Study On The Mechanism Of IL-8 Regulated Expression Of Matrix Remodeling Proteins In Corneal Cells Under Mechanical Stretching

Keratoconus is a corneal ectasia disease,which is mainly characterized by progressive thinning of the center of the cornea and protruding forward in a conical shape.As a typical load-bearing tissue,the change of the mechanical environment of the cornea is one of the important factors for keratoconus.The composition and structure of the extracellular matrix of the cornea are closely related to its biomechanical properties.Under pathological conditions,the amount of collagen in the corneal stroma layer is reduced,and the content of proteoglycans is changed.The disordered collagen arrangement as well as the reduced thickness of the cornea and its anti-deformation ability result in keratoconus.Keratoconus is generally considered to be a non-inflammatory ophthalmic disease,but more and more studies have found that the expression of pro-inflammatory factors in the tears of keratoconus patients is significantly higher than that of the normal population,indicating that the inflammatory signal in the keratoconus is activated and the keratoconus may be a chronic inflammatory corneal disease.Interleukin 8（IL-8）,also known as chemokine CXCL8,can promote the chemotaxis of neutrophils and the expression of adhesion molecules,regulating cell migration,cell adhesion,angiogenesis and other physiological processes.It participates in keratitis,corneal ulcers,corneal scars and other eye diseases.Studies have shown that IL-8 is highly expressed in the tears of patients with keratoconus,and is strongly positively correlated with the Pentacam parameter corneal dilatation comprehensive deviation analysis index BAD-D.It is speculated that IL-8 plays an important role in the process of keratoconus.However,its function in keratoconus is still unclear.In this study,the influence of mechanical stimulation on the expression of IL-8 in corneal fibroblasts and its regulation mechanism were analyzed by mechanically stretching and drug treatment of corneal fibroblasts in vitro.Furthermore,the expression of IL-8 in corneal fibroblasts was regulated by si RNA technology and overexpression vector transfection.The effect of IL-8 expression on extracellular matrix metabolism and the related signal pathways were analyzed in corneal fibroblasts.The results would provide a foundation for further elucidation of IL-8 function in corneal cell responded to mechanical stimulation,and provide references for revealing the pathogenesis of keratoconus and its clinical diagnosis and treatment.The main work and conclusions are as follows:（1）The expression of IL-8 in keratoconus and normal corneal fibroblasts were compared using real-time fluorescent quantitative PCR（qPCR）technology.Results showed that the expression of IL-8 in keratoconus fibroblasts was significantly higher than that in normal cornea fibroblasts,indicating that IL-8-mediated inflammatory process was involved in the occurrence and development of keratoconus.（2）Both the normal and keratoconus fibroblasts were treated with mechanical stretching,and then the expression changes of IL-8 and genes related to oxidative stress signaling pathway were analyzed.The results showed that IL-8 expression was induced in corneal fibroblasts at an early stage after mechanical stretching treatment.In addition,mechanical stretching can induce the expression of NOX4 and AQP1,genes related to hydrogen peroxide（H₂O₂）production and transport.It indicated that mechanical stretching can induce AQP1-mediated oxidative response in corneal fibroblasts.（3）Treat corneal fibroblasts were treated with H₂O₂ and acetazolamide respectively,and the changes of IL-8 expression were detected.The results showed that H₂O₂ can induce the expression of IL-8 and AQP1 in corneal fibroblasts.After azolamide treatment,the level of intracellular reactive oxygen species was reduced,with the significantly reduction of IL-8expression in corneal fibroblasts,indicating that AQP1-mediated oxidative stress was involved in the regulation of IL-8 expression in corneal fibroblasts.（4）The expression of IL-8 in corneal fibroblasts was regulated by si RNA interference and overexpression technology respectively,and the effect of IL-8 expression on the synthesis and degradation of extracellular matrix in corneal fibroblasts was analyzed by qPCR,ELISA and gelatin zymography.The results showed that after overexpression of IL-8 in normal corneal fibroblasts,the expression of collagen synthesis genes was decreased.While the total collagen content,the expression of genes related to collagen synthesis（COL1A2,COL3A1,COL5A3,COL6A1）and collagen cross-linking（LOX-4）were increased significantly in both normal and keratoconus fibroblasts after IL-8 silencing.Besides,the expression and activity of MMP-2,one of the enzymes related to collagen degradation were decreased,and the expression of proteoglycan genes（LUM,DCN and BGN）was increased significantly in IL-8silenced keratoconus fibroblasts.It showed that IL-8 negatively regulated the synthesis of collagen and proteoglycan synthesis in corneal fibroblasts,and participated in the MMP-2-mediated collagen degradation.（5）Proteins interacted with IL-8 were predicted using bioinformatics method.The effect of IL-8 on the involved signal pathways in corneal fibroblasts was analyzed by qPCR and western blotting.The results showed that gene expression of IL-6 and IL-1βin corneal fibroblasts decreased after IL-8 interference.In addition,the expression of VEGF-α/VEGFR2and the proteins in the downstream pathways including Akt,β-Catenin and Erk 1/2 were reduced.It suggested that IL-8 could regulate the expression of IL-6,IL-1βand VEGF in corneal fibroblasts,and participate in the regulation of corneal extracellular matrix metabolism through PI3K/Akt and Erk signaling pathways.