Change Of THBS1 In Serum Of Rats With High-salt Diet And Its Effect And Mechanism In Endothelial Dysfunction

BackgroundsIn recent years,with the improvement of living standards and changes in lifestyle,cardiovascular and cerebrovascular diseases have become the primary risk factor that seriously threatens human health.High-salt diet is one of the important risk factors for cardiovascular and cerebrovascular diseases.Approximately 1.65 million people worldwide die of cardiovascular disease every year caused by a high intake of salt.Increasing evidence suggests that high-salt intake impairs vascular reactivity,the regulation of peripheral vascular resistance as well as nitric oxide（NO）synthesis.Its damage to cardiovascular,cerebrovascular,glomerular capillaries,and mesenteric resistance vessels has been a research focus in recent years.Thrombospondin-1（THBS1）was originally defined as the first natural anti-angiogenic agent.In recent years,it has been discovered that it is also a secreted multifunctional glycoprotein.THBS1 regulates angiogenesis and cell proliferation,apoptosis,migration and adhesion,and participate in the maintenance of vascular system and homeostasis.It plays an important role in a variety of physiological and pathological processes,including thrombosis,angiogenesis,tumorigenesis,inflammation,apoptosis and fibrosis.Through interacting with cellular surface receptors,THBS1 inhibits intracellular NO signaling,such as inhibiting the activation and phosphorylation of endothelial nitric oxide synthase（e NOS）and reducing the production of NO.NO has many important functions,especially in maintaining vascular tension,regulating blood pressure and inhibiting inflammatory response.Store-operated calcium entry（SOCE）is an important way to regulate calcium concentration in EC.High salt induces a decrease in the bioavailability of NO,which in turn leads to EC dysfunction and reduces vasodilation.However,the mechanism of EC dysfunction and vascular damage induced by THBS1 in a high-salt diet has not been fully elucidated.Based on the above background,the present study intends to explore two research topics:1.The roles and mechanisms of key proteins and their related signaling pathways in cardiovascular diseases caused by a high-salt diet;2.The role and mechanism of THBS1in endothelial dysfunction in rats with high salt diet.Objectives1.To explore the key differentially expressed proteins and related signal pathways in the serum of high-salt diet rats;2.To verify the changes in the expression of THBS1 induced by high-salt diet and its role in the response of MAECs to high-salt,provide a new potential molecular mechanism for vascular damage caused by high-salt diet.Methods1.Serum collectionSD rats were divided into two groups:control group（normal diet group,0.4%Na Cl）and high-salt diet group（4%Na Cl）,fed for 4 weeks.Under aseptic conditions,the abdominal main venous blood was drawn.And the supernatant was extracted.2.Protein labelingIsobaric tags 113,114,115,and 116 were used to label the peptides in the control group,whereas isobaric tags 117,118,119,and 121 were used to label peptides in the high-salt diet group.3.High-p H HPLC classificationPeptides were separated by a linear gradient formed from buffer A and buffer B（98%ACN,p H 10）at a flow rate of 700μL/min.4.Liquid chromatography with tandem mass spectrometry（LC-MS/MS）analysisThe peptides were separated by a linear gradient formed from mobile phase A（0.1%FA,H2O）and mobile phase B（0.08%FA,80%ACN）at a flow rate of 600 n L/min.The separated peptides were placed into a mass spectrometer for detection,subsequently.5.Database retrieval and bioinformatics analysisWe used Proteome Discoverer Software to search the uniprot-rattus+norvegicus.20180702＿36089.fasta database.Gene ontology（GO）analysis and Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway analyses were then conducted to identify those pathways that were statistically significantly enriched with the peptides of interest.6.Immunohistochemistry experiment and western blottingImmunohistochemistry experiment and western blotting were used to detect the expressions of THBS1,e NOS,transforming growth factor-β1（TGF-β1）,Orai1,Orai2and Orai3.7.Mesenteric resistance artery（MRA）tension measurementAcetylcholine and sodium nitroprusside were used to detect MRA endothelium-dependent and endothelial-independent relaxation,respectively.8.Primary MAECs culture and identification0.2%type I collagenase was used to digest and separate cells.The EC marker protein VWF was used to identify the MAECs.9.Transfection of primary MAECsCells are transfected with four small interfering RNA（si RNA）sequences,which are scrambled si RNA,Thbs1 si RNA,Orai1 si RNA and Orai2 si RNA.10.Determination of NO content in MAECsDAF-FM DA（NO fluorescent indicator）was used to detect the NO content in the cells of each group.The green fluorescence intensity in the cells was observed under a fluorescence microscope.11.Detection of Smad4 entering the nucleus by confocal laserThe laser confocal microscope was used to observe the distribution of Smad4 in the MAECs.12.Measurement of intracellular calcium concentration([Ca²⁺]_i)Calcium imaging system was used to detect the calcium store-operated calcium influx（SOCE）of the primary MAECs.13.StatisticsData are shown as means±SEM and graphed in Graph Pad Prism（v8.3.0.538）software（Graph Pad Software,San Diego,CA,USA）.Two-way analysis of variance followed by Games-Howell post hoc tests or two-tailed Mann-Whitney test were performed to analyze statistical differences among groups.Statistical significance was considered as P<0.05.Results1.There were 111 serum proteins with concentrations significantly different between rats fed a high salt diet and those fed a regular diet:65 proteins were upregulated and 46proteins were downregulated.The fold change（FC）of THBS1 was 1.5,that is,the expression was significantly up-regulated.2.THBS1 was highly expressed in the serum and vascular endothelium of rats on a high-salt diet;3.Compared with normal diet rats,the endothelium-dependent relaxation of MRA was significantly weakened in high-salt diet rats.But high-salt diet had no effect on the endothelium-independent relaxation;4.High salt induced an up-regulation of THBS1 protein expression and down-regulation of e NOS expression in MAECs;5.THBS1 inhibited e NOS expression level and NO production in MAECs as well as reduced endothelium-dependent relaxation of MRA;6.THBS1 was responsible for down-regulation of e NOS in high-salt environment;7.TGF-β1 activation induced Smad4 to enter the nucleus,resulting in increased THBS1expression in high-salt environment;8.THBS1 suppressed the protein expressions of Orai1 and Orai2 in MAECs,and the amplitude of SOCE.Moreover,knockdown of Orai1 and Orai2 significantly inhibit e NOS protein expression,NO release and SOCE in the cells;9.High salt induced down-regulation of the expression of Orai1 and Orai2 in MAECs,but had no significant effect on the protein expression of Orai3.ConclusionThe present study effectively used the i TRAQ technique for protein identification and quantification and filtered the key proteins（such as THBS1）,wchich were closely related to vascular endothelial dysfunction.Then,we demonstrated that a high salt diet causes elevations of THBS1 expression and secretion,and the changes in THBS1 were associated with impairment of endothelial-dependent vasodilation in high-salt diet rats.In vitro experiments,we further proved the important role of THBS1 in the response of MAECs to high salt stimulation,that is,by activating TGF-β1 and downstream Smad4into the nucleus,high salt up-regulated the expression of THBS1 in cells.And the alterations of THBS1 lead to the down-regulation of Orai channel protein expressions and the weakening of SOCE,which resulted in the reduction of e NOS expression and NO production,and caused endothelial cell dysfunction.