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IntroductionCardiovascular diseases(CVD)are currently one of the leading causes of death and morbidity worldwide.The main cause of CVD is atherosclerosis(AS)due to endothelial injury or dysfunction,and leads to a chronic disease in which arteries stiffen by the accumulation of plaques in the vessel wall.Numerous epidemiological studies have shown that HHcy is an independent risk factor for CVD,with each 5?mol / L increase in plasma Hcy associated with a ? 1.6-fold increase in CVD risk.Recent studies suggest that,as an independent risk factor for CVD,HHcy induced impairment of vascular function may be the result of a complex set of mechanisms,including the initiation of prolonged chronic inflammation,oxidative stress,and mitochondrial damage.However,the underlying mechanism is unclear.In recent years,miRNAs,as regulators of different cell types and functions,during the initiation and progression of AS,have received much attention.The results of clinical trials with miRNAs mimics or inhibitors provide new therapeutic opportunities and means involving in CVD treatment,and a large number of basic studies also confirm that miRNAs can be new targets for early detection and therapeutic intervention of CVD.miRNAs,as potential ‘mini-secreted' factors,are readily available in body fluids and are tissue-specific,especially they are stably present in the circulation.miRNAs and miRNAs based therapeutics are among the most innovative advances in recent years and hold great promise for future clinical applications in CVDs.Although there is a clear relationship between Hcy and CVD,the underlying mechanisms are not fully understood.Our group's previous results also showed that Hcy inhibited the migration and angiogenesis of vascular endothelial cells.On a prophase basis,we showed that HHcy induced a decrease in miR-18 a expression during endothelial injury,which was accompanied by aberrant PTEN expression by transcriptomics.Taken together,we speculate that miR-18 a may have a therapeutic role in the process of HHcy induced vascular endothelial injury;the mechanism may be related to the regulation of PTEN,a downstream target gene,by miR-18 a,which in turn regulates the expression levels of related molecules,such as inflammatory response,oxidative stress and mitochondrial damage,and then alleviates the inhibitory effect of Hcy caused on endothelial cell migration and vascular regeneration,and prevents the alteration of vascular function.To confirm our speculation,the following studies were performed: first,by establishing an in vitro and in vivo model of hyperhomocysteinemia,its damage to the vascular endothelium was clarified,while the expression of miR-18 a was detected.Second,in in vivo and in vitro experiments,the expression levels of miR-18 a were overexpressed and silenced,to clarify its therapeutic role in HHcy induced vascular endothelial injury.Finally,the most valuable differentially expressed gene PTEN was found by transcriptomics,followed by validation of whether miR-18 a inhibited HHcy induced vascular injury by targeting PTEN.Section ? HHcy induces vascular endothelial injury and decreased expression of miR-18aObjective:HHcy stimulation induces inflammatory response,oxidative stress and mitochondrial damage in vascular endothelium,which in turn leads to damage of vascular endothelium with down-regulation of miR-18 a expression.Methods:1)C57BL/6J mice were fed a specially tailored high methionine,low vitamin,low folate diet for 16 weeks,and physiological parameters such as weight,blood pressure,and the concentration of plasma Hcy were measured every four weeks to clarify whether HHcy was successfully constructed in animal model;2)In vivo experiments to define the inflammatory response during HHcy induced vascular endothelial injury,we mainly examined the changes in the vascular endothelium of mouse aortas over a period of 16 weeks by HE staining and immunohistochemistry,and the expression of ICAM-1 and VCAM-1,as well as the changes in the m RNA expression levels of pro-inflammatory cytokines IL-1?,IL-6,and TNF? and anti-inflammatory cytokine IL-10 by RT-PCR,together with the detection the expression level of of miR-18a;3)In vivo experiment to determine the level of oxidative stress in the process of HHcy induced vascular endothelial injury,the levels of ROS in each group were mainly detected by DHE staining,the Plasma SOD and MDA were detected by Reagent test kits,and the expression changes of oxidation related protein NOX4 and antioxidant proteins SOD1 and SOD2 were detected by Immunofluorescence;4)In vivo experiments to determine the mitochondrial damage during vascular endothelial injury induced by HHcy,the expression of mitochondrial fusion protein Mfn2 and fission protein Drp1 in each group was mainly detected by immunofluorescence,meanwhile,the mitochondrial alterations in vascular endoth-elial of each group were observed by transmission electron microscopy,and the level of vascular apoptosis was detected by TUNEL staining;5)In vitro experiments confirmed that Hcy induced inflammatory responses during endothelial cell injury,mainly by detecting the m RNA levels of ICAM1 and VCAM1,IL-1?,IL-6,TNF? and IL-10 by RT-PCR,while detecting miR-18 a expression levels;6)In vitro experiments confirmed the level of oxidative stress during Hcy induced endothelial cell injury,mainly by collecting the supernatant of endothelial cells in each group,and detecting the expression of SOD and MDA with related kits,while detecting the expression levels of oxidized protein NOX4 and antioxidant proteins SOD1 and SOD2 by WB;7)In vitro experiments confirmed that Hcy induced mitochondrial damage during endothelial cell injury,the expression of mitochondrial fusion protein Mfn2 and fission protein Drp1,as well as the expression of anti-apoptotic protein Bcl2 and pro-apoptotic protein Bax were mainly detected by WB,and the level of apoptosis in each group was detected by flow cytometry.Results:1)Changes in body weight,blood pressure,and blood Hcy in each group were monitored every 4 weeks.We found that the systolic blood pressure had a slight increased in the HHcy group(P < 0.05),and no significant change in diastolic blood pressure;there is a significant increase in plasma Hcy level,without any significant difference in body weight,suggesting that the HHcy mouse model was successfully constructed.2)HE staining found that with longer feeding time,the aortic morphology of mice in the HHcys group was more irregular than that in the control group,with more disordered cell arrangement,worse size uniformity of nuclei,uneven cytoplasmic staining,and more obvious fiber fragmentation.Immunohistochemical staining to detect ICAM-1 and VCAM-1 expression in the aortic endothelial cells of mice at all stages revealed that the expression of ICAM-1 and VCAM-1 in the aortas of the HHcy group began to change at 8 weeks and was significantly higher than that in the control group after 16 weeks.We also found that the expression of IL-1 ?,IL-6,and TNF-? in the vascular endothelium was significantly increased and the expression of IL-10 was decreased in HHcy mice compared to the control(P<0.05).miR-18 a expression in the vascular endothelium of HHcy mice was significantly lower than that in the control group.3)DHE staining revealed that the level of ROS in vascular endothelium was significantly increased in HHcy group compared to control,meanwhile serum SOD and MDA were also significantly increased compared to control(P < 0.05).Immunofluorescence found that the expression of oxidative protein NOX4 was elevated,as well as the expression of anti-oxidant proteins SOD1 and SOD2 were significantly decreased in the HHcy group compared with the control(P < 0.05).4)The results of immunofluorescence found that HHcy induced an increase in the expression level of the fission protein Drp1 and a concomitant decrease in the expression level of the fusion protein Mfn2(P < 0.05).Transmission electron microscopy revealed severe edema of the endothelial cells and a low number of mitochondria(m),moderate swelling,and some weakening of the intramembranous matrix of the mitochondrial cristae in the HHcy group.Moreover,the percentage of Tunel fluorescence was significantly higher in the HHcy group compared with the control group(P < 0.05);5)In vitro Hcy treatment resulted in lower viability than control,but Hcy treatment also resulted in higher cytotoxicity.As an endothelial activator ICAM1 and VCAM-1 in the Hcy group were significantly higher than the control.At the same time,the expression of IL-1?,IL-6,and TNF-? was significantly increased in the Hcy group,and the expression of IL-10 was decreased,and Hcy stimulation was found to inhibit miR-18 a expression in endothelial cells(P < 0.05);6)Examining the cell supernatant of each group,we found that compared with the control,the expression of MDA increased,and the expression of SOD decreased;detecting the corresponding proteins of oxidative stress by WB,we found that HHcy could promote the expression of the oxidative protein NOX4 increased,and the expression of the antioxidant proteins SOD1 and SOD2 decreased significantly compared with the control(P < 0.05);7)Hcy promotes the elevation of Drp1 in endothelial cells compared with control,whereas Mfn2 is inhibited,and the expression of Bcl2 is decreased and Bax is increased in endothelial cells compared with control,corresponding to the increased level of apoptosis detected by flow cytometry(P < 0.05).Conclusions:1)Successfully constructed the HHcy mouse animal model;2)HHcy can induce the activation of vascular endothelium and stimulate the expression of inflammatory factors,which in turn damage vascular endothelium;3)HHcy can induce an increase in the level of oxidative stress of vascular endothelium,which in turn damages vascular endothelium;4)HHcy can induce the imbalance of mitochondrial fusion / fission and promote an increase in the level of apoptosis,which in turn damages the vascular endothelium.5)miR-18 a expression is downregulated during HHcy induced vascular endothelial injury.Section ? miR-18 a inhibits HHcy-induced vascular injuryObjective:1)To clarify that miR-18 a can inhibit vascular endothelial inflammation,oxidative stress and mitochondrial damage induced by HHcy in vivo,and further reduce vascular endothelial injury.2)In vitro Hcy experiment confirmed that miR-18 a can inhibit Hcy-induced vascular endothelial inflammation,oxidative stress and mitochondrial damage,thereby reducing endothelial cell injury.Methods:1)Design the endothelium-specific promoter TIE2,and carry the adenoassociated virus-sig serotype with specific affinity for vascular endothelial,so as to achieve specific regulation of miR-18 a in vascular endothelial,Observe the expression of GPF fluorescence intensity and RT-PCR to detect the expression of the corresponding transcription level of miR-18 a.2)The four experimental groups and interventions are: Control group,HHcy group,HHcy + Overexpression miR-18 a group(HOE group),HHcy + Knock down miR-18 a group(HKD group).Immunohistochemistry and immunofluorescence were used to detect the relative expression levels of ICAM-1 and VCAM-1;ELISA detected the serum collected from each treatment group to detect IL-1?,IL-6,TNF?,and IL-10,as well as by immunohistochemistry.3)DHE staining to detect the ROS levels of vascular endothelial in each group,and detect the plasma SOD and MDA levels,immunofluorescence to detect the expression changes of oxidation-related protein NOX4 and antioxidant proteins SOD1 and SOD2;4)Immunofluorescence was used to detect the expression of Mfn2 and Drp1 in each group,and Tunel staining was used to detect the level of vascular apoptosis in each group;5)In vitro verification that miR-18 a inhibits Hcy-induced endothelial cell injury,experimental groups and interventions are: Control group,Hcy group,Hcy + miR-18 a Mimic(HM group),Hcy + Mimic control group(HMC group),Hcy + Inhibitor miR-18a(HI group),Hcy + Inhibitor Control group(HIC group),mainly through RT-PCR to detect ICAM1,VCAM1,IL-1?,IL-6,TNF? and IL-10;6)Collect the endothelial cell supernatant of each group,detect the expression of SOD and MDA with related kits,and detect the expression levels of oxidized protein NOX4 and anti-oxidant proteins SOD1 and SOD2 by WB;7)WB detects the expression of Mfn2 and Drp1 in each treatment group,as well as the expression of Bcl2 and Bax,and detects the level of apoptosis in each group by flow cytometry.Results:1)The results showed that the GFP fluorescence intensity was significantly higher than the control group,and the corresponding transcription level of miR-18 a was also regulated,suggesting that the specific regulation of miR-18 a was achieved in vivo.2)Compared with the HHcy group,the expression of ICAM-1 and VCAM-1 in the vascular endothelium of the HOE group was significantly reduced;while the HKD group had a higher expression level of ICAM-1 and VCAM-1 than the HOE group(P<0.05).At the same time,compared with the HHcy group,the expression of the IL1?,IL-6,and TNF? in the HOE group significantly reduced and improved the expression of IL-10;the HKD group was compared with the HOE group's IL-1?,IL-6and TNF? were significantly increased and inhibited the expression of IL-10(P<0.05).3)The level of ROS in the HOE group was lower than that of the HHcy group,while the level of the HKD group was higher than that of the HOE group.The corresponding SOD and MDA levels also changed accordingly(P<0.05).Compared with the HHcy group,the SOD level of the HOE group increased,while the MDA level decreased slightly.Compared with the HOE group,the HKD group had a lower SOD level and a higher MDA level.At the same time,compared with the HHcy group,the HOE group NOX4 expression decreased,and the SOD1 and SOD2 expressions increased;while the HKD group had higher NOX4 levels than the HOE group,and the level of SOD1 and SOD2 decreased(P<0.05);4)Compared with HHcy group,the expression of Drp1 was lower in HOE group,and the expression of Mfn2 was higher.Compared with the HOE group,the HKD group had the opposite result(P<0.05);at the same time,compared with the HHcy group,the HOE apoptosis level was significantly reduced,while the HKD group was increased compared with the HOE group.High(P<0.05);5)Compared with the HMC group,the expression of ICAM-1,VCAM-1,IL-1?,IL-6,and TNF? decreased in the HM group compared with the HMC group,and the expression of IL-10 increased(P<0.05).The results of the HI group and the HIC group showed an opposite trend(P<0.05);6)The cell supernatant of each group was tested and it was found that the expression of SOD in the HM group was higher than that in the HMC group,and the expression of MDA was lower(P<0.05).At the same time,compared with the HMC group,the overexpression of miR-18 a in the HM group can inhibit the increase of endothelial cell endothelial oxidized protein NOX4 and promote the expression of antioxidant proteins SOD1 and SOD2,the opposite result when HI group is compared with the HIC group.(P<0.05);7)Compared with the HMC group,the overexpression of miR-18 a in the HM group can reduce the increase of Drp1 and promote the expression of Mfn2(P<0.05);at the same time,it was found that the expression of the Bcl2 was reduced and Bax was increased,and the level of apoptosis detected by cell flow cytometry decreased.Compared with the HIC group,the HI group had the opposite trend(P<0.05).Conclusions:1)Successfully regulated the specific expression of miR-18 a in vascular endothelium;2)miR-18 a can inhibit the activation of vascular endothelium and the expression of inflammatory factors induced by HHcy,thereby reducing the damage of vascular endothelium;3)miR-18 a can inhibit the increase in oxidative stress level of vascular endothelium induced by HHcy,thereby reducing the damage of vascular endothelium;4)miR-18 a can inhibit the imbalance of mitochondrial fusion/fission and the increase of apoptosis induced by HHcy,thereby reducing the damage of vascular endothelium.Section ? miR-18 a targets PTEN to inhibit HHcy-induced vascular endothelial injuryObjectives:1)To clarify that miR-18 a targets PTEN to inhibit HHcy-induced vascular endothelial inflammation,oxidative stress and mitochondrial damage,and further reduce vascular endothelial injury.2)In vitro Hcy experiments confirmed that miR-18 a targets PTEN to inhibit Hcy-induced endothelial cell inflammation,oxidative stress and mitochondrial damage,thereby reducing endothelial cell injury.Methods:1)Using bioinformatics and related miRNA research databases to predict the relationship between miR-18 a and PTEN,and then verify the relationship between the luciferase reporter gene and the cell level.2)Performed transcriptomics sequencing analysis between HHcy group and the normal control group,trying to find the differentially expressed genes in the process of HHcy-induced vascular injury,and further use bioinformatics methods to analyze protein interactions PTEN Association with related pathway proteins in this study.3)Use lentivirus to construct overexpression and silence PTEN expression vector,realized the regulation of PTEN expression in endothelial cells,and perform subsequent miR-18 a recovery experiments;4)miR-18 a targets PTEN to inhibit Hcy-induced inflammatory response.The experiment is divided into HM+LV-Con(Hcy+miR-18 a mimic + PTEN overexpressi on control)group,HM+LV-PTEN(Hcy+ miR-18 a mimic + PTEN overexpression),HI+Sh-Con(Hcy + miR-18 a Inhibitor + Sh-PTEN control)and HI+Sh-PTEN(Hcy +miR-18 a Inhibitor + Sh-PTEN)groups,mainly through RT-PCR to detect endothelial ICAM1,VCAM1,IL-1?,IL-6,TNF? and IL-10;5)Collect the endothelial cell supernatant of each group,and detect the expression of SOD and MDA with related kits,and detect the expression levels of oxidized protein NOX4 and anti-oxidized protein SOD1 and SOD2 by WB;6)WB detects the expression of Mfn2 and Drp1 in each treatment group,as well as the expression of Bcl2 and Bax,and detects the level of apoptosis in each group by flow cytometry;7)Test the related functions of endothelial cells in each group: migration and tubule formation.Results:1)Through m RNA sequencing results,We found that compared with the Control group,there were significantly different expressed genes,including 1722up-regulated genes and 707 down-regulated genes.Cross-analysis of each group of expressed genes,there are 10667 co-expressed genes.Subsequent analysis of enrichment information of related bioinformatics pathways for this shared gene revealed that its existence was significantly related to the PTEN Regulation and Regulation of PTEN stability and activity pathways(P<0.05).Protein interaction analysis revealed that PTEN is closely related to the relevant pathway proteins in this study.2)Using bioinformatics and related miRNAs research databases,the analysis found that there are three possible binding sites between miR-18 a and PTEN;Luciferase reporter assay showed that the mutant PTEN-MT Compared with wild-type PTEN-WT,the fluorescence intensity has been significantly restored,indicating that the regulation of PTEN by miR-18 a is regulated by specific binding sites(P<0.05);cell experiments show that Mimic(miR-18 a overexpression)Compared with Mimic Con(Mi R-18 a overexpression control group),the m RNA and protein levels of PTEN were suppressed;while Inhibitor miR-18 a,the results were opposite(P<0.05);3)Overexpression of LV-Con vector and Sh-Con vector,72 hours after transfection,observe the corresponding fluorescence density and intensity under a fluorescence microscope.Compared with the control group,the other two groups of GFP green fluorescence have higher density and intensity,suggesting high transfection efficiency;according to the three pairs of targets previously designed for Sh-PTEN,RT-PCR and WB detection were carried out,respectively.The results showed that each target can inhibit the expression of PTEN,and we chose Sh-PTEN-A as the intervention to silence PTEN(P<0.05).At the same time,the effectiveness of the overexpression of the PTEN vector was verified.Compared with the LV-Con group,the m RNA and protein levels of the LV-PTEN group were increased(P<0.05);4)Compared with the HM+LV-Con group,the expression of ICAM1 and VCAM1 in the HM+LV-PTEN group increased,and the expression of IL-1?,IL-6,and TNF? increased,but decreased the expression of IL-10(P<0.05);on the contrary,compared with the HI+Sh-Con group,the inflammatory response in the HI+Sh-PTEN group was significantly improved(P<0.05);5)Compared with the HM+LV-Con group,the endothelial cells of HM+LV-PTEN have a higher oxidative stress level,which is manifested by a decrease in SOD level and an increase in MDA level;at the same time,the expression of oxidized protein NOX4 is increased,Inhibit the expression levels of antioxidant proteins SOD1 and SOD2(P<0.05);at the same time,the HI+Sh-PTEN group and HI+Sh-Con group showed the opposite trend,which can reduce oxidative stress related indicators and increase antioxidant SOD and the expression of antioxidant proteins SOD1 and SOD2,inhibit the expression of oxidized MDA and oxidized protein NOX4(P<0.05);6)Compared with the HM+LV-Con group,the expression of Mfn2 in the HM+LV-PTEN group decreased,while the expression of Drp1 increased,so the ratio of mitochondrial fusion/ fission protein: Mfn2/Drp1 was significantly reduced(P<0.05);The final performance is to promote the decrease of anti-apoptosis/pro-apoptosis protein Bcl2/Bax level,and the increase of apoptosis level detected by flow cytometry(P<0.05);while the HI+Sh-PTEN group is compared with HI+Sh-The Con group showed an opposite trend compared to the results(P<0.05);7)Compared with the Control group,the Hcy group can significantly inhibit the migration of endothelial cells and the ability of tubule formation.Compared with HMC,the migration of endothelial cells and the number of tubule formation in the HM group were improved to a certain extent(P<0.05);compared with the HM+LV-Con group,HM+LV-PTEN can inhibit the migration of endothelial cells and the ability of tubule formation(P<0.05);On the contrary,compared with HIC group,HI group significantly inhibited endothelial cell migration and tubule formation;HI+Sh-PTEN group compared with HI+Sh-Con group,the endothelial cell migration and tubule formation ability were improved(P<0.05).Conclusions:1)miR-18 a targets PTEN to inhibit HHcy-induced vascular endothelial inflammation,oxidative stress and mitochondrial damage,and further reduce vascular endothelial injury.2)In vitro Hcy experiments also show that miR-18 a targets PTEN to inhibit Hcy-induced endothelial cell inflammation,oxidative stress and mitochondrial damage,thereby reducing endothelial cell injury.Section ? SummaryIn this study,we confirmed that under HHcy stimulation,miR-18 a can reduce vascular damage by inhibiting vascular endothelial inflammation,oxidative stress and mitochondrial damage.In addition,transcriptome sequencing revealed differences in the expression of PTEN and its pathways.We further explored the molecular mechanism of miR-18 a in inhibiting HHcy-induced vascular endothelial damage,and found that miR-18 a targets and regulates the expression of PTEN,reduces vascular endothelial inflammation,oxidative stress and mitochondrial damage,thereby reducing vascular endothelial damage.In conclusion,we explored the role of miR-18 a in HHcy-induced vascular endothelial injury and its molecular mechanism,which will provide new ideas and new targets for the prevention and treatment of HHcy in CVD. |
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