| Vascular remodeling is a structural and functional change of blood vessel.When hemodynamic changes or metabolic disorder cause damage to the vascular endothelium,growth factors and vasoactive substances are produced at the site of vascular injury and induce phenotype transformation,proliferation and migration of vascular smooth muscle cells(VSMC),accompanied by the synthesis,degradation of the extracellular matrix and rearrangement of the cytoskeleton.Physiological vascular remodeling leads to the development of vascular disease.Vascular endothelial cells,smooth muscle cells and mononuclear macrophages are involved in the process of vascular remodeling.The transformation of vascular smooth muscle phenotype and cell biological behavior changes such as proliferation and migration play an important role in vascular remodeling.From the theory of traditional Chinese medicine,blood stasis refers to whenever the circulation of blood is not smooth or the blood flow is stagnant and forms stasis.The lesion caused by the internal resistance of blood stasis is a blood stasis syndrome.Blood stasis is caused by various pathogenesis including that rapid blood flow and aggregate coagulation cause blood stasis;blood flow slowing and metabolite siltation cause blood stasis;vascular damage leads to blood stasis.Blood stasis can be divided into "tangible stasis" and "invisible stasis." "Tangible stasis" includes thrombosis,hematoma,agglomeration,abnormal growth of connective tissue,etc;"invisible sputum" includes blood rheology changes,inflammation caused by increased tissue fluids,etc.Furthermore,the scope of blood stasis is further divided into a broad sense of "stasis" and a narrow sense of "stasis".The narrow sense of blood stasis is represented by blood clot,such as poor blood stasis,stagnation,siltation in local region.Generalized blood stasis involves vascular lesions,as well as a comprehensive lesion of various pathological products.This is very similar to the clinical manifestations of the vascular remodeling disease.According to the theory and clinical experience of Chinese medicine,the main cause of intimal hyperplasia is poor blood flow caused by vessel stenosis after vascular endothelial injury.The main pathogenesis is circulation stagnation and blood stasis.Chinese medicine believes that "circulation is fundamental to blood".For the prevention and treatment of intimal hyperplasia,it is appropriate to promote blood circulation and remove blood stasis.In recent years,although great progress has been made in the study of the pathogenesis of vascular remodeling,there is still no ideal drug for reversing or reducing vascular remodeling.Therefore,it is one of the current research hotspots to find the effective components of anti-vascular remodeling from traditional Chinese medicine.Salvia miltiorrhiza belongs to the mint family of plants,is commonly used as a traditional Chinese herbal medicines,with accelerating blood circulation to remove blood stasis,stimulating the menstrual flow to relieve dysmenorrhea,nourishing blood to tranquillize the mind,cooling blood to alleviate pain.It is widely used in the treatment of cardiovascular diseases,such as dilating coronary artery,lowering blood pressure,preventing and treating atherosclerosis,anti-cardiac hypertrophy,etc.Studies have shown that the important active ingredients of Salvia miltiorrhiza are divided into two broad categories,namely lipophilic tanshinones and hydrophilic phenolic compounds.Recent studies have found that small RNAs(mi RNAs)in rice and some Chinese herbal medicines can be absorbed by human and animals.These plant-derived mi RNAs,because of their special structure,can pass through digestive tract after oral administration,and enter the blood circulation,and thus play a role in various target organs.These exogenous plant mi RNAs have been shown to not only regulate lipid metabolism in humans,but also have anti-viral and even tumor-inhibiting effects.However,whether the mi RNAs derived from Salvia miltiorrhiza can regulate VSMC migration and adhesionremains to be studied.Biological behaviors of VSMCs such as migration and adhesion are regulated by gene expression programs and cytoskeletal organization.The zinc finger transcription factor KLF4 regulates the expression of VSMC marker genes(including migration and adhesion-related genes)and cell cycle regulators,and plays an essential role in VSMC proliferation,migration and adhesion.Cell migration is dependent on the cytoskeleton(especially actin filament)polymerization,depolymerization and rearrangement.Non-muscle myosin heavy chain ?A(NMHC IIA)regulates the cytoskeletal organization by interacting with actin filaments,which in turn affects cell migration and adhesion.However,the relationship between KLF4 and NMHC ?A in regulating VSMC migration and adhesion is unclear.Does Salvia miltiorrhiza-derived mi RNAs and Salvia miltiorrhiza protect cardiovascular structure and function by regulating KLF4 and NMHC ?A expression and/or activity? How does Salvia miltiorrhiza and its derived mi RNAs regulate KLF4 and NMHC ?A expression and/or activity? And how dose NMHC ?A regulate the VSMC cytoskeleton structure? These issues need to be studied.This study utilized VSMCs cultured in vitro and intimal hyperplasia model induced by carotid injury in mice to explore the mechanism whereby Salvia miltiorrhiza-derived Sal-mi R-1 and Sal-mi R-3 modulate the biological behavior of VSMC through regulating KLF4 and NMHC ?A expression and/or activity,providing new strategy for the development of effective drugs against vascular remodeling.Part ? Identification of Salvia miltiorrhiza-derived Sal-mi R-1 and Sal-mi R-3 and investigation on their stability in mouse body and effects on VSMCsObjective: To investigate the stability of Salvia miltiorrhiza-derived Sal-mi R-1 and Sal-mi R-3 in VSMCs and their effect on VSMCs.Methods:1.q RT-PCR was used to detect Salvia miltiorrhiza-derived mi RNAs inblood and tissues of mice.2.VSMCs were treated with different concentrations of thrombin(0 ? 2U/m L)for 24 h or with thrombin(1 U/m L)for different times(0 ? 48 h),the expression of VCAM-1,ICAM-1 and TXA2 R was detected by q RT-PCR and Western blot.3.After treatment of VSMCs with thrombin(1 U/m L),the cytoskeleton and myofilament were observed by phalloidin staining.Immunofluorescence staining was used to detect the localization and expression of VCAM-1,ICAM-1 and TXA2 R.4.VSMCs were transfected with Sal-mi R-1,Sal-mi R-3,or Sal-mi R-1and Sal-mi R-3,respectively,VCAM-1,ICAM-1 and TXA2 R expression was detected by q RT-PCR and Western blot.5.VSMCs were transfected with mi R-Ctl and Sal-mi R-1+3 for 24 h,and then were treated with thrombin(1 U/m L),VCAM-1,ICAM-1 and TXA2 R expression was detected by q RT-PCR and Western blot,immunofluorescence staining was performed for detection of VCAM-1,ICAM-1 and TXA2 R expression.Scratch test and Boyden chamber transwell migration assay detected VSMC migration,immunofluorescence staining detected of adhesion of VSMCs to lymphocytes and macrophages.6.Mouse carotid artery was injured by ligation and transfected with mi R-Ctl or Sal-mi R-1+3 in situ,morphological changes of intimal hyperplasia were observed by hematoxylin-eosin staining,immunofluorescence staining,q RT-PCR and Western blot were used to detect the expression of VCAM-1,ICAM-1 and TXA2 R.Results:1.Identification of Salvia miltiorrhiza-derived Sal-mi R-1 and Sal-mi R-3and their stability in miceUsing high-throughput sequencing to determined mi RNAs present in Salvia miltiorrhiza,a total of 187 mi RNAs were screened.Among them,there are mi RNAs homologous to other plants,such as ath-mi R166a-3p,hbr-mi R156,gma-mi R172b-5p,etc.,and there are 41 specific mi RNAspresent in Salvia miltiorrhiza.Through KEGG and GO analysis,we selected several mi RNAs of interest for q RT-PCR detection.The results showed that the selected mi RNAs existed in Salvia miltiorrhiza and Salvia miltiorrhiza injections.The mice were intragastrically administered with Salvia miltiorrhiza,and the RNA in the blood and various tissues and organs was extracted and treated with sodium periodate,accompanied by q RT-PCR.Due to higher level of the unique Sal-mi R-1 and Sal-mi R-3 in VSMCs,we decided to use these two mi RNAs as research objects.VSMCs were transfected with Sal-mi R-1 and Sal-mi R-3,respectively,and RNA was extracted and treated with sodium periodate.The results of q RT-PCR showed that Salvia miltiorrhiza-derived Sal-mi R-1 and Sal-mi R-3 were not oxidatively degraded by sodium periodate and could be stably present in mouse VSMCs.2.Sal-mi R-1 and Sal-mi R-3 affect VSMC cytoskeletal organization and migration by inhibiting thrombin-induced VCAM-1,ICAM-1 and TXA2 R expressionFirst,we confirmed by q RT-PCR and Western blot analysis that different concentrations of thrombin or 1 U/m L thrombin treatment of VSMCs for different times,could up-regulate VCAM-1,ICAM-1 and TXA2 R expression in a concentration and time-dependent manner.VCAM-1,ICAM-1 and TXA2 R m RNA levels were at least 2-4 folds higher than those of the control group.Immunofluorescence staining showed that thrombin significantly induced VCAM-1,ICAM-1 and TXA2 R protein expression.Phalloidin staining showed that compared with the control group,the VSMC myofilament arrangement after thrombin stimulation was disordered,and the stress fibers were reduced and thinned.VSMCs were transfected with chemically synthesized Sal-mi R-1 and Sal-mi R-3,and the results of q RT-PCR and Western blot showed that Sal-mi R-1,Sal-mi R-3 and Sal-mi R-1 plus Sal-mi R-3 co-transfection could significantly inhibit the expression of adhesion factors VCAM-1,ICAM-1 and TXA2 R,Sal-mi R-1 and Sal-mi R-3 co-transfection had the most obvious effect on VSMCs.After that,we further studied the effect of Sal-mi R-1 andSal-mi R-3 co-transfection on thrombin effect.VSMCs were transfected with Sal-mi R-1+3 for 24 h followed by thrombin stimulation(1 U/m L),the results of q RT-PCR and Western blot showed that Sal-mi R-1+3 significantly inhibited the expression of VCAM-1,ICAM-1 and TXA2 R induced by thrombin.Immunofluorescence staining gave consistent results.Cell scratch assays and Boyden chamber transwell migration assay demonstrated that Sal-mi R-1+3 inhibited thrombin-induced migration of VSMCs.Fluorescence staining showed that thrombin promoted the adhesion of VSMCs to lymphocytes and macrophages,transfection of Sal-mi R-1+3 significantly inhibited the adhesion of VSMC induced by thrombin.Taken together,Sal-mi R-1+3 inhibits thrombin-induced VSMC migration and adhesion of VSMCs to monocytes.3.Salvia miltiorrhiza injection and Sal-mi R-1+3 inhibit intimal hyperplasia and the expression of adhesion factor induced by carotid artery injuryLeft common carotid artery of C57BL/6 mice was ligated and transfected with Sal-mi R-1+3 or treated with Salvia miltiorrhiza injection,q RT-PCR analysis showed that in Sal-mi R-1+3 transfected mice,the expression of two mi RNAs increased 3-fold in vascular tissues,whereas the mice transfected with mi R-Ctl did not detect Sal-mi RNAs.HE staining showed that the intima of the C57BL/6 mice was significantly thickened after 21 days of carotid artery ligation.After in situ transfection of Sal-mi R-1+3 or treatment with Salvia miltiorrhiza injection,neointimal formation and intima-media ratio(I/M)were significantly decreased.The therapeutic effect of Sal-mi R-1+3 was better than that of Salvia miltiorrhiza injection.The expression of VCAM-1 in the injured artery was significantly up-regulated compared with the unligated group.Sal-mi R-1+3 significantly inhibited the up-regulation of VCAM-1expression caused by ligation.The same results were obtained by immunofluorescence and histochemical staining.These results fully demonstrate that Sal-mi R-1 and Sal-mi R-3 significantly inhibit vascular injury-induced intimal hyperplasia and adhesion molecule expression.Summary:1.Sal-mi R-1 and Sal-mi R-3 are specifically present in Salvia miltiorrhiza and Salvia miltiorrhiza injections and can be stably present in mouse body.2.In VSMCs,thrombin significantly up-regulates the expression of adhesion molecule VCAM-1 and ICAM-1.3.Transfection of Sal-mi R-1+3 significantly inhibits thrombin-induced expression of adhesion molecules and migration of VSMCs.4.Transfection of Sal-mi R-1+3 inhibits the intimal hyperplasia induced by carotid artery ligation.5.Sal-mi R-1+3 inhibits the migration of VSMCs and its adhesion to monocytes/macrophages by promoting blood circulation and removing blood stasis.Part ? OTUD7 B mediates the regulation of KLF4 and NMHC ?A expression by Sal-mi R-1 and Sal-mi R-3Objective: To investigated mechanism whereby Sal-mi R-1 and Sal-mi R-3 regulate the expression of KLF4 and NMHC ?A.Methods:1.Using bioinformatics to predict target genes for Sal-mi R-1 and Sal-mi R-3.q RT-PCR,Western blot,and dual luciferase reporter gene assays were used to test whether OTUD7 B is a target gene of Sal-mi R-1 and Sal-mi R-3.2.q RT-PCR,Western blot,immunofluorescence techniques were used to detect the effect of thrombin on the expression of OTUD7 B.3.Label-free quantitative genomic screening of OTUD7B-interacting protein,co-immunoprecipitation and Western blot were used to detect the interaction between OTUD7 B and NMHC ?A or KLF4.4.Ch IP-q PCR and dual luciferase reporter gene assay detected KLF4 binding to NMHC ?A promoter.5.The interaction between KLF4 and NMHC ?A was detected by co-immunoprecipitation and Western blottingResults:1.In VSMCs,Sal-mi R-1 and Sal-mi R-3 can targetedly inhibit thrombin-induced OTUD7 B expressionVSMCs were stimulated with 1 U/m L thrombin for different times(0,6,12,24 h)or stimulated with different concentrations of thrombin(0,0.5,1,2U/m L)for 24 h.Total RNA and protein were extracted for q RT-PCR and Western blot analysis.The results showed that thrombin upregulated the expression of OTUD7 B in a time-and concentration-dependent manner.Immunofluorescence staining results also showed that the expression of OTUD7 B was significantly up-regulated at 24 h after stimulation with 1 U/m L thrombin.OTUD7B protein expression was reduced in cells transfected with Sal-mi R-1,Sal-mi R-3,and Sal-mi R-1+3 compared to mi R-Ctl.The most significant inhibition of OTUD7 B expression was abserved by Sal-mi R-1+3.The same result was obtained by q RT-PCR analysis.Immunofluorescence staining also showed that the fluorescence intensity of OTUD7 B was significantly attenuated after transfection of Sal-mi R-1+3.These results suggest that OTUD7 B may be a target gene for Sal-mi R-1 and Sal-mi R-3.The luciferase reporter gene assay showed that the OTUD7 B 3'UTR directed reporter gene activity was significantly inhibited after transfection of Sal-mi R-1 and Sal-mi R-3 compared with the transfected mi R-Ctl group,this inhibitory effect was abolished when Sal-mi R-1 and Sal-mi R-3 binding site in OTUD7 B 3'UTR was mutated.In situ transfection of Sal-mi R-1+3 in the injured carotid artery could down-regulate the expression of OTUD7 B in vascular tissues.2.OTUD7 B increases the stability of KLF4 protein by deubiquitinationIn order to clarify OTUD7 B function in VSMCs,we identified the proteins interacted with OTUD7 B by Label-free quantitative proteomics.The results revealed that 53 proteins could interact with OTUD7 B.After classification and enrichment clustering of these proteins,VSMC function-related NMHC ?A was selected for further study.Western blotresults showed that overexpression or knockdown of OTUD7 B in VSMCs resulted in increased or decreased KLF4 protein levels.However,q RT-PCR results showed that overexpression or knockdown of OTUD7 B had no significant effect on KLF4 m RNA levels,suggesting that OTUD7 B may increase KLF4 protein level by increasing its stability.To further determine whether OTUD7 B increases the stability of the KLF4 protein,we analyzed the half-life of KLF4.The results showed that the protein synthesis of KLF4 was inhibited after the cells were treated with cycloheximide,and the half-life was about 6 h.When MG132 was used to block the KLF4 proteasome degradation pathway,the half-life of KLF4 was significantly prolonged,suggesting that KLF4 is degraded by the proteasome.After knocking down OTUD7 B in VSMCs,MG132 treatment could eliminate the destabilization of KLF4 by knocking down OTUD7 B.The stability of KLF4 was also detected under the conditions of OTUD7 B overexpression combined with CHX treatment.The results showed that the half-life of KLF4 was significantly prolonged in cells overexpressing OTUD7 B.This suggested that OTUD7 B increases the stability of KLF4 protein by deubiquitination.Further experiments indicated that overexpression of OTUD7 B in VSMCs significantly reduced the level of KLF4 ubiquitination.In contrast,knockdown of OTUD7 B increased the level of ubiquitination of KLF4.3.NMHC ?A is negatively regulated by KLF4 and OTUD7BBioinformatics analysis revealed that there is the putative KLF4 binding site in the NMHC ?A promoter region.We further validated this prediction using Ch IP-q PCR and dual luciferase reporter assays.The results showed that KLF4 bound to-619 bp ?-388 bp upstream of the NMHC ?A transcription initiation site and inhibited NMHC ?A promoter activity.q RT-PCR and Western blot showed that KLF4 negatively regulated the expression of NMHC?A gene.These results indicate that KLF4 inhibits NMHC ?A expression by directly binding to tits promoter.In addition,OTUD7 B interacted with NMHC?A.Overexpression of OTUD7 B significantly inhibited the expression of NMHC ?A compared with the control group.In contrast,knockdown of OTUD7 B in VSMCs significantly up-regulated NMHC ?A protein levels compared to the control group.We further confirmed that knockdown of OTUD7 B obviously reduced ubiquitination of NMHC?A,whereas overexpression of OTUD7 B markedly increased ubiquitination of NMHC?A.Summary:1.In VSMCs,Sal-mi R-1 and Sal-mi R-3 suppress the up-regulation of OTUD7 B expression by thrombin through targeting OTUD7 B 3' UTR.2.OTUD7 B increases the stability of KLF4 protein by deubiquitination.3.KLF4 inhibits the expression of NMHC ?A by directly binding to the NMHC ?A promoter.Part ? NMHC ?A promotes the organization of VSMC cytoskeleton and inhibits VSMC migration and recruitment of monocytesObjective: To reveal the mechanism underlying the regulation of VSMC migration and adhesion activity by NMHC ?A.Methods:1.Phalloidin staining was used to observe the effect of NMHC ?A knockdown on cytoskeleton and myofilament.2.q RT-PCR,Western blot and immunofluorescence staining were used to detect the effect of NMHC ?A knockdown of on the expression of cytoskeleton protein and VCAM-1.3.Cell scratch test and Boyden chamber transwell migration assay detected VSMC migration.4.Using VSMC-RAW264.7 cell co-culture system to detect the effect of NMHC ?A knockdown on the interaction between VSMC and macrophages.5.Using anti-VCAM-1-antibody to neutralized VCAM-1 and determining the effect of NMHC ?A knockdown on macrophage activation and migration.Results:1.NMHC ?A participates in the organization of VSMC cytoskeletonWe knocked down NMHC ?A in VSMCs with si RNA and observed changes in cytoskeleton.Phalloidin staining showed that the myofilament arrangement was disordered and the stress fibers were reduced after knocking down NMHC ?A compared with the control group.After treatment of VSMCs with thrombin for 24 h,the myofilament was reduced and the cytoskeleton organization was disordered.At the same time,NMHC ?A knockdown further reduced stress fiber formation.These results suggest that NMHC ?A is involved in the cytoskeletal remodeling.To further elucidate the mechanism by which NMHC ?A regulates cytoskeletal organization,we examined the effect of NMHC ?A knockdown on VSMC marker gene expression.Western blot and q RT-PCR showed that knockdown of NMHC ?A significantly reduced the expression of SM?-actin and SM22?.This suggests that NMHC ?A may affect cytoskeletal remodeling by participating in the regulation of VSMC marker gene expression.2.Knockdown of NMHC ?A causes VCAM-1 upregulationWe further investigated whether NMHC ?A is involved in the induction of VCAM-1 expression by thrombin.Western blot analysis showed that knockdown of NMHC ?A resulted in a significant up-regulation of VCAM-1expression.When NMHC ?A expression was silenced by transfecting si-NMHC ?A,thrombin-induced up-regulation of VCAM-1 expression was further increased.Immunofluorescence staining get the same result.The above results indicate that NMHC ?A suppresses the induction of VCAM-1expression by thrombin.3.Knockdown of NMHC ?A facilitates migration and adhesion of VSMCsCell scratch assays and Boyden chamber transwell migration assay indicated that knockdown of NMHC ?A in VSMCs increased the induction effect of thrombin on VSMC migration compared to the control group.To further investigate whether NMHC ?A affects adhesion between VSMCs and macrophages,we used the VSMC-RAW264.7 co-culture system to examinethe effect of knockdown of NMHC ?A in VSMCs on macrophage transmembrane migration and adhesion.The results showed that thrombin significantly promoted transmembrane migration of RAW264.7 cells and their adhesion to VSMCs.Knockdown of NMHC ?A further increased their migration and adhesion to VSMCs compared to control group.To further clarify the role of VCAM-1 in the adhesion of macrophages to VSMCs,VCAM-1 on the surface of VSMCs was neutralized with anti-VCAM-1antibody,VSMC interaction with RAW264.7 cells was observed.Cell adhesion analysis showed neutralizing VCAM-1,significantly reduced adhesion between VSMCs and macrophages.These results suggest that NMHC ?A-regulated expression of VCAM-1 participates in the recruitment and activation of macrophages by VSMCs.Summary:1.NMHC ?A is involved in the organization of VSMC cytoskeleton.2.Knockdown of NMHC ?A leads to the up-regulation of VCAM-1expression.3.NMHC ?A-regulated expression of VCAM-1 participated in the regulation of adhesion between VSMCs and macrophages.Conclusion:1.Sal-mi R-1 and Sal-mi R-3 derived from Salvia miltiorrhiza significantly inhibit VSMC migration and adhesion induced by thrombin and attenuated the intimal hyperplasia induced by carotid artery injurey.2.OTUD7 B mediates the regulation of KLF4 and NMHC ?A expression by Sal-mi R-1 and Sal-mi R-3.3.NMHC ?A inhibits the migration and adhesion of VSMCs by regulating VSMC cytoskeletal organization. |
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