Mechanism Of Inhibition Effects Of Thyroid Stimulating Hormone On Expression Of Nitric Oxide Synthase In Human Micrivascular Endothelial Cells

Objective: Subclinical hypothyroidism is characterized by normal thyroid hormone levels and elevated thyroid stimulating hormone(TSH)levels.Epidemiological data confirm that high serum TSH levels are positively associated with the risk of atherosclerosis(AS).Endothelial dysfunction is a key link in the development of AS in which endothelial nitric oxide synthase(eNOS)plays an important role.Studies have shown that TSH can inhibit the expression of eNOS gene in human umbilical vein endothelial cells,but the exact mechanism remains unclear.The purpose of this study was to detect content of intracellular eNOS,P-AKT,and phosphor-extracellular signal regulated kinase(P-ERK)in change after intervention with human microvascular endothelial cells(HMEC-1),which is to investigate the regulation of eNOS by TSH and its major signaling pathways and to further understand the regulatory mechanism of TSH on vascular endothelial function.Methods: HMEC-1 cells were cultured in vitro to extract total cellular RNA.Reverse Transcription-Polymerase Chain Reaction(RT-PCR)was used to detect the mRNA expression of thyroid-stimulating hormone receptor(TSHR)in HMEC-1 and verify the expression of TSHR in HMEC-1 cells.HMEC-1 was treated with different concentrations of TSH(0,10,100mIU/ml)for 24 h.Thenfluorescence quantitative PCR was used to detect the expression of eNOS mRNA and Western Blot was used to detect the expression of eNOS protein to observe the regulation effect of TSH on endothelial eNOS.HMEC-1 was treated with different concentrations of TSH(0,10,100mIU/ml)for 24 hours to extract total cellular protein.Then the total AKT,total extracellular signal regulated kinase(ERK)and their phosphorylated protein levels were detected by Western Blot.And the cells were pretreated with phosphatidylinositol 3-kinase(PI3K)inhibitor LY294002 and ERK inhibitor PD98059 to block the activation of AKT and ERK induced by TSH.After adding 100mIU/ml TSH to the cells,24 hours later,the total protein of each group was extracted.Western blot was used to detect the levels of eNOS,p-AKT,AKT,P-ERK and ERK proteins,and the mechanism of TSH regulating HMEC-1 eNOS was explored.Results:(1)TSHR was expressed in HMEC-1 cells.(2)After intervention of HMEC-1 with different concentrations of TSH,TSH significantly inhibited the expression of eNOS in a dose-dependent manner,and TSH significantly promoted the expression of p-AKT and p-ERK in a dose-dependent manner.(3)After pretreatment of HMEC-1with PI3 K inhibitor LY294002 and ERK inhibitor PD98059 blocked AKT and ERK pathways,and the inhibitory effect of TSH on HMEC-1 eNOS was significantly reduced.Conclusion: TSH may inhibit the expression of HMEC-1 eNOS through PI3K/AKT and ERK signaling pathways.