The Role And Mechanism Of Keratinocyte Lipid Metabolism Dysfunction In The Occurrence And Development Of Obesity-related Atopic Dermatitis

Background:Atopic dermatitis(AD)is one of the most common inflammatory skin diseases around the world,clinically characterized by recurrent eczema-like dermatitis with severe pruritus.In the setting of genetic predisposition,lifestyle changes due to excess calorie intake has led to a dramatic increase in obese population worldwide,which further boosts the growing prevalence of AD.Research on obesity as an independent risk factor for AD has been widely concerned.Epidemiological studies have shown that patients with obesity have a higher prevalence of AD,more severe clinical symptoms and more difficult to treat.But the specific mechanism of AD induced or aggravated by obesity has not been fully elucidated,therefore,the prevention and treatment of obesity-related AD is still lack of theoretical guidance.Obesity is a generalized,multi-system lipid metabolic disorder.In obese state,lipid metabolic imbalance in intestinal epithelial cell,hepatocytes,cardiomyocytes,podocytes and so on could induce endoplasmic reticulum stress,inflammasome production and the ensuing inflammatory signaling pathway activation,which contribute to obesity-induced injury.Keratinocytes are the main cells that initiate AD by secreting inflammatory mediators and inducing a T helper type 2(Th2)inflammatory response.However,whether a lipid metabolic imbalance has happened on keratinocytes in the obese condition is not clear.The access accumulation of essential fatty acid including palmitic acid,palmitoleic acid,and oleic acid were confirmed on the skin of high-fat dietary-induced obese mice,which suggested that keratinocyte is exposed to a high-lipid environment.Based on results,the content of fatty acid in the keratinocyte of obese mice was dramatically increased,and multiple classic molecules of lipid metabolism were highly expressed in obese skin.However,whether the abnormal lipid metabolism of keratinocyte contributes to AD onset and aggravation remains to be further explored.Objective:To confirm the abnormal lipid metabolism of keratinocyte in obese state,and to explore the mechanism of lipid metabolism in keratinocyte in inducing and aggravating AD inflammation.Method:1.Study on the effect of obesity on AD-like inflammation in mouse model: The obese AD-like mouse model was established and the inflammatory changes in the ear tissue of mice were evaluated by recording the scratching behavior of mice,measuring ear thickness,and performing hematoxylin-eosin(H&E)staining.Changes in inflammatory molecules in the peripheral blood circulation of the model were detected by enzyme linked immunosorbent assay(ELISA),and infiltrating T-cell subsets in spleen tissue were detected by flow cytometry technology.The expression of inflammatory molecules in the skin lesions were detected using real-time quantitative PCR(q RT-PCR).2.Analysis on the lipid metabolism of keratinocytes:Skin samples were collected from obese patients,AD patients,and healthy controls.(1)BODIPY500/510 fatty acid staining coupled with immunofluorescence technique was used to detect the fatty acid accumulation on the skin surface.(2)BODIPY500/510 fatty acid staining combined with flow cytometry technique was used to detect fatty acid content in keratinocytes of obese AD mice.(3)Data from the public database were used to analyze the expression of classic molecules of fatty acid metabolism in the skin of AD patients.(4)The expression of the target molecules in the skin of obese patients and obese mice was detected by immunofluorescence technique.3.Study on key molecules of lipid metabolic imbalance in obesity-associated AD inflammation.In this part,we try to confirm the role of target molecules by topically applying inhibitors on the mouse skin.(1)QRT-PCR and immunofluorescence techniques were used to determine the inhibitory effect of topical inhibitors on lipid accumulation.(2)BODIPY500/510 staining combined with flow cytometry technique was used to detect the inhibitory effect of the inhibitor on the content of fatty acids in mouse epidermal cells.(3)The scratching behavior of mice was recorded by a camera.The ear thickness was measured,and H&E staining was used to evaluate the inflammatory infiltration of mice ears.Finally,q RT-PCR and ELISA were used to evaluate the level of inflammatory molecules after the inhibitor application.4.Exploration on the mechanism underlying the imbalanced lipid mechanism from obesity on AD inflammation:(1)Western Blot was used to detect the expression of target molecules after palmitate acid(PA)stimulation.(2)The expression of target genes was downregulated by its inhibitors and small interfering RNA(si RNA)and further analyzed by Western Blot technique.(3)Chromatin immunoprecipitation(Ch IP)coupled with luciferase reporter assays were used to verify the transcriptional activity of the targeted gene.Results:1.AD-like inflammation was aggravated in obese mice: The results showed that the AD-like inflammation induced by calcipotriene(MC903)was aggravated in Obese-MC903 mice as compared with Lean-MC903 mice.(1)Compared with Lean-MC903 mice,the inflammation in Obese-MC903 mice was significantly aggravated,manifested as more obvious redness and scaling in the ears;significant increase in epidermal thickness and infiltration of inflammatory cells;(2)ELISA results showed that thymic stromal lymphopoietin(TSLP)and total Ig E levels in the peripheral blood of Obese-MC903 mice were significantly higher.QRT-PCR results showed that the expression levels of inflammatory molecules Tslp,Il-1β,Il-4,Il-5,and Il-13 were higher in Obese-MC903 mice.2.The imbalanced lipid metabolism in keratinocytes of obese mice was revealed:(1)Compared with Lean group mice,a significant increase in fatty acid content was found in the epidermis of obese mice(Obese-Control,Obese-MC903)and MC903 induced AD mice(Lean-MC903,Obese-MC903).(2)Immunofluorescence and q RT-PCR results showed that both Lean-MC903,Obese-Control and Obese-MC903 had high expression of CD36,a classical fatty acid transporter.(3)The high expression of CD36,SREBP1 and their downstream molecules were discovered in the lesions of AD patients by data accessed form NCBI database.The results suggested that the enhanced CD36-SREBP1 signaling is not only associated with obesity status,but also may be involved in the inflammatory process of AD.3.CD36 is a key molecule responsible for the dysfunction of lipid metabolism by obesity:(1)We explored the optimal concentration of salvianolic acid B(SAB)for inhibiting inflammation in obese AD mice;(2)QRT-PCR and immunofluorescence showed that SAB could significantly inhibit the expression of CD36 in mouse skin.(3)The results of BODIPY500/510 combined with flow cytometry showed reduced fatty acid content in epidermal cells after SAB treatment.(4)The inflammation of Lean-MC903,Obese-Control,Obese-MC903 group mice were significantly alleviated by SAB.The ear swelling and rear thickness was reduced.Scratching counts was significantly decreased.QRT-PCR results presented that the expression of Tslp,Il-1β,Il-4,Il-5 and Il-13 was significantly decreased in the skin of SAB treated mice.These results suggest that inhibition of CD36 could inhibit MC903-induced and obesity-aggravated inflammation.4.CD36-SREBP1 axis mediates obesity-associated AD inflammation.(1)QRT-PCR results showed that Fatostatin could significantly inhibit the expression of Srebp1 and its downstream molecules in mouse ear.(2)Both SAB and Fatostatin treatment could inhibit SREBP1 activation;(3)Both SAB and Fatostatin treatment could alleviate AD-like inflammation: ELISA results showed that the levels of TSLP and total Ig E in peripheral blood of MC903 plus Fatostatin group were significantly lower than those of MC903 group.QRT-PCR results showed that the expression of Tslp,Il-1β,Il-4,Il-5 and Il-13 were significantly decreased in the tissue of MC903 plus Fatostatin group.These results suggest that inhibition of SREBP1 expression and maturation could significantly reduce AD-like inflammation.5.CD36-SREBP1 signaling enhanced TSLP expression in keratinocytes.(1)QRT-PCR results showed that IL-4 stimulation could promote the expression of CD36 in keratinocytes.Immunofluorescence results revealed that IL-4 could promote the uptake of palmitate(PA)in keratinocytes,which was reduced by inhibition of CD36 by SAB.(2)Western Blot results showed that PA increased the expression of CD36 and TSLP in a concentration-dependent manner.After treated with si RNA、SAB and Fatostatin,the expression of activated SREBP1 and TSLP was significantly decreased.SREBP1 overexpression could dramatically elevate the expression of TSLP.(3)Ch IP results showed that SREBP1 antibody could pull down the DNA sequence of the second and fifth segments of the TSLP promoter region.Luciferase Report assay revealed that the effect of SREBP1 on TSLP gene expression was significantly inhibited when the fifth segments of TSLP promoter were mutated.These results suggested that CD36-SREBP1 signaling could promote the expression of TSLP in keratinocytes,which may be the key mechanism mediating the obesity-related AD inflammation.Conclusion:This study focuses on the major mechanism of obesity-induced AD inflammation.The imbalance of lipid metabolism mediated by CD36-SREBP1 signaling was found in both obese state and AD inflammation.CD36-SREBP1 is involved in AD and obesityaggravated AD inflammation by promoting TSLP expression in keratinocytes.This study elucidates the vital role of keratinocyte lipid metabolism in AD inflammation induced by obesity and provides a new theoretical basis for obesity aggravated AD inflammation.CD36 and its downstream molecule SREBP1 may be potential therapeutic targets for obesityassociated AD,providing a new individual strategy for AD treatment.