The Regulatory Role And Mechanism Of Crosstalk Between Endoplasmic Reticulum Stress And TGF-β1 On Scleral Fibroblast Extracellular Matrix Remodeling Of Experimental Myopic Guinea Pig

Purpose: To investigate the function and mechanism of crosstalk between endoplasmic reticulum stress(ER stress)and transforming growth factor-beta1(TGF-β1)on scleral extracellular matrix remodeling.Methods: In vivo,guinea pigs were used to develop form-derivation myopia(FDM)by wearing a translucent diffuser.Ultrastructure of sclera was observed by transmission electron microscope(TEM),the expression levels of ER stress markers glucose regulated protein 78(GRP78)and C/EBP homologous protein(CHOP),TGF-β1 and type Ⅰ collagen(COL1A1)were detected by western blot.In vitro,scleral fibroblasts were primary cultured and identified by vimentin and keratin.Tunicamycin(TM)and 4-phenylbutyric acid(4PBA)were used to induce and alleviate ER stress,TGF-β1 protein and TGF-β1 neutralizer were used to up and down regulate TGF-β1.The morphology of ER was observed by TEM,expression levels of GRP78,CHOP,TGF-β1,and COL1A1 were detected by immunofluorescence,RT-PCR,and western blot.RNA interference(RNAi)sequence of calreticulin(CRT)was transmitted into scleral fibroblasts by lentiviral vectors,expression of CRT was detected by RT-PCR and western blot;after intervention by TM,4PBA,TGF-β1 protein and TGF-β1 neutralizer,GRP78,CHOP,TGF-β1,and COL1A1 were detected by immunofluorescence,RT-PCR,and western blot.In vivo,TM,4PBA,TGF-β1 protein and TGF-β1 neutralizer were injected to scleral tissue of FDM guinea pigs by subconjunctival injection.The Myopia degree and axial length were measured,and the expression of GRP78,CHOP,TGF-β1,and COL1A1 were detected by RT-PCR,and western blot.Results: After four week’s FDM,the myopia degree and axial length increased significantly in FDM group.The diameter and density of collagen fibrils were thinner and lower in FDM group,ER presented highly dilatation and swelling in FDM group.The expression level of GRP78 increased significantly in FDM group.Primary scleral fibroblasts were spindle-shaped,scleral fibroblasts were positively stained for vimentin and negatively stained for keratin.After intervention,the ER presented highly dilatation,swelling and degranulation in TM group and TGF-β1 neutralizer group,the protein and m RNA levels of GRP78、CHOP、TGF-β1 increased significantly in TM group,the m RNA level of COL1A1 increased in TM group.No significant change in TM + 4PBA group,but GRP78 increased in both TGF-β1 group and TGF-β1 neutralizer group.After infected by lentiviral vectors for 72 h and 96 h,the expression of CRT decreased significantly.After intervention,the expression of TGF-β1 and COL1A1 did not change in CRT(-)TM(+)group;the expression of GRP78 did not change in CRT(-)TGF-β1 neutralizer(+)group.In vivo,TGF-β1 neutralizer could induce ER stress in the sclera tissue of FDM guinea pig;moderate ER stress could promote the expression of TGF-β1 and COL1A1,and restrain the development of myopia and axial length.Conclusion: ER stress occurred in the scleral tissue of FDM guinea pigs.The decrease in TGF-β1 was the inducing factor of ER stress.The unfolded protein response could promote the expression of TGF-β1.CRT was the essential link between TGF-β1 and ER stress,CRT was an important role on scleral extracellular matrix remodeling.Moderate ER stress could promote the expression of TGF-β1 and COL1A1,and restrain the development of myopia and axial length.