| Inflammation,oxidative stress,and vascular injury can cause the biological behavior of vascular smooth muscle cells to undergo a series of changes,including phenotypic transformation,abnormal proliferation and migration,matrix synthesis,and inflammation.These changes can result in vascular intimal hyperplasia,which is essential for the occurrence and development of vascular remodeling-related diseases such as atherosclerosis,hypertension and restenosis after angioplasty.Micro RNA(mi RNA)is a type of endogenous small RNA with a length of approximately 20-24 nucleotides.Previous studies have shown that mi RNAs play an important role in the regulation of the phenotype of vascular smooth muscle cells and control the inflammatory responses of endothelial cells and macrophages.Studies have confirmed that plant-derived mi RNAs function similarly to endogenous mi RNAs after entering mammals,and play a biological role by regulating multiple target genes.Sal-mi R-58 is the highest abundance of specific mi RNA in Salvia miltiorrhiza Bge,which was found by our lab through sequencing and experimental verification.In order to clarify the role and mechanism of Salvia miltiorrhiza-derived Sal-mi R-58 in promoting blood circulation and removing blood stasis,this study intends to investigate the role and molecular mechanism of Sal-mi R-58 in suppressing intimal hyperplasia induced by carotid artery ligation.Objective: To explore the effects of Sal-mi R-58 derived from Salvia miltiorrhiza on vascular remodeling and underlying mechanism.Methods: 1.The model of intimal hyperplasia was established by carotid artery ligation in mice.C57BL/6 mice were randomly divided into three groups: unligated group,ligated + control agomir group,and ligated + Sal-mi R-58 agomir group.The expression of inflammatory factors in vascular tissues was detected by immunofluorescence staining,and the levels of inflammatory factors in serum were detected by ELISA.2.The bioinformatics was used to predict the target genes of Sal-mi R-58,quantitative real-time PCR(q RT-PCR),western blot,Dual-Luciferase reporter assay detected whether OTUD7 B is the target gene of Sal-mi R-58.3.Immunoblotting and ELISA were used to detect the effects of Sal-mi R-58 on the expression of TNF-?-induced inflammatory cytokines after VSMCs were transfected with Sal-mi R-58.4.The protein interaction network was used to predict the protiens interacting with OTUD7 B.Using q RT-PCR,western blotting,double immunofluorescence staining,and co-immunoprecipitation to detect the interaction of OTUD7 B with E2F1 in vascular smooth muscle cells.5.According to the website of transcription factor prediction,it was found that there exist E2F1 binding sites in the p62 promoter region,and the effects of OTUD7 B and E2F1 on p62 expression were detected by q RT-PCR and western blotting.Results: 1.Sal-mi R-58 agomir inhibits carotid artery ligation-induced intimal hyperplasia by reducing inflammation.According to the sequencing results,KLF3,OTUD7 B,SMAD5,and USP20 may be the target genes of Sal-mi R-58.In this study,we selected OTUD7 B for validation.The results of ELISA showed that the expression levels of IL-1?,IL-6 and TNF-? were significantly higher in ligation group than in the control group.The levels of IL-1?,IL-6 and TNF-? in Sal-mi R-58-treated group were significantly lower than those in the untreated group.The immunofluorescence staining showed that the vascular intima of the ligated group increased significantly and the fluorescence intensity(green)of the p62 increased significantly.2.OTUD7 B is a target gene of Sal-mi R-58 in VSMCs,Sal-mi R-58 inhibits the expression of OTUD7 B.The bioinformatics analysis showed that OTUD7 B might be a potential target gene of Sal-mi R58.We transfected vascular smooth muscle cells with Sal-mi R-58 and then detected the expression of OTUD7 B.Western blotting showed that the expression of OTUD7 B at protein level was markedly decreased in Sal-mi R-58-tranefected smooth muscle cells.The luciferase reporter assay showed that mi R-58 could bind to the 3' UTR of OTUD7 b and inhibit its expression.3.The expression levels of inflammatory factors and OTUD7 B were significantly up-regulated in TNF-?-treated VSMCs.Sal-mi R-58 greatly inhibited TNF-?-induced up-regulation of inflammatory factors and OTUD7 B expression.Western blot analysis showed that expression of OTUD7 B at the protein level was up-regulated in a concentration-dependent manner after VSMCs were treated with different concentrations of TNF-?.Next,VSMCs were transfected with Sal-mi R-58 and then stimulated with TNF-?.Western blotting and ELISA showed that the expression of OTUD7 B,p62,NF-?B p65,NF-?B p50 at protein level could be significantly inhibited after transfection with Sal-mi R-58.Meanwhile,Sal-mi R-58 could markdely inhibit TNF-?-induced inflammatory factor expression.4.OTUD7 B interacts with and deubiquitinates E2F1 in VSMCs.By using the protein interaction databases,we found that OTUD7 B can interact with E2F1.We knocked down or overexpressed OTUD7 B in VSMCs,respectively,Western blotting and q RT-PCR showed that E2F1 expression was decreased or increased at both the protein and m RNA levels.Also,knockdown or overexpression of OTUD7 B in VSMCs obviously decreased or increased the fluorescence intensity of E2F1.Moreover,OTUD7 B and E2F1 co-localized in the nucleus.At the same time,the Co IP results showed that E2F1 could be detected in the IP sediment of OTUD7B;OTUD7B could also be detected in the IP sediment of E2F1.These results revealed the presence of interactions between OTUD7 B and E2F1 in VSMCs.5.Sal-mi R-58 inhibits p62 expression and NF-?B signaling by reducing OTUD7 B and E2F1 levels.According to the transcription factor prediction databases,there exist E2F1 binding sites within p62 promoter region,which can activate p62 expression.q RT-PCR showed that p62 expression was significantly decreased or increased at the m RNA level after knocking down or overexpressing E2F1 in VSMCs.Western blotting showed a significant decrease or increase at p62 protein level after knocking down or overexpressing OTUD7 B.Conclusions: 1.Sal-mi R-58 inhibits intimal hyperplasia induced by carotid artery ligation by attenuating inflammation.2.In VSMCs,Sal-mi R-58 suppresses the expression of OTUD7 B protein by targeting the 3' UTR of OTUD7 b.3.TNF-? upregulates inflammatory factors and OTUD7 B expression in VSMCs.Sal-mi R-58 abrogates TNF-?-induced up-regulation of inflammatory factors and OTUD7 B expression.4.OTUD7 B interacts with and deubiquitinates E2F1 in VSMCs.5.Sal-mi R-58 inhibits p62 expression and NF-?B signaling by reducing OTUD7 B and E2F1 levels. |
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