Preliminary Study On TSH Promoting Aortic Smooth Muscle Proliferation By Up-regulating BK_Ca Channel Subunit Expression

Objective:Hypothyroidism can appear in a variety of thyroid disease in the natural course of the disease or treatment period.Hypothyroidism can be divided into dominant hypothyroidism and subclinical hypothyroidism according to the presence or absence of abnormal serum thyrotropin,which is characterized by abnormal elevation of serum thyrotropin.Hypothyroidism can be divided into clinical hypothyroidism and subclinical hypothyroidism according to the presence or absence of abnormal serum thyroid hormones,which common feature is abnormal elevation of serum thyroid stimulating hormone（TSH）.With the increase of the total number of people suffering from various thyroid diseases in recent years,the number of people with abnormal serum TSH is also increasing.Therefore,many health problems of the population with increased TSH need to be explored and solved.Abnormal proliferation of Vascular Smooth Muscle Cells（VSMCs）plays a central role in the development and progression of AS plaques.Large conductance Ca2+activated Potassium(BK_Ca)channel was found on VSMCs,which is very important to cell electrophysiological and biological functions.BK_Capathway has been proved to play a crucial role in the genesis and development of AS,and it is associated with abnormal proliferation of VSMCs,but the specific mechanism is not clear.An increasing number of clinical studies have found that TSH elevation is an independent risk factor for AS,and it may be related to its ability to promote abnormal proliferation of VSMCs.However,it is not completely clear how TSH affects the occurrence and development of AS,whether it is achieved by influencing VSMCs and its specific mechanism.The purpose of this study was to investigate:1.Whether hypothyroidism can cause early atherosclerotic changes in thoracic aorta in rats in the short term?2.Whether TSH can promote abnormal proliferation of VSMCs?3.Whether TSH works by affecting BK_Cachannels in VSMCs cells?4.A preliminary study of the signaling pathways that might be at work.Methods:Animal experiment:Wistar rats were used to establish hypothyroidism model.To verify the success of the model,we recorded the operation by microscope photographing,observed the histomorphology by HE staining,and detected the blood indexes by ELISA and colorimetry.Thoracic aorta of rats at the 16th week after surgery was stained with HE to observe the morphological changes of arterial wall;Western Blot,tissue immunofluorescence and tissue immunohistochemistry were performed to observe the expression of corresponding proteins;tissue oil red O staining was performed to observe the lipid deposition of arterial wall;RT-q PCR was used to detect the expression of related m RNA.Cell experiment:Human aortic smooth muscle cells（HASMCs）were used as experimental cells,control group was normal cultured cells（TSH concentration of 0ng/ml）,experimental group 1,experimental group 2 and experimental group 3 were stimulated with 1ng/m L,5ng/ml and 10ng/m L TSH respectively on the basis of control group,and experimental group 4 was stimulated with 5ng/ml TSH+100nmol/L IBTX on the basis of control group,the stimulation time of the four groups was 24 hours.CCK8,RT-QPCR and Western Blot were used to determine the relationship between TSH,BK_Cachannels and VSMCs.ELISA was used to detect the changes of c AMP after TSH stimulation.Results:1.To establish a rat model of hypothyroidism:Observation of the intraoperative area and postoperative tissue showed that the thyroid tissue of Wistar rats was completely excised,then HE staining confirmed that the peeled tissue from the neck was thyroid tissue,and there is no characteristic follicles around the trachea of the model group on the 28th day after surgery.On the 28th day after operation,serum TT3 and TT4levels in model group were significantly lower than those in sham group（P<0.001）,serum TSH level in model group was significantly higher than that in sham group（P<0.001）,there was no significant difference in serum Ca²⁺levels between the two groups.2.Hypothyroidism causes early changes of AS in thoracic aorta of rats:At the 16th week after surgery,the thoracic aorta of rats in the two groups were stained with oil red O and HE staining.Compared with the sham group,the hypothyroidism group had lipid deposition in blood vessels（P<0.01）,and the smooth muscle layer was thickened and the cells were disordered.3.TSH promotes proliferation of aortic smooth muscle cells:At the 16th week after surgery,immunofluorescence of thoracic aorta showed that the expression ofα-SMA protein in the smooth muscle cells of the vascular medium layer in hypothyroidism group decreased（P<0.001）,while the expression of OPN protein increased（P<0.001）.Wstern Blot results of thoracic aorta tissues of rats in both groups showed that the protein expressions of cyclin A,cyclin D1 and proliferating cell nuclear antigen（PCNA）in thoracic aorta of rats in hypothyroidism group were higher than those in sham group（P<0.05）.In cell experiments,after TSH stimulated HASMCs,CCK8 experiment results suggested that TSH could stimulate HASMCs proliferation,and the proliferation rate increased most obviously at 5ng/m L.However,when TSH concentration increased to 10ng/m L,the proliferation rate decreased,but it was still higher than the control group.Western Blot results showed that TSH promoted the expression of cyclin A,cyclin D1and PCNA proliferation-related proteins in HASMCs,and the expression was most obvious when TSH concentration was 5ng/m L,while when TSH concentration was 10ng/m L,the expression of the three proteins was lower than that in experimental group 2（P<0.05）.4.Preliminary study on the mechanism of early AS changes in thoracic aorta of hypothyroidism rats:At week 16 after surgery,the expression levels ofαandβ1 subunits of BK_Cachannel in smooth muscle cells of vascular medium layer in hypothyroidism group were increased compared with those in sham group（P<0.01）.In cell experiment,when TSH concentration was 5ng/ml,the expression levels ofαandβ1 subunits of BK_Cachannel were significantly increased compared with the control group（P<0.05）.ELISA results indicated that the intracellular c AMP concentration of HASMCs was significantly increased when 5ng/m L TSH stimulated HASMCs（P<0.01）.Finally,when BK_Cachannel specific blocker IBTX was added,the expression levels of cyclin A,cyclin D1 and PCNA proliferation-related proteins in experimental group 4 were significantly lower than those in experimental group 2（P<0.05）.Conclusion:1.Hypothyroidism can cause early changes of AS in thoracic aorta of Wistar rats.2.TSH can promote the proliferation of aortic smooth muscle cells in the short term.3.TSH can promote the proliferation of aortic smooth muscle cells by increasing the expression levels of BK_Cachannel proteinsαandβ1 subunits in the short term.4.TSH regulation of BK_Cachannels may be related to camp-related pathways.