| Purpose:Diabetic keratopathy is a common diabetic complication,which can lead to severe visual impairment in patients with diabetes mellitus.The advanced glycation end products(AGEs)accumulation plays a pivotal role in diabetic keratopathy.Traditional studies suggest that AGEs can combine with the receptor of advanced glycation end products(RAGE)and initiate the process of reactive oxygen species(ROS),inflammation and apoptosis,which will accelerate the development of diabetic keratopathy.While recent studies have shown that advanced glycation products receptor-1(AGER1),another receptor of AGEs,is extensively depressed in diabetic mellitus.This suggests that it may participate in the development of diabetic complications.But no research about this factor on the complication of diabetic keratopathy can be searched until now.Therefore,our research aims to explore the role and mechanisms of how AGER1 functions in diabetic keratopathy in vivo and in vitro,using streptozotocin(STZ)-induced type I diabetic mice and human corneal epithelial cells(HCECs)as models respectively.Methods:1.In order to explicit whether AGER1 was involved in the development of diabetic keratopathy,immunohistochemistry,immunofluorescence,real-time quantitative PCR(RT-PCR)and Western blot(WB)were taken for the detection of expression of AGEs and the AGER1 pathway related genes in the cornea of diabetes mellitus,type I(STZ and INS2Akita/+)and type II(BKS-db/db)diabetic mice.2.In the in vitro research,HCECs were dealt with exogenous AGEs-BSA or received gene modification of AGER1-silence or AGER1-overexpression,and then we used MTT method to detect the influence on HCECs proliferation,cell-scratch-test to detect the influence on HCECs migration,AV / PI-staining to detect apoptotic changes of HCECs,DCFH-DA-staining to compare levels of ROS,and RT-PCR and WB to detect the gene expression of AGER1,NAD+-dependent deacetylase sirtuin 1(Sirt1),RAGE and so on in the AGER1 pathway related genes,aiming at investigating the specific role and mechanisms of AGER1 in diabetic keratopathy.3.In the in vivo research,subconjunctival injection of AGEs-BSA was given in C57 BL / 6J mice model,and the corneal epithelium was scraped in order to observe the effect of AGEs on corneal epithelial wound healing.Then immunofluorescence and RT-PCR were used to detect the alteration of AGER1 pathway related genes expression including AGER1,Sirt1,RAGE and so on.In order to make out the specific role and mechanisms of AGER1 in vivo,we used lentivirus to transfect the cornea of STZ-induced mice and established the AGER1 overexpression model,and RT-PCR was applied to detect the AGER1 pathway related genes expression.Results:1.Compared with the control,relatively more AGEs accumulation and significantly less AGER1 expression were observed in the epithelial basement membrane of corneas in diabetic patients and STZ,INS2Akita/+ and BKS-db/db mice.Significantly increased expression of RAGE was observed in STZ,INS2Akita/+ and BKS-db/db mouse corneas and STZ-mice corneas.Significantly reduced expression of Sirt1 was observed in STZ and INS2Akita/+ mice corneas,while the expressions of acetyl-nuclear factor-B(Acl-NF-B)and the proinflammatory factor of tumor necrosis factor-?(TNF-?)(P<0.01)were significantly increased,but the expression of antiapoptosis factor of B-cell lymphoma-2(Bcl-2)(P <0.01)was significantly reduced in the entire cornea of STZ mice.Further research was conducted on the corneal epithelium of STZ mice,and the results revealed that proinflammatory factor TNF-?(P<0.01),interleukin-1(IL-1)(P<0.05)and interleukin-6(IL-6)(P<0.01)expressions were all significantly increased.It was also found that in the entire cornea of BKS-db/db mice,the proinflammaroty cytokine IL-6 expression was increased.2.In HCECs,after AGEs-BSA treatment,the HCECs proliferation(P < 0.05)and migration ability(P < 0.05)decreased significantly,while ROS generation increased.The AGER1 and Sirt1 expressions were significantly reduced,while RAGE and AclNF-B expressions were significantly increased.After transfected with silence-RNA of AGER1,the HCECs proliferation also decreased significantly(P < 0.01),but the ROS generation and cell apoptosis increased.Further molecular research found that AGER1 interference made Sirt1 protein expression significantly decrease and the expressions of RAGE,Acl-NF-B,proinflammatory cytokine IL-1(P < 0.01)and proapoptosis cytokine Bcl-2 associated X protein(Bax)(P < 0.05)significantly increase.However,AGER1 overexpression depressed the protein expression of RAGE(P < 0.01),Acl-NF-B and m RNA expressions of proinflammatory and proapoptosis cytokines of TNF-?(P < 0.01),IL-1(P < 0.01),IL-6(P < 0.01)and Bax(P < 0.01).The Sirt1 protein expression was not significantly affected.3.The corneal epithelium of STZ mice was scraped in a diameter of 2.5 mm,and the corneal epithelial defect area was investigated and found to be significantly increased,compared with the control(P < 0.05)at 24 h and 36 h after the operation.AGEs-BSA was subconjunctivally injected in C57BL/6J mice,and we found that AGEs-BSA delayed the corneal epithelial wound healing(P < 0.05)at 12 h,24 h and 36 h after the corneal epithelium scraption.Meanwhile,the m RNA level of AGER1(P < 0.01),Sirt1(P < 0.01)and Bcl-2(P < 0.01)expressions was detected from the cornea,and all was found to be significantly reduced.The RAGE(P < 0.05),IL-1(P < 0.01),IL-6(P < 0.01)and Bax(P < 0.05)expressions were found to be significantly increased.The expression of Acl-NF-B protein also increased significantly.In the in vitro cultured cornea of STZ mice with medium containing AGER1 overexpressed lentivirus,the expressions of proinflammatory factor TNF-?(P<0.05)and proapoptosis factor Bax(P<0.01)were both significantly reduced.Conclusion:1.AGEs accumulation,increased expression of AGER1 and decreased expression of RAGE existed in the cornea of type II diabetic mellitus and type I and type II diabetic mice models.Exogenous AGEs can significantly delay the corneal epithelial wound healing in the mice model.2.The increased expression of proinflammation and proapoptosis cytokines is considered as their involvement in the pathogenesis of diabetic keratopathy.3.AGEs can significantly depress the HCECs proliferation,migration,and promote the ROS generation.AGEs activates inflammation and apoptosis in vitro and in vivo,and may function through signal pathways of both AGER1/Sirt1 and RAGE/Acl-NF-B in vivo.4.AGER1 interference can make the expression of RAGE increase,aggravating the adverse effects of AGEs on HCECs,while AGER1 overexpression decreases the expression of RAGE and make it alleviate.Receptor AGER1 and RAGE may competitively integrate with AGEs,as two antagonistic pathways in diabetic keratopathy.5.AGER1 receptor plays a protective role in diabetic keratopathy,which may reduceHCECs inflammation and apoptosis,and is a potential preventive and therapeutic target in diabetic keratopathy. |
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