| BackgroundThe mortality of cardiovascular disease (CVD) is the highest in the world. Atherosclerosis is the common basis of cardiovascular disease. Thyroid dysfunction is associated with an increased risk of CVD morbidity and mortality. Thyroid dysfunction has been implicated in the development of endothelial dysfunction and atherosclerosis. It is known to all that destruction of endothelium lead to the development of atherosclerosis. The apoptosis of endothelial cells is one of the important mechanisms which contribute to the atherosclerosis development process. The mechanisms that mediate the development of atherosclerosis and endothelial dysfunction in hyperthyroidism or hypothyroidism are unclear.Osteoprotegerin (OPG) is a secreted glycoprotein, which can inhibit osteoclastogenesis and increase bone mineral density and volume. However, there is additional evidence that OPG has been performed the role in angiogenesis. The effects of OPG on endothelial cells in thyroid dysfunction remain to be identified.NF-κB has played an important role in a large number of life activities. The activation of NF-κB is regulated through the I κB kinase (IKK), a complex of three subunits:IKKα、IKKβ'IKKγ. It is noted that IKKβ is essential for NF-κB activation. The tuberous sclerosis tumor suppressor complexl serves as a repressor of the mammalian target of rapamycin (mTOR). mTOR plays its essential role in cell growth and survival, nutrients, cell mass gain and cellular stress. mTOR activates S6K1or4EBP1, which are involved in the regulation of protein synthesis. The potential role of the IKKβ/mTOR pathway in activating an apoptotic signal cascade in thyroid dysfunction remains to be identified.Therefore, the main hypothesis is as follows:The apoptosis of vascular endothelial cells will increase in thyroid dysfunction and OPG will protect the vascular endothelial cells from apoptosis via IKKβ/mTOR pathway.ObjectiveThe effect of OPG on the apoptosis of human umbilical vascular endothelial cells (HUVECs) induced by different concentrations of thyroid hormone in vitro and its underlying mechanism will be determined in this study. Its aim is to provide experience basis for the effects of OPG on treatment and the prevention of angiopathy in thyroid diseases.Methods1. Human umbilical vein endothelial cells were cultured with DMEM containing10%calf serum.2. MTT assays:The cell-growth in different thyroxine concentrations (0、0.1、1、10、50.100nmol/L,T3) was analyzed by MTT assays at24h,48h,72h time points. The best time point will also be explored in this experiment.3. The cell apoptosis in different concentrations of thyroxine was measured with both flow cytometry and hochest33258at48h time point.4. Human umbilical vein endothelial cells (HUVECs) were cultured and divided into control group (0nmol/L,T3), control group+OPG (100ng/ml), low-concentration group(0.1nmol/L,T3), low-concentration group+OPG (100ng/ml), high-concentration group (10nmol/L,T3), high-concentration group+OPG (100ng/ml), physiological-concentration group, physiological-concentration group+OPG(100ng/ml) at48h time point. The cell apoptosis was confirmed by cytometry and Hochest33258.5. The expression of phosphorylation NF-κB inhibiting protein kinase β(p-KKβ), NF-κ B inhibiting protein kinase β(IKKβ), phosphorylation tuberous sclerosis complex-1(p-TSCl), t-tuberous sclerosis complex-1(t-TSCl), p-S6kinase (p-S6K), t-S6kinase (t-S6K), p-AKT, t-AKT, Bcl-2and Bax were analyzed by Western blot.6. Statistical analysis:All data are expressed as mean±SD; Data were analyzed with SPSS13.0. Factorial design analysis of variance and SNK test for MTT results. Data were analyzed with t-test for the results of cytometry and hochest3325. Comparisons between experimental groups and OPG treated group were analyzed with t-test. A P value<0.05was considered as significance.Results: 1. Endothelial cells:HUVECs begin to adhere to the flask and to stretch out pseudopodia at24h. After3-4days, all the cells can adhere to the culture bottle and present rounded and flat polygons shape. Cells begin to grow slowly after5-6days.2. MTT assyas:There is significant difference in total about HUVECs cell growth (F=299.27, P=0.000). Besides, The effects on HUVECs cell growth by thyroid hormone were time-dependent (F=1237.19, P=0.000).T3protects endothelial cells from apoptosis at concentrations ranging from0nmol/L to1nmol/L in a dose dependent manner. T3could induce endothelial cells apoptosis at concentrations of more than1nmol/L in a dose-dependent manner (p <0.05) at the time spot of24h,48h,72h,(p<0.05). We choose48h as the best time for follow-up experiment.3.The apoptosis of HUVEC:(1)Hoechst33258:At the physiological concentration,the apoptosis rates of HUVECs were13.67%±1.85%.The apoptosis rates of HUVECs at different concentration were48.22%±1.06%(0nmol/L,T3),34.13%±0.85%(0.1nmol/L,T3),31.32%±2.51%(10nmol/L,T3),62.74%±3.40%(50nmol/L,T3),82.47%±6.37%(100nmol/L,T3),respectively(compared with the physiological concentration, p<0.05).(2) Flow cytometry:The early apoptotic rates were28.24%±7.57%,29.92%±6.07%.26.13%±2.28%,8.57%±1.54%,at0、0.1、10nmol/L and physiological concentration, respectively(compared with the physiological concentration, p<0.05).Besides, the total apoptotic rates were44.90%±6.34%,38.95%±7.78%,31.86%±1.06%,13.52%±1.66%, respectively(compared with the physiological concentration, p<0.05).While the late apoptotic rates were59.19%±5.86%,75.45%±7.39%,4.95%±1.26%, at50、100nmol/L and physiological concentration, respectively(compared with the physiological concentration, p<0.05).Besides, the total apoptotic rates were67.04%±5.93%,84.66%±6.40%,13.52%±1.66%, respectively(compared with the physiological concentration, p<0.05).(3) Hoechst33258(OPG treated or untreated):The apoptosis rates of HUVECs were49.27%±4.80%,36.11%±2.57%,18.43%±4.91%,35.91%±1.86%, at control group、low-concentration group. physiological-concentration group. high-concentration group, respectively; After incubation with OPG, apoptosis rates of HUVECs are24.47%±2.16%,15.97%±0.87%,16.16%±2.33%,24.05%±3.04%, respectively. The P value of comparisons between OPG pretreatment and without treatment in control group, low-concentration group and high-concentration group were0.044,0.040,0.043, respectively. There are no difference between physiological concentration group and physiological concentration group pretreated with OPG (P=0.591).(4)Flow cytometry(OPG treated or untreated):The early apoptotic rates were35.33%±2.51%,28.63%±0.68%,27.09%±3.29%, at control group, low-concentration group, high-concentration group, respectivelyAfter incubation with OPG, the early apoptotic rates were19.51%±1.33%,11.05%±4.00%,18.40%±7.67%, respectively.The P value of comparisons between OPG pretreatment and without treatment in control group, low-concentration group and high-concentration group were0.035,0.048,0.019.Besides, the total apoptotic rates were47.97%±5.11%,39.79%±0.88%,35.14%±2.58%, at control group, low-concentration group, high-concentration group, respectively.After incubation with OPG, the total apoptotic rates were30.68%±1.97%,19.30%±3.63%,24.64%±8.78%.The P value of comparisons between OPG pretreatment and without treatment in control group, low-concentration group and high-concentration group were0.048,0.028,0.045, respectively. The early and total apoptosis in physiological concentration group were12.80%±2.62%,16.55%±2.36%, respectively.After incubation with OPG (100ng/ml), the early and total apoptosis in physiological concentration group were11.98%±2.71%,17.97%±1.55%, respectively. There are no difference between physiological concentration group and physiological concentration group pretreated with OPG in the early and total apoptosis(P=0.885, P=0.206, respectively).4. IKKβ/mTOR pathway:The expression of p-IKKβ、p-TSCl、p-S6K、Bax in control group, low-concentration group and high-concentration group were higher than those in physiological concentration group, however, the expression of p-AKT. Bcl-2in the group above were lower than those in physiological concentration group (all,p<0.05).(3) After OPG pretreatment and being cultured in control group, low-concentration group and high-concentration group, the expression of p-IKK β、p-TSCl、p-S6K. Bax decresed significantly (all,p<0.05) as compared with groups without treatment and the expression of p-AKT, Bcl-2increased significantly (all,p<0.05).There are no difference in the expression of p-IKKβ、p-S6K、 p-TSCl、p-AKT、Bcl-2、Bax between physiological concentration group and physiological concentration group pretreated with OPG. After incubation with rapamycin (10ng/ml) or BMS345541(20umol/L), the expression of p-AKT、Bcl-2in low-concentration group and high-concentration group were higher than those without pretreatment (all,p<0.05).Besides, the expression of Bax were lower than those without pretreatment (all,p<0.05).Conclusions1. T3has the best protective effects in endothelial cells at physiological concentration.2. Osteoprotegerin protects HUVECs from apoptosis in low and high concentration of thyroxine.3. The mechanism of OPG in protecting the HUVECs from apoptosis maybe associated with inhibition of IKK β/mTOR signaling pathway. |
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