Effect Of Orbital Adipose IGF-1/IGF-1R On The Thyroid-associated Ophthalmopathy And Mechanism Study

Background:With the change of social environment,psychological state and living habits,the incidence of thyroid-associated ophthalmopathy shows a gradual upward trend.It is also known as Graves' orbitopathy or thyroid eye disease,including Graves' ophthalmopathy(GO),which is an autoimmune disorder of the orbit.There are about annual 37,000 new cases in United States,and it is estimated to get prevalence rates of 16 per 100,000 in women and 2.9 per 100,000 in men.TAO is a type of complicated disease which involve the endocrine system disorder and immune system imbalance.To data specific pathogenesis remains elusive and the treatment means are also deficient.The clinical features of TAO mainly contain eyes proptosis,eyelid swelling,upper eyelid late fall and multiple bilateral injury than bilateral damage.Pathologically increased adipose tissue in orbit and thicken in extrinsic eyeball muscles are involved in the development of TAO.Particularly the increased adipose tissue plays a role in the disease.So far the primary autoantigens and exact mechanisms of thyroid-associated ophthalmopathy remain unclear.Though TSHR(TSH receptor)was reported to be likely the main autoantigen in the recent twenty years,we could not be sure how many kinds of autoantigens are participating in the process of TAO.In the early studies,it has been found that the antigen-antibody complexes of TSHR can effective combine with the IGF-1R receptor,which may be involved in the disease process.In recent years,Shanli Tsui and his mates proposed the TSHR/IGF-1R composite community theory by experiment.They found the co-existing of IGF-1R subunit with TSHR in orbital fibroblast by precipitation method.In the experiment IGF-1R antagonistic agent can obviously inhibit the signaling way of GD-Ig G.In addition,the animal experiment also confirmed the critical role of IGF-1R in the pathogenesis of TAO.IGF-1 as IGF-1R ligands is important for tissue growth and metabolism,which can promote the synthesis of hyaluronic and mucopolysaccharides in orbital fibroblast,moreover there is a direct relationship between glycosaminoglycan deposition and pathological development of GO.Apart from liver IGF-1 is also generated by other tissues.For example,IGF-1 was reported to increase significantly in the adipose tissue of IGF-1R knockout mice,suggesting a probable secretion from other tissues.Besides,some clinical studies have revealed that the level of serum IGF-1 was significantly higher in thethyroid-related eye disease group than in the control group.It is so interesting that autocrine of IGF-1 was happened in orbital fibroblasts of Graves' ophthalmopathy in Delu experiment.All of the above indicates that IGF-1R and IGF-1 ligand maybe play a role in the pathogenesis of TAO.Since IGF-1 was discovered in 1957,it was gradually confirmed to be related with the proliferation and secretory function of the most human tissue.As a family resemblance with the insulin protein,IGF-1 has some similar biological effect as insulin in vivo.It was showed that insulin can promote adipocyte to secrete plasminogen activator inhibitor-1(PAI-1).PAI-1 is a type of important cytokine which regulates balance between the body's tissue-type plasminogen activator(t-PA)and urokinase-type plasminogen activator(u-PA)metabolic,then subsequently affects collagen metabolic in the extracellular matrix.PAI-1has been confirmed to be connected with the fibrosis of lung,liver,skin and other tissue.After analyzing tears from the patient,it was also founded that PAI-1 protein is more in GO group than in GD group significantly.Results from other studies have shown that the PAI-1 was positively correlated with the disease activity score in GO patients.Although the specific mechanisms is still unclear,it can give us hints that GO orbital tissues maybe have abnormal secretion of PAI-1.Except immunizing autoantigen,the pathogenesis of TAO is also related with other disorder or abnormal physiological mechanisms which could not be ignored.As an important internal balancing mechanism,autophagy is a genetically programmed behavior in tissue growth and metabolism.The special self-protection played an important role in the tissue growth.Under physiological conditions,autophagy sustains the cell's own metabolic balance through recycling organelle and proteins and provides the necessary raw materials for repair and regeneration of organelles in pathological cases.Autophagy is involved in many diseases including some autoimmune diseases.It was shown that Atg5 which is one of autophagy protein was over expressed abnormally in the organization of demyelination and multiple sclerosis disease.It was also found that autophagy took part in the process of adipocyte differentiation,and more "autophagy body" could be detected by electron microscopy in mouse embryonic fibroblasts during differentiation.IGF-1 as an important cytokine in vivo joins in the regulation of autophagy in some disease.Nevertheless,the connection between autophagy and thyroid associated ophthalmopathy is unclear.Under above background conditions,our research is aimed to make basic explorebased on basic experiments of adipose tissue and cells.In the part one we mainly detected the expression of IGF-1R protein and RNA in the orbital adipose tissue to investigate whether there is a significant difference between the TAO patients and control group.After extracting,culturing and identifying primary preadipocyte cells from orbital adipose tissue we investigated the effect of IGF-1 on cells proliferation and differentiation.We also had observed the changes of IGF-1R in the process of cells differentiation and IGF-1 autocrine situation.In the part two we inspected the PAI-1 expression and distribution in the orbital adipose tissue of TAO patients and explored role and mechanics of IGF-1 in preadipocytes differentiation concerning PAI-1.Finally in the part three we researched the expression of LC3 protein in orbital adipose tissue from TAO patients group and control group,and then made a comparison.Further we performed an in-depth exploration in regard to how IGF-1affects autophagy in differentiation of orbital preadipocytes.Methods:1.Western blot and RT-PCR were used to test the expression of IGF-1R,PAI-1,and LC3 in orbital adipose tissue samples from TAO patients and control groups.2.Immunohistochemical techniques were applied to analyze IGF-1R,PAI-1 protein localization in TAO patients and control groups orbital adipose tissue through frozen sections and semi-quantitative detection.3.Primary adipocytes were extracted and cultured from orbital adipose tissue.Adipocytes were identified using pref-1 specific antibodies by flow cytometer.4.CCK8 method was used to detect the ability of preadipocytes proliferation and regeneration under varied concentrations of IGF-1(0n M,1n M,5n M,10 n M,20 n M).5.Western blot technique was utilized to detect the changing of LC3,Atg5 and IGF-1R protein in different stages of differentiation of preadipocytes.Simultaneously m RNA of PAI-1,PPAR?and IGF-1 was measured by RT-PCR.6.IGF-1(C0n M,C1 n M,C5 n M,C10 n M,C20 n M)was add in preadipocytes of TAO group with different concentrations(C0n M,C1 n M,C5 n M,C10 n M,C20 n M)during the cells differentiation and then to test the expression of LC3,Atg5 protein,PAI-1,PPAR?.Using oil red staining we observed the degree of fat.7.After add with LY294002(PI3k inhibitor)group and MK2206(AKT inhibitor)we observed the variation of LC3,Atg5,PAI-1and PPAR? in preadipocytes of TAO by western blot and RT-PCR.Results:1.The Western Blot and immunohistochemistry shown that the expressions of IGF-1R,PAI-1 were increased;but LC3 decreased.2.There is no significant difference in morphology and proliferative capacity of the preadipocytes between TAO group and the control group.3.IGF-1 can promote the proliferation of preadipocytes in TAO patients group obviously.4.IGF-1R is over expressed in the process of preadipocytes differentiation from TAO patients.5.The expression level of LC3,Atg5,PAI-1and PPAR? are not significantly different in preadipocytes between TAO Group and control group but the level of IGF-1 m RNA is higher in TAO group;6.IGF-1 inhibits expression of LC3,Atg5 in differentiation of preadipocytes in vitro,and promotes expression of PAI-1 and PPAR?.7.IGF-1 regulates LC3,Atg5,PAI-1 and PPAR? via PI3K/AKT signaling pathway during the process of preadipocytes differentiation.Conclusion:1.IGF-1R was over expressed in orbital adipose tissue and adipocyte in TAO patients,IGF-1 can promote preadipocytes proliferation and differentiation.2.The autocrine of IGF-1 is increased significantly in orbital preadipocytes during differentiation.3.The level of autophagy is obviously increased during the early differentiation stage in vitro but decreased in orbital adipose tissue of TAO patients.4.The orbital adipose tissues express more PAI-1 and IGF-1 that can accelerate the synthesis of PAI-1.5.IGF-1 can regulate expression of LC3,Atg5,and PAI-1 via PI3K/AKT signaling pathway during preadipocytes differentiation.