| ObjectiveWe first established a lens-induced myopia(LIM)model with-6.0D lenses in guinea pigs,then investigated the effect of intravitreal injection of nitric oxide synthase inhibitor L-NMMA acetate on the expression of hypoxia-inducible factor-1α(hypoxiainducible factor-1α,HIF-1α)through regulating NOS expression,and the effect of miR-138-5p regulating HIF-1α expression to influence NO level on the choroidal fibrosis of LIM guinea pigs.We aimed to elucidate the underlying molecular mechanism of controlling the development of myopia by delaying the choroidal fibrosis of LIM guinea pigs,providing a theoretical basis for the prevention and treatment of myopia.MethodsThe experiment consists of two parts.Part I:To investigate the effect of nitric oxide synthase(NOS)inhibitor L-NMMA acetate on choroidal fibrosis in LIM guinea pigs.A total of 120 healthy 2-week-old male tricolor guinea pigs were randomly divided into a normal control(NC)group,a lens-induced myopia(LIM)group,a NOS inhibitor(LNMMA)injection group,and a NOS inhibitor control(PBS)group.The guinea pigs in the NC group received none-intervention,while those in the LIM,L-NMMA,and the PBS groups wore-6.0D lenses on the right eye to induce myopia,and the left eye was as self-control received no intervention.In addition,the animals in the L-NMMA group were treated with intravitreal injection of NOS inhibitor(L-NMMA acetate)at a dose of 5 μl(20 nmol)every other day for a total of 3 injections a week.Meanwhile,those in the PBS group were given an injection of 5 μl of 1×PBS as a control,and the treatment was the same as in the NOS inhibitor group.After myopia induction for 2 and 4 weeks,the refractive status and other ocular parameters of the guinea pigs were measured.Further,the expression of neuronal nitric oxide synthase(nNOS or NOS1),endothelial nitric oxide synthase(eNOS or NOS3),hypoxia-inducible factor(HIF)-1α,transforming growth factor(TGF)-β,collagen I(COLI),and alpha-smooth muscle actin(α-SMA)in choroidal tissues was investigated.Part II:This study aimed to investigate the effect of miR-138-5p on choroidal fibrosis in myopic guinea pigs via regulating the HIF-1α signaling pathway.First,guinea pigs were randomly divided into a normal control(NC)group,a lens-induced myopia(LIM)group,a LIM+miR-138-5p-carried Lentivirus treatment(miR-138-5p)group,and a LIM+miR-138-5p-Vector treatment(VECTOR)group.All animals were induced experimental myopia with a-6.0 diopter lens except those in the NC group.Meanwhile,animals in the miR-138-5p group were supplemented with 5 μl of miR-138-5p-carried lentivirus,while those in the VECTOR group were only supplemented with the same volume of lentivirus vector.After myopia induction for 2 and 4 weeks,the refractive status and other ocular parameters of the guinea pigs were measured.Further,the expression of NOS1,NOS3,HIF-1α,TGF-β,COLI,and α-SMA in choroidal tissues was investigated.ResultsPart I:1.After 2 weeks and 4 weeks of myopia induction,compared with the NC group,the refraction and choroidal thickness in the LIM group evidently decreased,whereas the axial length increased significantly,and the choroid showed a fibrotic trend.The expression of NOS1,NOS3,HIF-1α,TGF-β,COLI,and α-SMA at mRNA and protein levels was increased significantly in the choroid(all P<0.05).After intravitreal injection of NOS inhibitor L-NMMA,compared with the LIM group,the refraction and the choroidal thickness significantly increased,whereas the axial length reduced significantly,accompanied by an increase of choroidal thickness and an improvement of choroidal fibrosis.The expression levels of choroidal NOS1,NOS3,HIF-1α,TGFβ,COLI,and α-SMA mRNAs were significantly reduced,and the differences were statistically significant(all P<0.05).Part II:After intravitreal injection of miR-138-5pcarried lentivirus,compared with the LIM group,the axial length was decreased significantly,the refraction and choroidal thickness were elevated,and choroidal fibrosis was improved.The expression choroidal NOS1,NOS3,HIF-1α,TGF-β,COLI,and α-SMA at mRNA and protein levels were significantly reduced,and the differences were statistically significant(all P<0.05).Conclusions1.The trend of choroidal fibrosis in LIM guinea pigs is positively correlated with the increase of axial length.NOS inhibitor can alleviate the process of choroidal fibrosis in myopia guinea pigs by inhibiting NO signaling transduction and effectively reduce NOS 1,NOS3 and HIF-1α,TGF-β,COLI,and α-SMA expression.2.MiR-138-5p can alleviate the process of choroidal fibrosis by inhibiting HIF-1αSignaling transduction in myopic guinea pigs and effectively reduce NOS1,NOS3,HIF-1α,TGF-β,COLI,and α-SMA expression.3.Through intravitreal injection of NOS inhibitor L-NMMA acetate and miR-1385p to interfere with experimental myopia model,we found that NO can regulate HIF1α in the development of myopia.The expression of NOS affects choroidal fibrosis,and NOS inhibitor L-NMMA and miR-138-5p can delay the development of choroidal fibrosis in LIM guinea pigs by reducing the expression of NOS molecules,exhibiting a certain control effect on the development of myopia. |
