| Objective:In terms of epigenetic pathogenesis,investigated the pathogenesis of deep vein thrombosis(DVT)by microRNA(miRNA)mediated the regulation of apoptosis of vascular endothelial cells,providing new targets for the diagnosis and new approach for the treatments of clinical DVT.At the same time,the key targets and mechanisms of Xiaoshuantongmai decoction(XSTM)for treatment of DVT are expounded,and the theoretical and scientific basis of this therapy is provided for the clinical treatment of DVT.Methods:Peripheral blood mononuclear cells(PBMCs)were collected from outpatient or inpatient patients of Peripheral Vascular Disease Department,Affiliated Hospital of Shandong University of Traditional Chinese Medicine.Western blot was used to determine the protein levels of Caspase-3 and cleaved Caspase-3.The real-time quantitative polymerase chain reaction(qRT-PCR)was used to analyse the expression of miR-6740-5p and Caspase-3 mRNA expression.The miRNA microarray kit for whole-transcriptome high-throughput detection of RNAs extracted from PBMCs,combined with bioinformatic analysis for the screening of Caspase-3 upstream regulatory miRNA.The interaction between miR-6740-5p and Caspase-3 mRNA was tested by a dual-luciferase reporter gene assay.The expression of cleaved Caspase-3 was measured by Immunofluorescence(IF).The changes in mitochondrial membrane potential were detected by JC-1 staining.Apoptosis rate was measured by flow cytometry and TUNEL assay.DVT mice models were established,Hematoxylin-eosin(HE)staining and doppler ultrasound were used to assessment of thrombosis.The fluorescence in situ hybridization(FISH)was to observe the localization and expression of miR-6740-5p.Results:1.The qRT-PCR and Western blot results stated that Caspase-3 mRNA and protein levels were increased in PBMCs of 30 DVT patients,compared with healthy controls(P<0.05),which suggested that Caspase-3-mediated apoptosis participates in DVT formation.2.High-throughput chip detection combined with TargetScan 7.2 and DIANA bioinformatics analysis showed that the expression of miR-6740-5p was reduced in DVT patients,and there was a potential binding site between miR-6740-5p and Caspase-3.The dual-luciferase reporter gene experiment verified that miR-6740-5p directly binds to the 3’ UTR of Caspase-3 mRNA by base complementation.3.The qRT-PCR verified that miR-6740-5p expression was significantly reduced in PBMCs of DVT patients,compared to the healthy controls(P<0.05),Pearson correlation analysis stated a negative correlation between miR-6740-5p and Caspase-3 expression(P<0.05).ROC analysis showed that miR-6740-5p could sensitively identify DVT patients from normal healthy people.4.Up-regulation of miR-6740-5p in HUVECs was able to reduce the apoptosis rate by inhibiting the expression of Caspase-3(P<0.05),white down-regulation of miR-6740-5p showed the opposite effect(P<0.05).5.miR-6740-5p was decreased and Caspase-3 mRNA and protein levels increased in DVT mice,compared with normal and sham mice(P<0.05).HE staining showed that Caspase-3 inhibitor significantly reduced thrombosis in the DVT model mice(P<0.05).6.In vitro experiments showed that the XSTM-containing serum inhibited endothelial cell apoptosis by reducing Caspase-3 mRNA and protein levels through up-regulated miR-6740-5p(P<0.05).And miR-6740-5p inhibitor reversed the effect of XSTM-containing serum on inhibition endothelial cell apoptosis in vitro(P<0.05).7.HE staining and doppler color ultrasound showed that XSTM significantly reduced the inferior venae cava(IVC)thrombosis in DVT mice(P<0.05).In vivo,XSTM alleviated the formation of DVT by increasing the expression of miR-6740-5p in vascular tissues and inhibiting the expression of Caspase-3(P<0.05).However,down-regulation of miR-6740-5p significantly increased IVC thrombosis in DVT mice(P<0.05).These results suggest that XSTM alleviate DVT formation by inhibiting Caspase-3-mediated apoptosis through up-regulation of miR-6740-5p.Conclusions:1.The miR-6740-5p/Caspase-3 signaling axis promotes vascular endothelial cell apoptosis and participates in DVT formation,and targeted regulation of miR-6740-5p/Caspase-3 signaling axis to inhibit vascular endothelial cell apoptosis may be an effective treatment way to DVT;2.miR-6740-5p directly binds with Caspase-3 mRNA 3’ UTR by base complementation and subsequently suppresses its mRNA and protein expression;3.XSTM reduces vascular endothelial cell apoptosis through miR-6740-5p/Caspase-3 signaling axis,it is one of the mechanisms for the treatment of DVT. |
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