| Objective:Explore the potential targets of vascular restenosis through bioinformatics,and construct endothelial cell injury model and vascular restenosis rat model,use hematoxylin extract to intervene,and explore the mechanism of hematoxylin extract inhibiting vascular inflammation and vascular restenosis.Methods:Part 1: Bioinformatics mining related targets of vascular restenosis: The data of the vascular restenosis miRNA chip GSE60959 was obtained from the GEO public data platform of NCBI in the United States,and the differential miRNAs were screened using online analysis tool matrix and probe annotation files.The miRNA with significant differences in the chip was selected as the research goal.Part 2: The effect of hematoxylin extract on the proliferation of endothelial cells.HUVECs were divided into blank group,miR-1183 overexpression group,miR-1183 overexpression plus hematoxylin-containing serum group.Gene transfection of endothelial cells was used for model replication.The blank group endothelial cells were incubated in complete medium for 48h;the miR-1183 overexpression group cells were transfected for 36 h + endothelial cell complete medium was incubated for 12h;The group was transfected with 36 h + 1?g / ml hematoxylin-containing serum for 12h;RT-PCR method was used to detect the expression of miR-1183 m RNA,MTT method was used to detect the cell proliferation level;flow cytometry was used to detect the cell apoptosis level;TNF-?,IL-1?,IL-6,IL-18 and cytokines MCP-1,MMP-2,CXCL16 levels;the target gene is analyzed by the online tool Targctscan,verified by the dual luciferase experiment,the predicted Functional analysis of genes;RT-PCR method to detect PTEN m RNA expression;Western-blot method to detect PTEN,Akt,p-Akt,Bax,Bcl-2,NF-?B p65 protein expression.Part 3: The effect of hematoxylin extract on vascular restenosis and its molecular mechanism.In this experiment,40 SD rats were randomly divided into a blank group,a model group,a hematoxylin extract group and an atorvastatin group.Except for the blank group,the remaining three groups constructed a rat femoral artery restenosis model by wire contact method.From the 28 th day of model replication,each group was given corresponding drug intervention.The blank group and the model group were given an equal volume of sodium carboxymethylcellulose solution,and the hematoxylin extract group and the atorvastatin group were given separately.Both hematoxylin extract suspension and atorvastatin suspension were treated by gavage,once a day for 28 consecutive days.After the intragastric administration,observe the general status and body weight change of each group of mice;RT-PCR method to detect the expression level of miR-1183 m RNA in femoral artery tissue;HE staining to observe the pathological morphology,lumen diameter and intima thickness of femoral artery in mice;Immunohistochemical method was used to detect the expression levels of PTEN,Akt,p-Akt protein in femoral artery tissue;ELISA method was used to detect the inflammatory factors TNF-?,IL-1?,IL-6,IL-18 and cytokines MCP-1,MMP-2,CXCL16 level;Western blot detection of PTEN,Akt,p-Akt,Bax,Bcl-2,NF-?B p65 protein expression in femoral artery tissue.Results:First part:According to bioinformatics prediction,a total of 198 differentially expressed miRNAs were screened in the chip database,of which 89 miRNAs were significantly up-regulated;109 miRNAs were significantly down-regulated.In this study,miR-1183,which had significant differences,was studied.The second part:1.miR-1183 overexpression plus hematoxylin-containing serum group can significantly inhibit the expression of miR-1183 m RNA in HUVECs.Compared with the miR-1183 overexpression group,the difference was statistically significant(P<0.01).2.The miR-1183 overexpression plus hematoxylin-containing serum group can significantly increase the cell viability of HUVECs.Compared with the miR-1183 overexpression group,the difference was statistically significant(P<0.01).3.The miR-1183 overexpression plus hematoxylin-containing serum group can significantly inhibit the total apoptosis level of HUVECs.Compared with the miR-1183 overexpression group,the difference was statistically significant(P<0.01).4.The target gene was analyzed by the online tool Targctscan,and it was found that miR-1183 had a binding site with PTEN.The predicted gene was used for functional analysis.The dual luciferase reporter gene experiment confirmed that PTEN gene is a potential target gene for miR-1183.5.miR-1183 overexpression plus hematoxylin-containing serum group can significantly inhibit the expression level of Bax protein in HUVECs and up-regulate the expression of Bcl-2 protein.Compared with the miR-1183 overexpression group,the difference is statistically significant(P<0.01).6.miR-1183 overexpression plus hematoxylin-containing serum group can significantly reduce the inflammatory factors TNF-?,IL-1?,IL-6,IL-18 and cytokines MCP-1,MMP-2,HUVECs cell supernatant Compared with the miR-1183 overexpression group,the content of CXCL16 was statistically significant(P<0.01).7.miR-1183 overexpression plus hematoxylin-containing serum group can significantly inhibit the expression level of NF-?B p65 protein in HUVECs.Compared with the miR-1183 overexpression group,the difference was statistically significant(P<0.01).8.miR-1183 overexpression plus hematoxylin-containing serum group can significantly inhibit the expression of p-Akt protein in HUVECs and increase the expression of PTEN protein.Compared with the miR-1183 overexpression group,the difference is statistically significant(P<0.01);but no significant effect on the expression level of Akt protein,compared with the miR-1183 overexpression group,the difference was not statistically significant(P>0.05).9.The miR-1183 overexpression plus hematoxylin-containing serum group can significantly increase the expression of PTEN m RNA in HUVECs.Compared with the miR-1183 overexpression group,the difference was statistically significant(P<0.01).The third part:1.Hematoxylin extract group can improve the pathological changes of femoral artery in vascular restenosis model.2.The hematoxylin extract group was able to down-regulate the expression of miR-1183 m RNA in animal models.Compared with the model group,the difference was statistically significant(P<0.01).3.The hematoxylin extract group can significantly inhibit the expression level of Bax protein in rat femoral artery and up-regulate the expression of Bcl-2 protein.Compared with the model group,the difference is statistically significant(P<0.01).4.Hematoxylin extract group can significantly reduce the content of inflammatory factors TNF-?,IL-1?,IL-6,IL-18 and cytokines MCP-1,MMP-2,CXCL16 in rat femoral artery serum The difference between the groups was statistically significant(P<0.01).5.The hematoxylin extract group can significantly inhibit the expression level of NF-?B p65 protein in rat femoral artery.Compared with the model group,the difference was statistically significant(P<0.01).6.The hematoxylin extract group can significantly inhibit the expression of rat femoral artery p-Akt protein and increase the expression of PTENprotein.Compared with the model group,the difference is statistically significant(P<0.01);The expression level had no obvious effect,compared with the model group,the difference was not statistically significant(P>0.05)Conclusion:1.miR-1183 is expressed at high levels in patients with vascular restenosis.2.miR-1183 can promote the apoptosis of vascular endothelial cells and promote the secretion of inflammatory factors and cytokines.3.Hematoxylin extract can down-regulate the expression of miR-1183 in endothelial cells,and then promote the apoptosis of endothelial cells through the PTEN/Akt signaling pathway to achieve the effect of inhibiting vascular restenosis,which may be its mechanism of prevention and treatment of vascular restenosis One. |
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