooth muscle cells as the research object, establish a model in which AngⅡ stimulates VSMC proliferation. Incubating the cells for 48 h with final AngⅡconcentration in the medium at 0mol/L,10-8mol/L,10-7mol/L, 10-6mol/L,10-5mol/L. Cell viability was detected by MTT to pick out the best concentration of AngⅡ.Cells were incubated with AngⅡat the best concentration for 0,12,24,48,72 hours, then measure the cell viability.2) Detect the level of intracellular reactive oxygen species(ROS) and the expression of SOCS1 in the VSMCs after treated with the best concentration of AngⅡfor 0,12, 24,48,72 hours.3) Design and synthesize 3 pairs of SOCS1-si RNA,then pick out the optimum one by q PCR and Western-blot.4) Transfect SOCS1- si RNA into VSMC using by cationic liposome,then treated them with AngⅡ.Experiment groups:Experiment 1: The blank control group, SOCS1-si RNA group,Ang Ⅱgroup,AngⅡ+ SOCS1-si RNA group,Ly294002 group,Ly294002+Ang Ⅱgroup;Experiment 2:The blank control group,SOCS1-si RNA group, Natural vitamin E group,Ang Ⅱgroup,Ang Ⅱ+ SOCS1-si RNA group, Natural vitamin E +AngⅡgroup.5) Cell viability and apoptosis rate were detected by MTT and Flow Cytometry,respectively; The level of intracellular reactive oxygen species(ROS) was determ-inated by DCFH-DA probe; the protein level of SOCS1,Bax,Bcl-2,total Akt and p-Akt were measured by Western blot technique.6)All the experiments above were repeated 3 times. The measured data and transformed data were expressed as mean±sd and analyzed with SPSS 17.0 software.P < 0.05 was considered statistically significant difference.Results:1)All the 4 concentration of AngⅡpromote the proliferation of VSMC, the best one is 10-7mol/L,the best time is 48 h.2) AngⅡcan promote the formation of ROS and the expression of SOCS1 in VSMC.3) The pair of SOCS1-si RNA3 silenced the expression of SOCS1 most effectively.4) Compared with the blank control group, cell proliferation rate increased significantly in AngⅡ group(P<0.01), however decreased in SOCS1-si RNA group and Ly294002 group(P<0.01) and the rate of apoptosis in the two group increased significantly(P<0.05), the effects of SOCS1- si RNA and Ly294002 on the survival of VSMC are similar(P>0.05); cell proliferation rate of SOCS1-si RNA+Ang Ⅱgroup and Ly294002+Ang Ⅱ group was significantly lower than that in Ang Ⅱgroup(P<0.01), the apoptosis rate had no obvious difference compared with the blank control group( P>0.05); the survival rate between the two compared with no significant difference(P>0.05).5) Compared with the blank control group, the expression of SOCS1 protein in SOCS1-si RNA group was significantly decreased and the Bax/Bcl2 ratio increased(p<0.01) and Bax/Bcl2 ratio decreased in AngⅡ group(P<0.05); compared with the Ang Ⅱgroup, The expression of SOCS1 protein in SOCS1-si RNA + Ang Ⅱ group was decreased and the Bax/Bcl2 ratio increased(P<0.01);compared with the blank control group, the difference of the expression of SOCS1 protein in Ly294002 group was not statistically significant(P>0.05), while the Bax/Bcl2 ratio increased(P<0.01);compared with Ly294002 group, the Bax/Bcl2 ratio of Ly294002+AngⅡ group decreased significantly(P<0.01).6) For each experimental group,the expression of total Akt protein showed no statistically significant difference(P>0.05); compared with the blank control group,the expression of p-Akt protein of Ang Ⅱ group was increased(P<0.05),that of SOCS1-si RNA group and Ly294002 group decreased significantly(P<0.01), and the expression of p-Akt protein in the latter two groups have no significant difference(P>0.05); compared with the Ang Ⅱ group, the expression of p-Akt protein of SOCS1-si RNA+Ang Ⅱ group and Ly294002+ Ang Ⅱ group was reduced(P<0.05),and there was no statistically significant difference between the two groups of p-Akt protein(P>0.05).7) Compared with the blank control group, in SOCS1-si RNA group, the mean fluorescence intensity(MFI) decreased(p<0.05),the MFI of AngⅡ group increased significantly(P<0.01),the MFI of natural vitamin E group had no statistically significant difference( P>0.05); compared with the Ang Ⅱ group, the MFI of SOCS1-si RNA+ AngⅡgroup, vitamin E+AngⅡgroup decreased significantly(P<0.01). The change of survival viability of VSMC is consistent with the change of ROS.Conclusion:1) The expression of SOCS1 in the VSMC was inhibited effectively by RNA interference.2) Down regulation of SOCS1 inhibited VSMC’s proliferation which induced by AngⅡvalidly, SOCS1 mediated the proliferation of VSMC induced by AngⅡdue to the change of Bax/Bcl2 ratio.3) This effect of SOCS1 might be mediated by Akt signaling pathway and ROS. |
