The Observational And Mechanism Study On The Relationship Between TSH And Atherosclerosis

Background:Subclinical hypothyroidism(SCH)refers to an endocrine disorder with no obvious symptoms or only mild hypothyroidism symptoms,which characterized by the increased thyroid stimulating hormone(TSH),but normal serum free triiodothyronine(FT3),free thionine(FT4)level.SCH is a common thyroid disease,of which the prevalence is getting higher and higher,and its incidence gradually increased with age.The United States Third National health shows that compared with those in younger people(20-29 years old),the serum TSH level is increased by about 40%in the older people(60-69 years old).The phenomenon that SCH is often accompanied by atherosclerosis has become a hot topic in recent years.SCH may be an independent risk factor for coronary heart disease,such as hypercholesterolemia,hypertension,smoking,and diabetes et al SCH is also associated with an increased risk of congestive heart failure among older adults.A large cross-sectional study in Rotterdam,the Netherlands,showed an increased prevalence of myocardial infarction among SCH women older than 55 years.The Whickham Survey which examined the association between thyroid dysfunction and cardiovascular disease in an unselected community-based sample also proved this idea.Recent years many meta-analysis studies supported these conclusions from the perspective of evidence-based medicine.A meta-analysis from Taiwan,and also another one based on previous studies of the 11 countries in the America,Europe,Asia and other data showed that SCH was associated with coronary heart disease incidence and mortality,especially in the group of TSH higher than 10 mIU/L.Atherosclerosis(AS)is the initial cause of cardiovascular disease and a chronic disease,characterized by the formation of plaque on the arterial wall.Atherosclerosis lesions form mainly in the following three processes:first of all,endothelial cell injury,smooth muscle cell proliferation,migration and infiltration of macrophages,lymphocyte infiltration,and thus inflammation and chemokines are released.Second,collagen fibers and elastin are secreted by smooth muscle cells,to form the connective tissue.Third,the lipid(including both modified and free lipoproteins)accumulates in the matrix and foam cells.At present,there are many hypotheses about the mechanism of atherosclerosis,such as inflammation,lipid oxidative stress,and immune response.However,to date there is still no conclusive evidence that any process plays a decisive role in the formation of porridge.Chronic inflammation hypothesis is now one of the more accepted hypotheses of atherosclerosis.The development of atherosclerosis develops with pathological processes characterized by chronic inflammation.Traditionally,hypothyroidism and SCH associated with atherosclerosis is due to the role of thyroid hormones.In recent years,our group found that TSH,independent of thyroid hormone,has some of the non-thyroid target organs and important roles.TSH acts as a pituitary hormone,and works on the TSH receptor(TSHR).Traditionally,TSH acts on thyroid TSHR and plays a role in promoting thyroid secretion of thyroid hormones.In addition,TSHR has been found in many other cells and tissues distributed in the body except the thyroid.TSH could play an important role besides thyroid by binding these TSHRs.In the previous study,our group also confirmed that TSH played an important role in liver tissue and adipose tissue.Based on the above theory,we propose a hypothesis that TSH may be an independent risk factor for atherosclerosis.TSH may play a direct role in affecting the development of atherosclerosis by acting on TSHR in porcine-related cells or tissues.The impact of TSH on porridge may be related to the inflammatory response.This study intends to discover the problems from clinical studies,animal experiments,which explore the mechanism of two aspects to explain the impact of TSH on atherosclerosisResearch Aims:1.To observe the correlation between serum TSH level and atherosclerosis index2.To study the association between inflammatory cytokines and TSH level.3.To observe the role of TSH in the development of atherosclerosis.4.To explore the mechanism of TSH effects on atherosclerosis.Research Methods:Clinical research1.Population selection and data acquisition:The population came from the 2011-2012 National Metabolic Disease Epidemiology Survey.In 2011,a total of 2480 people were collected in Ningyang County,Shandong Province.After a rigorous criteria screening,a total of 1103 people was included into our study for cross-sectional analysis.General information was recorded through the questionnaire,including gender,age,past disease history,medication history,smoking and drinking history.Through the examination,blood pressure,weight,height,waist circumference,hip circumference and other information were asssessed.2.Serum thyroid function and biochemical indicators:Fasting blood was collected and centrifuged to obtain serum.The blood lipids,blood glucose and other indicators were determined by biochemical methods.Thyroid hormone levels(FT3,FT4,TSH)were determined by chemical luminescence.These are all done by Shandong Provincial Hospital.3.Carotid artery ultrasound measurement of carotid intima-media thickness:All patients underwent carotid intima-media thickness(CIMT)measurements.Carotid intima-media thickness diagnosis was referred to China's guidelines for prevention and treatment of hypertension(2010).Carotid ultrasound was performed by the Department of Ultrasound,Shandong Provincial Hospital.4.Luminex multi-factor kit for detecting serum inflammatory cytokines:The inflammatory cytokines in human serum were assessed using Elisa kit and RD's Luminex Multi-Factor kit(TNF-?,IL-6,IL1,IL-10,IL-12,CD40L,CX3CL1,CCL2,GM-CSF).5.Statistical analysis:Continuous variables are expressed as mean ± standard deviation.Multiple groups were compared using one-way ANOVA analysis.Multivariate linear regression was used to test the effect of various factors on CIMT.All data were analyzed using SPSS 18.0 software,the definition of P<0.05 as a significant difference.Mechanism research1.Knockout mice:Eight-week-old male ApoE-/-mice were purchased from Beijing Weitonglihua Company and Tshr+/-mice were purchased from Jackson Lab,USA.ApoE-/-mice were crossed with Tshr+/-mice.Tshr-/-ApoE-/-double knockout mice and littermate Tshr+/+ ApoE-/-were selected as control group.2.Atherosclerosis model:The double knockout and control mice were fed with Western high-fat diet at 6 weeks of age.After high-fat diet feeding for 12w,16w,mice were sacrificed after high-fat feeding and samples were taken.3.Tissue sample collection:After high fat diet for 12w,16w,sacrifice mice and take the whole blood,centrifuge to obtain serum.Take the full length aorta.The aortic root is fixed directly in the glue,or fixed in formalin,for preparing frozen sections or paraffin sections.4.Histological staining and immunohistochemical staining:Aortic root tissue is embedded,then frozen or paraffin sections are prepared.Aortic root sections were sectioned,pathological staining,HE staining,oil red O staining were done.Immunohistochemical staining:smooth muscle marker ?-SMA,macrophage marker F4/80,Sirius red staining to evaluate the collagen,the analysis of plaque components.5.Blood lipids and thyroid function level determination:The serum of mice was measured by chemical luminescence(TC,TG,LDL-c,HDL-c).Serum thyrotropin(TSH)was measured by Elisa method and thyroid hormones(T3,T4)were measured by radioimmunoassay.6.Mouse serum inflammatory cytokines:The levels of inflammatory cytokines IL-6 and TNF-? in mice serum were determined by Elisa method.7.Inflammatory cytokines(IL-6,TNF-?)expression in RNA levels.Mouse vascular inflammation factor gene level:Mouse aorta RNA extraction,then reverse transcribed to cDNA,and do PCR to detect changes in vascular levels of inflammatory cytokines.8.Mouse primarymacrophage:Macrophage primary cells were obtained from the abdominal cavity of mice and cultured in vitro.9.Mouse primary macrophage experiment:Macrophages were cultured in vitro,treated with TSH,and the changes of inflammatory cytokines in medium were observed by Elisa.10.Statistical analysis:Quantitative data are expressed as mean ± standard deviation.Data were analyzed using SPSS,independent sample t-test was used for comparison between the two groups,and one-way ANOVA test was used for multiple groups.P<0.05 was defined as statistically significant.Results:1.TSH is an independent risk factor for atherosclerosisA total of 1103 people were included in the final analysis,including normal thyroid function group of 787 people,279 mild hypothyroidism group,37 severe hypothyroidism group.Carotid intima-media thickness(CIMT)increased with TSH increased,according to TSH levels.Compared with the control group,mild SCH and severe SCH were statistically different(P<0.001).The incidence of plaque and the number of plaques in each group increased with increasing TSH.Compared with the control group,mild SCH and severe SCH were significantly different(P<0.01).Multivariate linear regression models showed that TSH,along with other recognized risk factors for atherosclerosis,such as blood lipids and blood pressure,entered the multivariate linear regression equation and was an independent influencing factor for CIMT.2.Serum AS-related inflammatory cytokines increased with elevated TSHA series of inflammatory factors related to atherosclerosis were determined in some populations.TNF-? was found increased with TSH level,the level was higher in mild SCH and severe SCH group,than in control group(P<0.001).In addition,serum.CCL2 level,comparing with control group,increased in mild SCH group(P<0.01),severe SCH group also significantly increased(P<0.05).Serum GM-CSF,comparing with control group,increased in mild SCH group(P<0.01).3.Tshr knockdown attenuated the occurrence of AS in ApoE-/-miceCompared with control group(Tshr+/+ ApoE-/-),there was no significant change in blood lipids(TC,TG,LDL-c,HDL-c)in double knock mice group(Tshr-/-ApoE-/-).There was also no significant difference in thyroid function(T3,T4,TSH)between the two groups of mice.Gross Red-Oil staining showed a decrease in atherosclerotic plaque formation throughout the aorta in double knock out(Tshr-/-ApoE-/-mice,compared to control(Tshr+/-ApoE-/-).In high-fat diet 12w and 16w,they both showed significantly difference.Oil red O staining of the aortic root showed that in the Tshr-/-ApoE-/-mice,the aortic root plaque was reduced,compared with the control group.And 16 weeks had also the same significant difference.F4/80 staining of macrophages in plaques showed a decrease in macrophages in the double knock group(Tshr-/-ApoE-/-)plaques,as compared to the control group(Tshr+/+ ApoE-/-).4.Tshr knockdown attenuated the inflammatory response in ApoE-/-miceInflammatory cytokines in serum of mice:compared with Tshr+/+ ApoE-/-mice,Tshr-/-ApoE-/-mice showed decreased serum inflammatory cytokines(IL-6,P<0.01;TNF-?,P<0.05).RNA expression level of mouse vascular tissue:The expression of a series of inflammatory cytokines in the blood vessels of double knock group(Tshr-/-ApoE-/-)mice decreased compared with the control group(Tshr+/+ApoE-/-).The levels of a series of factor all reduced.(F4/80,P<0.05;IL-1?,P<0.05;IL-6,P<0.05;TNF-?,P<0.05;Ccl2,P<0.01).The expression of inflammatory cytokines in vascular plaque of the mice:Immunohistochemistry staining showed that,compared with the control group,IL-6 and TNF-? were decreased in Tshr-/-ApoE-/-mice(IL-6,P<0.05;TNF-?,P<0.01).The above results showed that the inflammatory response in the serum,tissue and blood vessel of double knock group(Tshr-/-ApoE-/-)mice were alleviated,which may be the reason of porridge reduction in porcine knockout mice.5.TSH can promote the primary macrophage inflammatory reaction.Primary macrophages were obtained and treated with TSH.TSH was found to promote the secretion of IL-6 and TNF-?,the key inflammatory factors of primary macrophages.And in a dose-dependent manner,0.1 ?mol,0.4?mol,1 ?mol of TSH treatment,inflammatory cytokines IL-6,TNF-? secretion gradually increased.These inferred TSH might directly promote cell inflammatory response.Conclusion:1.TSH is an independent risk factor for atherosclerosis.2.Serum inflammation factors increased with TSH increased.3.TSH can directly promote the occurrence of atherosclerosis.4.The mechanism may be related to TSH pro-inflammatory effect.