| BackgroundMyocardial ischemic disease caused by atherosclerotic plaque stenosis is called coronary heart disease,which is a common and frequent disease all over the world.In addition,other arterial lesions are also particularly serious.At present,there are two problems in the diagnosis and treatment of arterial diseases: first,a considerable number of patients have serious complications before diagnosis,which resulting in significantly increased uncertainty of treatment and prognosis in these patients;second,the mechanism of many types of cardiovascular disease is still unclear,despite the equipment gained considerable advancement including coronary angiography and coronary artery CT.Although the treatment is constantly improving,the long-term effect still needs to be improved.Therefore,the molecular regulation mechanism of arterial disease is critical in preventing and curing the disease to improve the prognosis.The interaction between the constituent cells of the arterial vessels is essential to maintain the normal morphology and function of the vessels.Cumulating studies have shown that there are complex signaling interactions between vascular smooth muscle cells,endothelial cells and adventitia fibroblasts which composed of the blood vessels.However,the signal transduction networks and its mechanisms involved have not been fully investigated,the reasons for which are that blood vessels are both regulated by biological and mechanical signals and respond to chemical changes in the environment.Under the combined action of biological,physical and chemical factors,vascular diseases occur once the homeostasis between the constituent cells of blood vessels is destroyed.The relationship between these cells and the mechanism is needed to be further studied.microRNA(miRNA)is a type of non-coding RNA which is about 19-25 bases in length.Due to the advancement of microarray and next generation sequencing technology,tens of thousands of miRNA have been identified in many types of diseases including tumor,cardiovascular disease,and cerebral vascular disease.A large number of studies have confirmed that miRNA can affect pyroptosis in different cells by regulating different targets.We found that co-culture of vascular endothelial cells and vascular smooth muscle cells can lead to a decrease in the expression of inflammatory factors in smooth muscle cells.Therefore,we speculated that vascular endothelial cells may regulate the pyroptosis of smooth muscle cells by paracrine.Recent studies have found that exosomes could be secreted and uptake in vivo and in vitro by almost all types of cultured cells.Exosomes is a double membranous vesicle which could be secreted by almost all types of cells.The diameter of exosomes mainly ranged from 30 nm to 100 nm,and exosomes can be stable in extracellular fluid which makes it possible for the diagnosis of cardiovascular disease.With the deepening of understanding of exosomes,it is found that exosomes can stably transport cytokines,mRNA,miRNA and other nucleic acids,to achieve the purpose of intercellular information transmission.Focal death of vascular endothelial cells can increase vascular permeability,leading to endothelial injury and promote the development of arteriosclerosis.microRNA-181b(miR-181b)plays an important role in the occurrence and development of cardiovascular disease.A large number of studies have shown that miR-181 b can regulate the expression of cytokines,proteases and receptors by regulating the relevant target genes in cardiovascular tissue,thus participating in the regulation of the physiological and pathological state of the cardiovascular system.miR-181 b plays an important regulatory role in cardiovascular diseases such as hypertension,atherosclerosis,arterial valve calcification,myocardial fibrosis,myocardial hypertrophy,and myocarditis.In addition,miR-181 b is a mechanical sensitivity miRNA,its expression level is affected by shear force in the aortic valve.However,the regulatory mechanism of miR-181 b under low shear stress and its relationship with pyroptosis are still not clear.Methods1.To establish a mono-culture system of smooth muscle cells and endothelial cells and smooth muscle cells co-culture system,respectively.The effects of low shear stress on pyroptosis in mono culture smooth muscles and endothelial cells and smooth muscle cells co-culture system were observed.Western blot assay was used to detect the expression level of Caspase-1?NLRP3 at protein level.RT-PCR method was performed to measure the expression of miR-181b?Caspase-1?NLRP3?IL-1? and IL-18 at mRNA level.ELISA method was used to detect the expression level of IL-1? and IL-18.2.The mono-cultured endothelial cells were cultured in serum without exosomes and the supernatant of mono-cultured endothelial cells treated with low shear stress or static state treatments was collected,respectively.After removal of cell fragments from culture medium by ordinary centrifugation,the exosomes in the cell culture supernatant was separated by differentiated ultracentrifugation method.The exosomes collected by ultracentrifugation were stored at-80 ? or prepared for the following experiment.The morphology of extracted exosomes was verified by transmission electron microscopy technology.The surface protein markers,including CD63 and CD9,of exosomes were detected by western blot assay.Calnexin was also used to differentiate exosomes from apoptotic bodies and cell debris.The microarray technique was used to detect the expression of miRNA in exosomes derived from endothelial cells under low shear stress or static treatment.We further selected the target miRNA to verify by RT-PCR method according to the fold change of microarray results and related references.3.To further observe the effect of exosomes on the pyroptosis of smooth muscle cells,we added exosomes derived from endothelial cells with static treatment.The protein level of Caspase-1and NLRP3 was evaluated by Western blow assay.The expression level of miR-181 b,Caspase-1,NLRP3,L-1? and IL-18 was detected by RT-PCR method.The expression level of IL-1? and IL-18 was detected by ELISA method.In further to evaluate the anti-pyroptosis effect of miR-181 b in exosomes on smooth muscle cells,smooth muscle cells were transfected with miR-181 b inhibitor and endothelial cell derived exosomes.The protein level of Caspase-1and NLRP3 was evaluated by Western blow assay.The expression level of miR-181 b,Caspase-1,NLRP3,L-1? and IL-18 was detected by RT-PCR method.The expression level of IL-1? and IL-18 was detected by ELISA method.4.Using miR-181 b as the key word,the target genes of miR-181 b were predicted by Target Scan?Pic Tar and miRanda website.The target genes were finally identified by the prediction results of these websites and related references.We validated the interaction of STAT3 and miR-181 b by double luciferase reporter method.The mRNA expression level of STAT3 was detected to confirm STAT3 as the direct target of miR-181 b.5.We next investigated that miR-181 b regulated pyroptosis in smooth muscle cells by STAT3 signal pathway.By transfecting smooth muscle cells with miR-181 b inhibitors and siSTAT3 or negative scramble control to observe the effect of miR-181 b on the expression level of pyroptosis related genes.The protein level of Caspase-1and NLRP3 was evaluated by Western blow assay.The mRNA expression level of Caspase-1,NLRP3,L-1? and IL-18 was detected by RT-PCR method.The expression level of IL-1? and IL-18 was detected by ELISA method.6.Statistical analysis: the data obtained in this experiment were calculated by mean ±standard deviation,each set of data was repeated at least 3 times.Comparisons of differences between the two groups using unmatched t tests,differences between multiple groups were analyzed using ANOVA analysis.Data entry and analysis was performed using Graph Pad Prism 7.0 software.Chip data and bioinformatics analysis were carried out by R 3.4 software.p < 0.05 indicated the difference was statistically significant.Results1.We first used western blot method to detect the expression of pyroptosis related protein,including Caspase-1 and NLRP3,in smooth muscle cells.The results showed that the protein expression level of Caspase-1 and NLRP3(p<0.05)in smooth muscle cells was significantly increased by low shear force.RT-PCR results also suggested that the mRNA expression of pyroptosis-related markers,including Caspase-1,GSDMD-N,IL-1? and IL-18,was significantly higher than that of the control group.ELISA assay also suggested that the expression of inflammatory factor IL-1? and IL-18 was much higher than that of the control group.Further analysis showed that endothelial cells in the co-culture system could decrease the pyroptosis of smooth muscle cells.In the co-culture system,the expression level of pyroptosis-related protein including Caspase-1 and GSDMD-N in smooth muscle cells was significantly lower than that in the smooth muscle cells monoculture system.RT-PCR results also suggested that the mRNA level of Caspase-1,GSDMD-N,IL-1? and IL-18 was significantly lower than that in the mono smooth muscle cell culture system.ELISA results displayed the same trend of IL-1? and IL-18 between different smooth muscle cells group and the difference was statistically significant.2.The exosomes was isolated and characterized by differential centrifugation method after collecting supernatant from mono-culture endothelial cells system.Transmission electron microscopy showed that the exosomes displayed characteristic teacup-like or oval bilayer membrane vesicles.The results also showed that the diameter of exosomes was between 60-110 nm and the median diameter was 80 nm.The results showed that the expression of CD63 and CD9 was positive and the expression of Calnexin was negative.According to the above results,the exosomes was successfully collected.3.The microarray analysis of exosomes derived from endothelial cells indicated that there were 16 miRNAs that were differentially expressed with fold change over 2 and p value below 0.05.Among of these differentiated expressed miRNAs,12 miRNAs were upregulated and four miRNAs were downregulated.GO analysis suggested that the physiological functions of these differentially expressed miRNAs mainly enriched in intercellular communication,regulation of nucleic acid metabolism,signal transduction and so on.According to the results of expression analysis and literature retrieval,miR-181 b with the greatest fold change in the microarray analysis was selected for follow-up analysis.RT-PCR results showed that low shear force significantly reduced the expression of miR-181 b in exosomes.4.By adding exosomes derived from endothelial cells treated with static to monoculture smooth muscle cells in vitro,RT-PCR indicated that the expression level of miR-181 b was significantly higher than that in the control group.By adding exosomes or exosomes inhibitors GW4869,Western blot was used to detect pyroptosis-related protein expression in smooth muscle cells.The results indicated that exosomes can inhibit the mRNA and protein expression level of Caspase-1 and NLRP3 in smooth muscle cells.After adding GW4869 to the co-culture system,the protective effect of endothelial cells in co-culture system on pyroptosis was significantly decreased.The results showed that the co-cultured endothelial cells affected the pyroptosis of smooth muscle cells through exosomes method.By transfecting miR-181 b mimics or inhibitors into smooth muscle cells,Elevated or downregulated miR-181 b expression in smooth muscle cells,RT-PCR detection of miR-181 b expression in smooth muscle cells after transfection,The results showed that the miR-181 b in smooth muscle cells could be up-regulated by transfection miR-181 b mimics.Transfection of miR-181 b inhibitors could inhibit the anti-pyroptosis effect of exosomes on smooth muscle cells.Based on these evidences,it suggested that miR-181 b in the exosomes was involved in the pyroptosis regulation of endothelia cells on smooth muscle cells.5.By combined with the prediction results of Target Scan,Pic Tar and miRanda website,the commonly used prediction tool of target genes of miRNAs,STAT3 was screened out and identified as the target gene of miR-181 b.To verify that STAT3 is the target gene of miR-181 b,we constructed the reporter gene plasmid containing seed region of 3' UTR of STAT3 that could bind to miR-181 b and the mutant sequence of 3' UTR of STAT3 was also constructed simultaneously.The results showed that miR-181 b could inhibit the fluorescence level of p GL3-STAT3-wildtype plasmid,whereas the fluorescence level p GL3-STAT3-mutant plasmid does not displayed statistically difference.The mRNA expression of STAT3 was then detected by RT-PCR method.The results showed that upregulation of miR-181 b inhibited STAT3 expression at the mRNA level.These effects were observed at both shear stress and static condition.It suggested that STAT3 is the target gene of miR-181 b.6.STAT3 signal pathway promotes pyroptosis of smooth muscle cells.The effect of STAT3 on the pyroptosis of smooth muscle cells was first observed by transfection siSTAT3 smooth muscle cells.The results showed that siSTAT3 transfection can reduce the occurrence of pyroptosis of smooth muscle cells.The expression level of pyroptosis-related proteins Caspase-1 and NLRP3 was detected by Western Blot method in smooth muscle cells after transfection with siSTAT3.The results showed that the expression level of Caspase-1 and NLRP3 in smooth muscle cells was down-regulated by transfection with siSTAT3.RT-PCR detection also suggested that Caspase-1,NLRP3,IL-1? and IL-18 were significantly lower than those in the control group.ELISA detection suggested the same trend in IL-1? and IL-18 expression with statistically significance.We continued to validate that miR-181 b regulated pyroptosis through STAT3 signal pathway by co-transfected with miR-181 b inhibitors and siSTAT3 in smooth muscle cells.The results suggested that down-regulation of STAT3 expression can reverse the effect of miR-181 b inhibitors on the pyroptosis of smooth muscle cells.The above results showed that miR-181 b regulated the pyroptosis of smooth muscle cells through STAT3 signaling pathway.ConclusionLow shear stress can inhibit the expression level of miR-181 b in endothelial cells.Whereas,co-culture system including endothelial cells and smooth muscle cells indicated that pyroptosis in smooth muscle cells may be suppressed by endothelial cells.Endothelial cells can secrete exosomes;the diameter of these exosomes was about 100 nm.According to the results of chip data analysis,it suggested that miR-181 b was low expressed in exosomes with low shear stress treatment.Bioinformatics analysis suggested that the physiological functions of differential expressed miRNAs in exosomes mainly enriched in intercellular communication,regulating nucleic acid metabolism,signal transduction and so on.Endothelial exosomes can inhibit the pyroptosis of smooth muscle cells by increasing the miR-181 b in smooth muscle cells.The further analysis showed that STAT3 was a candidate target gene of miR-181 b and was verified by dual luciferase reporter system.Up-regulation of miR-181 b expression level can inhibit the occurrence of pyroptosis in smooth muscle cells,and down-regulation of STAT3 expression can counteract the inhibitory effect of miR-181 b inhibitors on pyroptosis in smooth muscle cells. |
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