| Objective:The cornea is located in the outermost layer of the eyeball wall,and its shape and function play an important role in the shape and visual maintenance of the eyeball.There is a dynamic balance in the corneal epithelium,which is broken when diseases such as ocular trauma,dry eye,and drug poisoning occur,causing corneal epithelial dysfunction(CED),and persistent CED leads to corneal epithelial defects,ultimately leading to visual defects or loss.At present,epidermal growth factor(EGF)and fibroblast growth factors(FGFs)are mainly used to treat CED.FGFs have been paid more and more attention in the treatment of CED.FGF2 in FGFs family promotes the healing of corneal epithelium by binding to fibroblast growth factor receptor 1(FGFR1).FGF2 currently available has been used in the treatment of ocular surface diseases.However,more and more studies have shown that FGF2 can activate corneal stromal cells while promoting corneal epithelial repair,leading to corneal stromal scar formation,while promoting corneal endothelial-mesenchymal transition,which can lead to posterior corneal membrane formation and blindness,fibroblast growth factor5(FGF5)is a member of the fibroblast growth factor(FGFs)family,and other members of the family such as FGF2,FGF7,and FGF9 are found to be involved in regulating corneal epithelial turnover and balance,while FGF5 is also found to be highly expressed in limbal epithelial stem cells by sequencing the limbal stem cell gene.Based on this,we carried out this experiment to explore the expression of FGF5 and its receptor FGFR1 in different locations of the cornea,the effect of FGF5 in promoting corneal epithelial proliferation and turnover and its mechanism of action,and providing a theoretical basis for providing new potential targets for corneal epithelial dysfunction(CED)in clinical practice.Methods:Part I: Expression of FGF5 and its receptor FGFR1 in corneal epitheliumCorneal epithelium within 2 mm of the central cornea and corneal epithelium within about 1 mm of the limbus were extracted from C57BL/B6 mice,respectively,and their RNA was extracted to observe the normal morphology of the cornea of C57/B6 mice by HE staining,and the expression of fibroblast growth factor 5(FGF5)and its receptor FGFR1 in the limbus and central cornea of mice was verified by immunohistochemistry,WB and RT-q PCR.PartⅡ:To investigate the effects of FGF5 at optimal concentrations on epithelial healing in a human corneal epithelial cell line(TKE2)and mouse corneal epithelial injury modelIn order to verify that FGF5 may maintain the stemness of limbal stem cells and activate stem cells during the healing process,thereby promoting corneal wound healing,the CCK8 assay was used to find the most suitable concentration range of FGF5 to promote corneal epithelial cell(TKE2)healing,and corneal epithelial cell lines and C57BL/B6 mice were selected as study subjects to establish epithelial injury models and perform related project experiments.(1)A scratch model was established in the cells.The scratch cells were treated with FGF5 solution at the optimal concentration,corresponding epidermal growth factor(EGF)solution and blank control.The scratch repair status was observed at 0,4,8,12 and 24 hours,and the expression of each proliferation and differentiation marker gene in TKE2 treated with rh FGF5 at the optimal concentration was verified by RT-q PCR.(2)Epithelial injury models were established on the corneas of C57/B6 mice and treated with the optimal concentration of FGF5 and the corresponding EGF and 1×PBS,respectively,and the defect area of the mouse corneal epithelium was assessed with 0.25% fluorescein sodium under a slit lamp(BQ900H Haag-Streit,Bern,Switzerland),and the expression of FGF5 in the corneas during corneal injury was observed by immunohistochemical,WB,and PCR experimental methods,while the repair ability of the optimal concentration of rh FGF5 on corneal injury was observed compared with the same concentration of EGF and 1×PBS.(3)A mouse dry eye model was established to observe the ocular surface dryness under microscope,and the status of corneal epithelial cells,stromal cells and endothelial cells in dry eye mice and FGF5-treated dry eye mice was observed under confocal microscope.HE staining was used to observe the corneal morphology of normal mice,dry eye mice and FGF5-treated dry eye mice.RT-q PCR and immunofluorescence staining were used to verify the expression of PI3K-AKT pathway genes and proliferation marker genes in normal mice,dry eye mice and FGF5-treated dry eye mice.PartⅢ:Explore the downstream pathways of FGF5 promoting cell proliferationRNA sequencing was performed in the TKE2 treatment group treated with rh FGF5 at the optimal concentration and the untreated normal control group,followed by WB and RT-q PCR to verify the sequencing results,and FGF5 was used to promote corneal epithelial repair through the PI3K-AKT signaling pathway by pathway inhibitor reversion.FGF5 knockdown cell lines were constructed by transfecting TKE2 cells with Si RNA,and RT-q PCR was used to verify the expression of each proliferation and pathway gene after knockdown of FGF5.Results:PartⅠExpression of FGF5 and its receptor FGFR1 in corneal epithelium:Immunohistochemical staining(IHC)showed that FGF5 was expressed in the limbal region,mainly in the basal layer,and some stromal cells also expressed FGF5.For FGFR1,there were also minor differences between limbus and central cornea on the stromal layer of the cornea.Real-time polymerase chain reaction(RT-q PCR)results showed that FGF5 and FGFR1 expression was higher on the limbus than in the central cornea in mice,and Western blot results confirmed the same results for PCR.PartⅡ(1)FGF5 stimulated TKE2 proliferation: We selected eight concentrations of rh FGF5(0,2.5,5,10,20,40,80,160 ng/m L)to treat TKE2 cells.The results of CCK8 assay showed that the number of cells slightly increased with increasing concentration in the treatment group after the same time of treatment,and 40 ng/m L appeared to be the optimal concentration in all other groups after 24 hours of drug treatment,notably,rh FGF5 did not show cytotoxicity even at a high concentration of160 ng/m L,and RT-q PCR results showed that the gene expression of FGF5,Ki67,K14,K15,and P63 was significantly increased in TKE2 cells treated with 40 ng/ml FGF5(P<0.05),while no significant increase in the expression of Sprr1 b gene labeled for abnormal differentiation was observed.(2)FGF5 promotes corneal epithelial wound healing: In a mouse corneal epithelial injury model,FGF5 expression in the mouse cornea gradually increases from 0 to 24 hours,gradually decreases after peaking at 24 hours,and decreases below normal levels at 48 hours.In the injury repair model,the healing promoting ability of 400 ng/ml FGF5 group was significantly enhanced compared with 400ng/ml EGF group and control group within 24 hours,and the cornea of mice in 400ng/ml FGF5 group was completely repaired at 48 hours,while 400 ng/ml EGF group and control group still did not heal.(3)FGF5 promotes corneal epithelial wound healing in dry eye model: mouse dry eye model was successfully constructed,normal mice were observed under microscope,PBS control dry eye mice,FGF5 treatment dry eye mice found that PBS control mice corneal punctate staining,while FGF5 group mice corneal surface was smooth,confocal microscopy showed that PBS control mice corneal epithelium had a large number of necrotic cells,corneal stromal edema,endothelial morphology changes,while FGF5 group and blank control mice epithelial,stromal and endothelial cells showed no significant abnormalities,HE staining results showed that PBS control mice corneal inflammatory cells increased,stromal edema.3,RT-q PCR was used to verify the expression of AKT1,AKT2,AKT3,BCL-2,CAK,Ccnd1,CDK1,CDK4,CDK6(PI3K pathway gene),FGF5,K14,K15,Ki67,and P63(proliferation marker gene),and immunofluorescence staining K10,K14,K17,and Ki67 results suggested that gene expression in the PI3K-AKT signaling pathway as well as proliferation marker gene expression were significantly enhanced(P<0.05),and these gene expression was also enhanced in the FGF5 group,but weaker compared with the PBS control group.PartⅢ(1)FGF5 may be expressed through the PI3K-AKT pathway: the main regulatory genes in the FGF5-treated group were mostly enriched on the PI3K-AKT signaling pathway,and RT-q PCR results showed that the expression of AKT1,AKT2,AKT3,BCL-2,CAK,CDK1,CDK4,CDK6,and Ccnd1 genes in the pathway increased after FGF5 treatment of TKE2 cells(P<0.05).(2)AKT pathway promotes corneal epithelial cell proliferation through FGF5:pathway inhibitor reversion assay results demonstrated that BCL-2,AKT,P-AKT,PI3 K,and P-PI3 K protein expression was significantly increased(P<0.05);after Si RNA transfection of TKE2 to knockdown FGF5,RT-q PCR results indicated that the corresponding proliferation marker genes(FGF5,Ki67,K14,K15,K17,P63)and pathway genes were also significantly decreased(AKT1,AKT2,AKT3,BCL-2,CDK1,Ccnd1)(P<0.05).Conclusions:(1)FGF5 and FGFR1 expression was higher on the limbus than in the central cornea.(2)FGF5 can effectively promote the growth of corneal epithelial stem cells,and the proliferation of TKE2 cells treated with FGF5 is accelerated.FGF5 can significantly promote the wound healing of corneal epithelium in a mouse corneal epithelial injury model;In the dry eye model,it resists the damage caused by dry eye to the ocular surface.(3)FGF5 can effectively promote the proliferation of corneal epithelial stem cells through PI3K-AKT signaling pathway. |
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