| Background:Thyroid associated ophthalmopathy(TAO)is an organ-specific autoimmune disease with the highest prevalence of orbital disease in adults,mainly manifesting as unilateral or bilateral proptosis,eyelid recession,diplopia,and exposed corneal ulcers,and in severe cases,compressive optic neuropathy,leading to vision loss.Proptosis is the most common and most difficult symptom to relieve in TAO.Imaging and basic research further suggest that the increase of intraorbital adipose tissue makes the most significant contribution to this pathological change in proptosis.Adipogenesis is an extremely complex process,and adipose mesenchymal stem cells(Ad-MSCs)are the primitive progenitor cells in the process of adipogenesis,with high self-replication capacity and differentiation potential.It has been shown that dysregulation of adipose MSC recruitment and differentiation,as well as the inflammatory environment in adipose tissue,can contribute to abnormal adipogenesis.Inflammation is,in turn,an important factor mediating the pathogenesis of TAO,and interleukin-4(IL-4)is considered to be one of the major effector cell molecules involved in the pathogenesis of TAO.Therefore,we hypothesized that IL-4 may also be involved in the pathological process of orbital adipose tissue infiltration in TAO.Therefore,we hypothesize that inflammatory factors in the orbital tissue of TAO patients may affect orbital fat mesenchymal stem cells,thereby inducing their differentiation and ultimately leading to increased orbital fat formation in TAO.Purpose:To investigate the changes in lipogenic function of orbital fat mesenchymal stem cells in TAO and the effect of inflammatory factors on the regulation of lipogenic function of orbital fat mesenchymal stem cells to explore a new potential strategy for the treatment of TAO.Menthods:1.Culture and identification of orbital adipose mesenchymal stem cells from thyroid-associated ophthalmopathy(TAO)in vitroOrbital adipose tissue obtained from TAO orbital decompression and eyelid cosmetic surgery was extracted and cultured with primary orbital Ad-MSCs by digestion enzyme method and tissue block method.flow cytometry was used to examine the expression of markers on the surface of the primary cells,and the ability of the primary cells to differentiate by three lineages was verified by conditional induction.2.To investigate the lipogenic differentiation function of TAO orbital adipose mesenchymal stem cellsRT-PCR was performed to detect the expression of lipogenesis-related genes in orbital Ad-MSCs of TAO group and control group under the conditions of lipogenesis factor stimulation.Quantification of lipid droplet formation in orbital Ad-MSCs of TAO and control groups under the stimulation of lipogenic factors by oil red staining.3.To investigate the effects of disease-associated inflammatory factors IL-4,IL-1β and IL-17 on lipogenic differentiation of TAO orbital adipose stem cellsWe stimulated orbital Ad-MSCs with IL-4,IL-1β and IL-17 alone,and examined the expression of lipogenesis-related genes C/EBPα,FABP4 and ADIPOQ in orbital Ad-MSCs of TAO group and control group.Next,the orbital Ad-MSCs were stimulated with IL-4,IL-1β,and IL-17 in combination to detect any synergistic effect of the three inflammatory factors.4.The mechanism of IL-4 promoting lipogenic differentiation of TAO orbital Ad-MSCs through upregulation of FABP4The expression of FABP4 and lipogenic key protein PPAR-γ in TAO orbital adipose MSCs after IL-4 stimulation was examined.The expression of PPAR-γ was detected by knockdown and overexpression of FABP4 gene.The formation of lipid droplets in TAO orbital Ad-MSCs after IL-4 stimulation was also detected by oil red staining assay.Results:1.TAO and normal human orbital Ad-MSCs positively expressed CD29,CD44,CD73,CD90,and CD105,and negatively expressed CD34 and CD45.under differentiation induction culture conditions,they had the ability to tri-lineage differentiation.2.RT-PCR results showed that the expression of lipogenesis-related genes C/EBPα,FABP4,and ADIPOQ was increased in orbital Ad-MSCs of TAO patients compared with normal orbital Ad-MSCs under lipogenic factor-stimulated conditions.It was also found that under the condition of lipogenic factor stimulation,lipid droplet formation was increased in orbital Ad-MSCs of TAO patients compared with the normal group.This demonstrates that the orbital Ad-MSCs lipogenic capacity is abnormally enhanced under TAO disease conditions.3.RT-PCR results showed that the expression of lipogenesis-related genes was significantly increased in TAO orbital Ad-MSCs after stimulation with IL-4,IL-1β,and IL-17.combined stimulation with IL-17 had a synergistic effect on the expression of lipogenesis-related genes C/EBPα and ADIPOQ in TAO orbital Ad-MSCs,and the expression of IL-4,IL-1β,and This synergistic effect was further amplified when the three inflammatory factors IL-4,IL-1β and IL-17 were stimulated simultaneously,and the difference was significant compared with normal subjects.4.Western blotting showed that the lipogenesis-associated protein FABP4 and PPAR-γ,a key protein for lipogenic differentiation,were elevated in orbital Ad-MSCs after IL-4stimulation.The expression of PPAR-γ decreased and increased after knockdown and overexpression of FABP4 gene.Oil red staining showed that IL-4 promoted the formation of lipid droplets in TAO orbital Ad-MSCs.Conclusions:We successfully cultured and amplified Ad-MSCs and verified their surface markers and trilineage differentiation ability.We found that the lipogenic ability of TAO orbital Ad-MSCs was significantly increased compared with normal subjects,and the inflammatory factors IL-4,IL-1β and IL-17 were found to promote the expression of lipogenic genes in TAO orbital Ad-MSCs,and there were synergistic effects among the three inflammatory factors.IL-4 was also found to promote the expression of PPAR-γ,a key protein for lipogenic differentiation,through upregulation of FABP4,thereby inducing lipogenic differentiation in TAO orbital Ad-MSCs. |
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