Experimental Study On The Changes Of RNF10 Expression And The Biological Characteristics Of Smooth Muscle Cells In The Process Of Atherosclerosis

Objective:To establish a stable animal model of atherosclerosis and explore the dynamic changes of RNF10(Ring Finger Protein 10)expression level and the changes in the biological characteristics of smooth muscle cells at different stages of atherosclerosis development,in order to provide a theoretical basis for the study of the molecular mechanism of RNF10 participating in the atherosclerosis regulation.Methods:Selected 30 ApoE knockout mice,half male and half female,and damaged the carotid artery of the mice by air drying.Selected 24 mice randomly from 30 mice and randomly divided into A,B,C,D four groups(6 in each group),the A group was as normal control group(mice did not do carotid artery injury treatment and use the common feed),and group B mice were fed with continuous high fat forage for 8weeks after the carotid artery injury,and group C mice were fed with continuous high fat forage for 12 weeks after the carotid artery injury,and group B mice were fed with continuous high fat forage for 16 weeks after the carotid artery injury.Mice in each group were sacrificed at the corresponding time points,and then primary smooth muscle cells were isolated and cultured.The carotid artery samples were stained with HE and oil red and observed.The expression of RNF10,SMA,SMHC and VEGF were detected by immunohistochemistry.The primary cells were used to performed cellular identified,cell proliferation ability changes in each group were identified by CCK8 experiment,flow cytometry were used to detect cell apoptosis and cell cycle,the scratch test was used to detect each cell group migration ability change,western blot method was used to detect RNF10,SMA and SMHC protein expression level in each group of cells,cell immunofluorescence test was used to cell phenotypic changes in each group of smooth muscle.Results:HE staining showed a small amount of thickening of the intima in group B,withfoam cell aggregation and lipid deposition.Collagen fibers were observed in group C,with hyaline changes,large amounts of lipid deposition and formation of lipid fiber caps in the intima.In group D,there were significant thickening of the intima,cholesterol crystals,calcium salt deposition,and a large number of foam cells.Oil red staining showed that group A had no obvious staining,while group B,C and D had obvious staining,and the proportion of staining area in the total area gradually increased.This suggested that the animal model of atherosclerosis was successfully constructed,and the groups B,C and D presented different degrees of atherosclerotic lesions.Immunohistochemical staining results showed that in group A,RNF10,SMA and SMHC staining were strongly positive,while VEGF staining was negative.In group B,RNF10,SMA and SMHC staining were positive and VEGF staining was positive.In group C,SMA and SMHC staining were weakly positive,while VEGF staining was strongly positive.In group D,SMA and SMHC staining and VEGF staining were positive.CCK8 experimental results showed that: compared with the control group,the proliferation ability of cells in groups B,C and D was significantly improved,and the cell activity was gradually increased in groups B,C and D,with statistically significant differences(P < 0.05).Flow cytometry results showed that compared with the control group,the apoptosis rate of cells in group B and group C was significantly reduced,and the number of G0/G1 phase cells were significantly reduced.Cells in S phase increased significantly,and the difference was statistically significant(P < 0.05).The scratch test showed that at 12 h,cells in each group began to migrate significantly.The scratch in group A was about 0.90 cm,the scratch in group B was about 0.65 cm,the scratch in group C was about 0.55 cm,and the scratch in group D was about 0.5cm.At 24 h,the scratches in group A were about 0.60 cm,group B about 0.40 cm,group C about 0.20 cm,and group D almost closed.Western Blot results showed that compared with group A,the expression of RNF10,SMA and SMHC in the other three groups was significantly reduced,with A statistically significant difference(P < 0.05).The expression of RNF10,SMA and SMHC decreased gradually in group B and C,and increased slightly in group D,but the expression level was still lower than that of group A.Compared with group A,the expression of VEGF in other groups was significantly increased,with statisticallysignificant differences(P < 0.05).Moreover,its expression gradually increased in groups B and C,decreased in group D,but was still higher than group A.Cell immunofluorescence assay showed that: compared with group A,the expression of RNF10,SMA and SMHC was significantly reduced and the cell phenotype was changed.The expression levels of RNF10,SMA and SMHC in tissues and smooth muscle cells all gradually decreased and then increased,but they were still significantly lower than normal levels.Conclusion:the treatment of ApoE gene knockout mice with dry air injury combined with high-fat diet can quickly obtain a stable atherosclerosis animal model.Compared with normal tissues,the expressions of RNF10,SMA and SMHC in atherosclerotic tissues were significantly decreased,and the expression of VEGF was increased.With the increasing degree of atherosclerosis,the cell phenotype of smooth muscle cells was changed,and the cell proliferation and migration ability were gradually improved.The expression levels of RNF10,SMA and SMHC in cells all gradually decreased and then increased,but were still significantly lower than the normal level.RNF10 may be a key target involved in the regulation of the development of atherosclerosis,and its decreased expression is associated with the formation of atherosclerosis.