Role Of Ranibizumab In Regulating The Repair Of Early-stage Diabetic Retinopathy Retina Damage Via VEGF/STAT3/GFAP Signaling Pathway And Its Mechanistic Study

Objective:To explore and elucidate the role of ranibizumab in the repair of early-stage diabetic retinopathy（DR）retinal tissue damage and its molecular mechanism.Methods:Diabetes Mellitus（DM）rat model was established by intraperitoneal injection of streptozotocin 60 mg/kg.One hundred DM rats were selected and randomly divided into five groups:group A,ranibizumab（+）+strict glycemic control（+）;group B,ranibizumab（+）+strict glycemic control（-）;group C,ranibizumab（-）+strict glycemic control（+）;group D,ranibizumab（-）+strict glycemic control（-）;group E,sterile saline（+）+strict glycemic control（-）.Twenty normal SD rats matched for age,weight,and ocular health without any treatment were used as group F.The rats were intravitreal injected on the 8th day after induction.The rats in each group were sacrificed after fundus fluorescence angiography（FFA）at the 4th week（N=5）,6th week（N=5）,8th week（N=5）and 10th week（N=5）after successful modeling,respectively.The vitreous and retinal tissues were isolated after removal of the eyeballs.The fundus fluorescence angiography technique was applied to observe the changes of retinal vessels in SD rats to clarify whether there was vascular leakage;VEGF-A ELISA kit was used to detect the vitreous VEGF-A concentration in each group of SD rats at each time point.Retinal tissues were used for:1)hematoxylin-eosin staining to observe the changes in retinal tissue morphology,retinal ganglion cells（RGCs）and vascular network system,as well as to count RGCs and measure RGCs area;2)periodic acid-Schiff staining to count the ratio of vascular endothelials/pericytes（E/P）and the number of acellular strands in retinal tissues,and to observe the presence or not of new vascular buds;3)immunofluorescence confocal imaging to observe the retinal vascular network system,count the number of anti-IV+collagen strands,and measure the number and area of retinal cells in SD rats;（4）q RT-PCR to detect the expression of VEGF m RNA,IL-6 m RNA,CD18 m RNA,ICAM m RNA,TNF-αm RNA and STAT3 m RNA in retinal tissues of SD rats;（5）Western Blot to determine the expression of GFAP,p STAT3 and STAT3 proteins in retinal tissues;to clarify the changes of RGCs,vascular network system and various cytokines in each group of SD rats at each time point.Results:（1）.FFA examination results showed that the vascular tortuous dilatation of retinal vessels in groups C,D and E presented earlier,which worsened with time.The obvious vascular tortuous dilatation and peripheral grossness were performed at the8^th week,and vascular leakage at the 10^th week.（2）.In the observation of HE-stained sections of retinal tissues,RGCs in groups D and E began to show a significant decrease at the 4^th week after induction and gradually worsened with the progression of the DR,and there was no difference between group D and group E compared with the same time point.The RGCs of group C began to show a significant decrease at the 8^th week after successful modeling.Vascular abnormalities（abnormal vascular dilatation and/or new vascular buds）of retinal tissues were observed in groups C,D and E in the 10-week observation period.（3）.Compared with group F at the same time,the number of acellular strands in retinal tissues of PAS-stained pavement started to increase significantly at the 4^th week,the E/P ratio became higher at the 6^thweek,and the formation of new vascular buds began to appear at the 8^th week in groups C,D and E.（4）.Vitreous VEGF-A concentrations began to appear gradually increased at the 4^th week of the DM course in groups C,D and E,but reached a peak at the 8^th week in groups D and E.（5）.Immuno-fluorescence confocal imaging of retinal tissue observed that the number of anti-IV₊collagen strands in retinal tissue and the reduction of retinal tissue pavement cells increased over time in groups C,D and E,and the phenomenon of vascular fluoresc-ence leakage and new vascular buds were also observed.There was no difference in retinal tissue cell area compared with each other at the same time point during groups C,D and E.（6）.It was found that VEGF m RNA,IL-6 m RNA,ICAM m RNA,CD18 m RNA and TNF-αm RNA in retinal tissues of groups C,D and E showed high expression levels starting from the4^th week of DM course,but the expression of STAT3 m RNA in groups D and E was at low expression level,and the expression level of STAT3 m RNA was increased in group C.As the disease progressed,dynamic changes in retinal tissue cytokines were observed in groups C,D and E.（7）.Western Blot showed that the expression levels of GFAP protein in retinal tissues of groups C and D were higher than those of group F at the 4^th week after induction,while there was no significant difference between group E and group F at the same time point.The expression of STAT3 and p STAT3proteins in retinal tissues of group C was not significantly different from group F at the 4^th week of DM course,while the expression of STAT3 and p STAT3 proteins was elevated in groups D and E compared with group F.The expression levels of GFAP,STAT3 and p STAT3 proteins in the retinal tissues of groups C,D and E showed dynamic changes over time.（8）.Intravitreal injection of ranibizumab in groups A and B could induce the vitreous VEGF concentration to be maintained at normal levels,down-regulate the expression of VEGF m RNA,IL-6 m RNA,CD18 m RNA,ICAM m RNA and TNF-αm RNA in retinal tissue,up-regulate the expression level of STAT3m RNA,and inhibit the expression of STAT3 protein,promoting the upregulation of GFAP protein and p STAT3 protein expression,all of which would significantly improve the amount of RGCs reduction,the number of acellular strands,the number of anti-IV₊collagen strands and the amount of retinal tissue pavement cells loss in early-stage DR retinal tissue.Conclusion:（1）Intravitreal injection of ranibizumab can promote the repair of damage to retinal tissue nerve cells and vascular network system,effectively delaying or even stopping the occurrence and progression of DR;（2）Ranibizumab may mediate the expression of various cytokines in the retina at the early stage of DR through the VEGF/STAT3/GFAP signaling pathway and participate in the repair of DR damage,thus delaying or even stopping the occurrence and progression of the early-stage DR;（3）Intravitreal injection of ranibizumab under insulin glucose control can better promote the damage repair of DR retinal tissues and significantly delay or prevent the development of DR;（4）Insulin glucose control can inhibit the expression of VEGF,IL-6,GFAP,STAT3,and other factors in DR retinal tissues,which can delay the progression of early DR to some extent.