| Background High-salt diet and excessive salt intake have great harm.High-salt diet can increase the risk of various cardiovascular diseases,and can also increase the risk of gastric cancer and the burden on the kidneys.At the same time,studies have shown that high-salt diets Can cause high blood pressure.Excessive salt intake is a great threat to health.Coro1 a may be an important component of the cytoskeleton of highly motile cells,playing a role in the invagination of bulk plasma membranes and the formation of plasma membrane protrusions involved in cell motility.Coro1 a also plays an important role in cell proliferation and migration.Clinical studies have reported that the mutation and dysfunction of Coro1 a are associated with immune function,and animal experimental studies have also found that the Coro1 a gene may be involved in the occurrence and development of autoimmune diseases.Coro1 a is a calcium-dependent leukocytespecific regulator involved in signaling processes that is essential for the maintenance of peripheral naive T cells.In this study,we treated rat mesenteric arterial endothelial cells（MAECs）with high salt,and found that the expression of Coro1 a was significantly changed by RNA-seq,which was consistent with the results of plasma protein profiling in rats fed a high-salt diet.It has been reported that Coro1 a can protect endothelial cells from TNFα-induced apoptosis by regulating p38β expression and activation in human umbilical vein endothelial cells（HUVEC）.Based on our findings in RNA-seq and plasma mass spectrometry,this study investigated the role and mechanism of Coro1 a in rat MAECs.Purpose 1.The differentially expressed genes of rat MAECs in high-salt environment were found by RNA-seq,and compared with the plasma mass spectrometry results of highsalt diet rats,and key genes and proteins were found.2.To verify the expression of Coro1 a in high-salt rat MAECs cells,to study the regulatory effect of Coro1 a on endothelial cell function and its mechanism,and to provide a new molecular mechanism for high-salt-induced vascular endothelial injury.Methods 1.RNA-seq detection of differential genes after high salt treatment Rat MAECs cells were extracted and cultured,treated with 102.67 m M,122.67 m M,and 142.67 m M Na Cl for 24 h,the supernatant was discarded,and samples were collected using Trizol for detection.2.Comparison of RNA-seq results and high-salt diet rats The 122.67 m M,142.67 m M Na Cl-treated groups and the 102.67 m M Na Cl-treated group were subjected to differential analysis to obtain up-regulated differential genes,and then cross-analyzed with the high-salt diet rat plasma up-regulated differential proteins using Venn diagram,and finally the key genes of Coro1 a were obtained.3.RT-q PCR The expression of Coro1 a in MAECs after high-salt treatment was verified at the m RNA level using RT-q PCR.4.Primary cell culture and identification Rat MAECs cells were extracted and cultured,and the primary cultured cells were verified as endothelial cells using v WF antibody.5.Western blotting experiments Western blot was used to detect the expression of Coro1 a protein.6.NO fluorescent probe NO fluorescent probes were used to label intracellular NO products in each group,and NO fluorescence was detected by Flexstation multi-plate reader.7.Cell scratch After the cells reached 100% confluence,they were scratched and cultured in serumfree medium to observe the cell migration rate between different treatment groups.8.Cell proliferation After transfection,the cells were seeded in 96-well plates,and treated with Na Cl for 24 h to detect cell proliferation using CCK-8 reagent.Results 1.The key gene Coro1 a was obtained by comparing the up-regulated genes in the RNAseq results with the up-regulated genes in plasma mass spectrometry of high-sodium diet rats.2.After treating MAECs with high sodium for 24 h,the results of RT-q PCR and western blotting showed that the expression of Coro1 a was significantly increased under high sodium conditions.3.After high sodium treatment of MAECs for 12 h,the migration ability of rat MAECs was enhanced,and there was no significant change after 24 h.After knockdown of Coro1 a,high sodium treatment for 12 h increased cell proliferation in normal group.After 24 h,high sodium and Coro1 a si RNA were simultaneously treated Decreased cell migration ability.4.The amount of NO synthesized in MAECs cells was reduced in high sodium environment for 24 h,and the NO synthesis in MAECs was significantly reduced after using si RNA to knock down Coro1 a.5.The proliferation of cells was attenuated in the high sodium environment for 24 h,and the knockdown of Coro1 a had no significant effect on cell proliferation.Conclusion Coro1 a was elevated in endothelial cells in a high-sodium environment and in the plasma of rats fed a high-sodium diet.And our study found that Coro1 a can regulate endothelial cell migration and NO synthesis,providing a new molecular mechanism for vascular endothelial injury induced by high sodium diet. |
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