The Role And Mechanism Of Adaptor Protein P66Shc In Diabetic Retinopathy

Background and ObjectiveOne of the most serious microvascular complications in diabetes mellitus is diabetic retinopathy(DR)is.It is one of the main causes of adult blindness in developed countries.High glucose can induce various stress responses in the retina,which will activate three major pathways to apoptosis:mitochondria,death receptors and endoplasmic reticulum.Ultimately,it induces apoptosis of retinal cells and neurons,aggravates glial cell proliferation,and causes visual function decline.Mitochondrial pathway is the most important apoptosis pathway,and is known as the"regulating center" of apoptosis.In the process of activating intracellular pathway,excessive consumption or inactivation of intracellular reductive substances or enzymes by extracellular information,such as high glucose,will result in the production of reactive oxygen species(ROS)in the respiratory chain of mitochondria,which can activate oxidative stress and lead to apoptosis.p66Shc protein can be used as a receptor of ROS concentration,promote cell apoptosis and regulate cell life.p66Shc can mediate oxidative stress which is induced by diabetes,and mediate oxidative stress dependent kidney injury as well.Just like diabetic nephropathy,diabetic retinopathy is also one of the most common microvascular complications of diabetes.They both have a common pathogenesis.However,the role and mechanism of p66Shc protein in diabetic retinopathy have not been reported.We speculate that p66Shc may play an important role in the development of diabetic retinopathy.In this study,we intend to observe the expression of p66Shc and phosphorylated p66Shc under high glucose,and their relationship with oxidative damage and apoptosis in vivo and in vitro,which will confirm our hypothesis from organ,cell and molecular level.It will propose a new way to reduce oxidative stress and apoptosis in retina under high glucose,and provide a new idea for the prevention and treatment of diabetic retinopathyPart 1 P66Shc expression in diabetic rat retinaPurpose:p66Shc is partially localised within the mitochondrial fraction.It is primarily related to the generation of mitochondrial reactive oxygen species and apoptosis.Based on previous studies,we hypothesize that in the retina,p66Shc may exist and affect the development of diabetic retinopathy.The purpose of this study was to investigate p66Shc expression in retinal in streptozotocin-induced diabetic(SD)rats,which may provide a pathway to study the pathogenesis of diabetic retinopathyMethods:According to the DM course,all of the rats were assigned to 3 groups:15 rats of D4w(4 weeks after diabetes onset)and 15 rats of D12w(12 weeks after diabetes onset)and 15 rats of not-diabetic control.Reverse transcription-polymerase chain reaction(RT-PCR)and western blot were used to detect retinal p66Shc mRNA and protein expression in SD rats,respectively.Immunohistochemical staining was applied to detect the location of rat retinal p66Shc expression.TUNEL assay was applied to detect the number of apoptotic cellsResults:p66Shc expression was found in the retina of normal and diabetic rats,and the level of mRNA and protein expression increased with the progression of diabetes mellitus(DM).P66Shc expression was mainly located in the retinal ganglion cell layer and inner nuclear layer.Compared with the normal group,retinal cell tissue apoptosis rate in the D12w group was significantly increased.Conclusion:Rat retinal p66Shc expression was mainly in the ganglion cell layer and inner nuclear layer.As the degree of DM progressed,p66Shc expression gradually increased,and the number of apoptotic cells also increasedPart 2 Effect of high glucose on the expression of p66Shc in R28 cellsPurpose:The purpose of this study was to investigate effect of high glucose on the expression of p66Shc and phosphorylation of p66Shc Ser36 in R28 cellsMethods:R28 cells were cultured with different concentrations of D-glucose(5.5mM,15mM,30mM,45mM)for 12h,then,the expression of p66Shc mRNA was detected by RT-PCR.The expression of p66Shc and phosphorylated p66Shc protein was detected by Western blot.Meanwhile,R28 cells were cultured with 30mM D-glucose or 30 mM mannitolonly for indicated time points.Then,the expression of p66Shc and phosphorylation of p66Shc Ser36 were detected.Results:With the increase of glucose concentration,the expression of p66Shc mRNA and p66Shc protein gradually increased.The expression of p66Shc mRNA and p66Shc protein was strongest in 45mM glucose group.P66Shc mRNA,p66Shc protein and phosphorylated p66Shc Ser36 protein increased with the prolongation of the action time.Stimulated by 30mM mannitol(isotonic control group),the expression of p66Shc mRNA,p66Shc protein and phosphorylated p66Shc S36 protein did not change.Conclusion:High glucose increased the expression of p66Shc in R28 cells in a time dependent and concentration dependent manner.High glucose induced phosphorylation of p66Shc Ser 36.Part 3 The role of p66Shc in high glucose concentration-induced oxidative damage in R28 cellsPurpose:The aim of this study was to investigate the effects of p66Shc in high glucose induced oxidative damage and apoptosis in R28 cellsMethods:R28 cells were divided into five groups:(1)normal control group(untransfected R28 cells+5.5mM D-glucose);(2)high glucose group(untransfected R28 cells+30mM D-glucose);(3)high glucose over expression group(transfected plasmid p66Shc R28 cells+30mM D-glucose);(4)high glucose p66Shc slience group(Small interfering RNA slience p66Shc R28 cells+30mM D-glucose);(5)high glucose S36A mutant group(transfected pcDNA3.1 his p66Shc S36A R28 cells+30mM D-glucose).MitoSox Red was used to detect mitochondrial ROS content.Laser confocal microscopy was used to detect membrane potential.Flow cytometry was used to detect cell apoptosis.Western blot was used to detect caspase 9 proteins,cytochrome C and p66Shc protein in mitochondria.PCR was used to detect mitochondrial DNA damage after mitochondrial DN A was extracted.Results:With the increase of p66Shc level in the mitochondria of R28 cells,ROS content in mitochondria increased,mitochondrial membrane potential decreased,mitochondrial DNA damage was exacerbated,cytochrome C content in mitochondria decreased,caspase-9 protein expression in cytoplasm decreased,and apoptosis increased.On the contrary,compared with the high glucose group,in high glucose p66Shc slience group and high glucose S36A mutant group,p66Shc in mitochondria decreased,ROS content in mitochondria decreased,mitochondrial membrane potential partially recovered,mitochondrial DNA damage was recovered,cytochrome C content in mitochondria increased,caspase-9 protein expression in cytoplasm partially recovered,and apoptosis decreased.Conclusion:p66Shc affected oxidative damage and apoptosis in R28 cells via mitochondrial pathway under high glucose condition.Over expression of p66Shc aggravated high glucose induced oxidative damage and cells apoptosis in R28 cells.On the contrary,p66Shc slience or p66Shc 36 serine mutation decreased oxidative damage and cells apoptosis in R28 cells induced by high glucose.