Effects Of Hypoxia On Autophagy Of Human Fetal Scleral Fibrolasts

Background and objectiveMyopia has become a public health problem of visual impairment around the world,which is one of the major eye diseases causing irreversible visual impairment.In recent years,the incidence of myopia has been increasing,which poses a serious threat to human health and quality of life.The pathogenesis of myopia are still unknown,and some studies have shown that the development of myopia is closely related to the improper elongation of the eye axis,that is,sclera remodeling.The decrease in scleral thickness and strength in myopia is related to the up-regulation of proteases,resulting in the thinning of the collagen framework and changes in the composition of the extracellular matrix.Matrix metalloproteinase(MMP)-2 is one of the known proteases that mediate these changes.In the FD eyes,the expression levels of α-SMA were significantly increased,and the differentiation and metastasis of myofibroblasts in the sclera of myopic showed that the over-expression of α-SMA was involved in myofibroblast remodeling.The axial elongation of high myopia is associated with choroidal thinning and decreased choroidal blood flow,which may leave the retinal and sclera of myopia in a hostile environment of ischemia and hypoxia.Recent studies have found that hypoxia signaling pathway is activated in the sclera of myopia in mice,and hypoxia plays an important role in scleral ECM remodelling and myopia progression.However,how hypoxia affects the occurrence and development of myopia still needs further investigation.Autophagy is the process of degradation of self-damaged organelles and macromolecules by lysosomes under the regulation of autophagy-related genes,so as to maintain the natural metabolism of normal cells and the renewal of organelles,thus regulating the homeostasis of the body.Autophagy can be caused by many factors in cells.Previous studies have found that autophagy has a close and complex relationship with hypoxia,and autophagy plays different roles in different diseases caused by hypoxia.Autophagy is involved in the metabolism of collagen in the extracellular matrix of different fibroblasts in different ways.It is still unclear that the level of autophagy in myopic sclera fibroblasts and its relationship with MMP-2 and Collagen I in hypoxia environment.Therefore,the purpose of this study is to explore hypoxic environment on the collagen metabolism and autophagy of HFSFs.MethodHFSFs were exposed at a low O2 level of 5%,lasting for 6h,12h,or 24h,and cells maintained under the normoxic condition(21%O2)served as the control.The morphological changes of cells were observed by light microscope.The cell viability was detected by CCK8 assay.Western Blot and q-PCR are to determine the relative expression level of Collagen I,MMP-2,α-SMA,and HIF-1 alpha.The expression of autophagy-related factors(LC3II/I,ATG5,P62)of HFSFs under hypoxic treatment are determined by q-PCR and Western blot.ResultsIn hypoxia environment,the cellular morphology of HFSFs becomes flat and irregular polygon.The results of CCK-8 showed that the activity of HFSFs decreased gradually with the extension of hypoxia time(P<0.05).It was found by Western blot that HIF-1α expression in human scleral fibroblasts increased with the extension of hypoxia intervention time(P<0.05).Through q-PCR and Western-blot,it was found that with the extension of hypoxia time,the expression of Collagen I gradually decreased(P<0.05),the expression of mmp-2 gradually increased(P<0.05),and the expression of α-SMA gradually increased(P<0.05).By western-blot and q-PCR,it was found that,compared with the control group,the autophagy level of HFSFs increased and the expression of autophagy-related factors increased in the hypoxic environment(P<0.05)and autophagy increased with the extension of hypoxia time.ConclusionsHypoxia can inhibit the synthesis of type I collagen by scleral fibroblasts,and promote the degradation of collagen and promote the transdifferentiation of scleral fibroblasts into myofibroblasts,which further verify the important role of hypoxia in the development of myopia.In addition,hypoxia activates autophagy of HFSFs.