Expression Of Adamts1 And Adamts5 In Mouse Scleral Fibroblasts And Its Role In The Formation Of Myopia

Background:The increasing prevalence of myopia will inevitably bring a great medical burden and other far-reaching negative effects to society.Myopia has become a public health problem that needs urgent attention.In recent years,studies related to the pathogenesis of myopia have received increasing attention.Scleral extracellular matrix remodeling is one of the important mechanisms of myopia formation.The Adamts family of proteases is anchored in the extracellular matrix and is involved in the remodeling of the extracellular matrix.Adamts1 and Adamts5 are hyaluronidases that directly degrade type I collagen fibers and are also involved in the metabolism of glycoproteins in the sclera.Several glycoproteins in the sclera can influence myopia formation by regulating the expression level of matrix metalloproteinase 2.But the relationship between the expression of Adamts1 and Adamts5 in scleral fibroblasts and the formation of myopia is still unclear.Purpose:To explore the expression of Adamtsl and Adamts5 in mouse sclera fibroblasts and their role and possible mechanism in myopia.Method:Scleral fibroblasts were divided into hypoxia group and control group.The concentration of oxygen in the incubator of hypoxia group(HO)was 1%,and the concentration of oxygen in the incubator of control group(NO)was 21%.Scleral fibroblasts were collected for Drug-RNA-seq after 48h of treatment to screen related transcriptome changes,Use the Metascape website to perform GO and KEGG analysis on sequencing results and visualize the analysis results.Expressions of Adamts1,Adamts5,Mmp2,Collagen Ⅰ and Hif-1 in scleral fibroblasts under hypoxia are detected by qRT-PCR and Western Blot.The expressions of Adamts1 and Adamts5 in sclera fibroblasts were inhibited by RNA interference in vitro,and the effect of that on the expression of Collagen I and Mmp2 were detected by cell immunofluorescence technique.The effects of reduced expression of Adamts1 or Adamts5 on cell migration and cell proliferation were observed by wound healing test and CCK8 proliferation assay,respectively.40 mice were divided into two groups.30 mice in form deprivation group were sutured with right eyelid for 4weeks,and the right eyes served as form deprivation eye(FDM group)and the left eyes served as self-control eye(SC group).10 mice in normal control group(NC group)received no treatment.The diopter was measured by static retinoscopy,the eyeball was enucleated and the length of the eye axis was measured by vernier caliper,and the thickness of the sclera was observed by Masson staining.The mRNA and protein expression levels of Adamts1,Adamts5,Collagen Ⅰ and Hif-1 a in sclera of mice in each group were detected by qRT-PCR and Western blot respectively.ResultDrug-RNA-seq and bioinformatics analysis results showed that compared with the control group,410 genes were up-regulated and 454 genes were down-regulated in the hypoxic group(p<0.05).Genes related to extracellular matrix formation,such as Adamts1,Adamts5,Plodl,Plod2,Loxl,underwent significant modifications,and Adamtsl and Adamts5 had direct interactions with CollagenⅠ and various other proteoglycans.The results of qRT-PCR and Western blot showed that the expressions of Hif-1α,Adamtsl and Adamts5 increased,and the expression of Collagen Ⅰdecreased in hypoxia group.Results of cell immunofluorescence showed that after inhibiting the expression of Adamtsl or Adamts5 in the scleral fibroblasts under hypoxia,the expression of CollagenⅠ increased and MMP2 decreased,but did not reach the expression level of normal control group.Wound healing test showed that hypoxia accelerated cell migration,and Adamts1 knocking down or Adamts5 knocking down could slow down that.Results of CCK8 proliferation assay showed that Adamtsl knocking down or Adamts5 knocking down had no significant effect on the cell proliferation ability.The model of form deprivation myopia in mice was successfully established:the diopter of mice in FDM group was significantly smaller than that in SC group(p<0.001);The eye axis of FDM group mice was significantly longer than that of SC group and NC group(p<0.05);The thicknesses of sclera in FDM group were thinner than of NC group and SC group(p<0.0001).There was no significant difference in the axial and scleral thicknesses between the NC and SC group.The results of qRT-PCR showed that the expression of Adamts1,Adamts5 and Hif-1 at mRNA level in the sclera of mice in FDM group were higher than those in NC group and SC group(p<0.05),with no significant difference between NC group and SC group.Western blot showed that the expression of Adamts1 and Adamts5 in sclera of mice in FDM group increased,while the expression of Collagen Ⅰ decreased with no significant difference between SC group and NC group.ConclusionAdamtsl and Adamts5 are involved in the development of myopia in mice,and the expression of Adamtsl and Adamts5 in scleral fibroblasts of myopia mice is up-regulated.Knocking down of Adamts1 or Adamts5 in scleral fibroblasts decreased the expression of Mmp2,increased the expression of Collagen Ⅰ,as well as slowed down the migration speed.Inhibitors targeting Adamtsl and Adamts5 may become a new attempt to develop drugs for the prevention and treatment of myopia.