| Objective:Diabetic retinopathy is one of the common complications of diabetes,and severe cases can lead to blindness in patients,of which retinal ganglion cell apoptosis and microangiopathy are the main causes.Diabetes can induce apoptosis of ganglion cells and other cells,which may be related to the changes of microglia reactivity and the decrease of neuroprotective factors.There are thinning phenomenon of ganglion cell layer and inner plexiform layer in diabetic patients,and obvious regional deletion has occurred even before the appearance of visible microvascular lesions.Therefore,it is of great clinical significance to study the effective neuroprotective treatment of diabetic retinopathy.JQ1 is a bromide structured extraterritorial end protein inhibitor with therapeutic effects on tumor proliferation,inflammation,and cardiovascular disease.In this study,we investigated the effects of JQ1 on retinal ganglion cell apoptosis and inflammatory factors through in vivo and in vitro experiments,proposed hypotheses and analyzed their molecular mechanisms in depth.Methods:1.SD rat diabetic model was constructed by intravenous injection of STZ,The experimental animals were divided into three groups: blank control group:healthy SD rats,bilateral intravitreal injection of 10% dimethyl sulfoxide + phosphate buffer solution;diabetic rats group: right eye as experimental group: Intravitreal injection of 10% dimethyl sulfoxide + phosphate buffer solution;left eye as treatment group: Intravitreal injection of 10% dimethyl sulfoxide + JQ1.After JQ1 injection,rat eyeball specimens were collected on days 1,3,7,and 14 to make retinal sections.RGC loss was observed under high glucose environment,and the specific marker proteins,NRN1、SNCG、Thy1,inflammatory factors,RANTES、TNF-α,apoptotic factor-related proteins Caspase-3 and m RNA expression levels of RGC were detected to investigate the protective effect of JQ1 by Western blotting and Real-time PCR on RGC in normal and diabetic retinal rats.Objective to investigate the protective effect of JQ1 on ganglion cells in diabetic retinopathy rats.2.Bioinformatics analysis of differential gene expression between diabetic patients and healthy people,GO and KEGG analysis of gene and pathway enrichment.The DR animal model was established using STZ,JQ1 was injected into the vitreous cavity of rats in control group and diabetic group.The eyeballs of rats were taken on the days 1,3,and 7 respectively,and the retinas were stained with HE to observe the retinal structure and count the retinal GCL.To further verify the protective effect of JQ1 on the retina,the PI3K phosphorylation level of retina was observed by immunofluorescence staining.3.The BV2 microglia high glucose model was established in vitro,and the effects of JQ1 on cell migration and invasion were analyzed using scratch assay and Transwell.Western blotting was used to analyze the PI3K/AKT pathway protein expression level in each group of cells,and immunofluorescence staining was used to analyze the PI3K phosphorylation level in each group of cells to deeply explore the molecular signaling pathway by which JQ1 exerts its efficacy.Results: 1.DAPI staining results showed that high glucose stimulation resulted in loss of RGC death in rats,and RGC death cells were reduced in the JQ1 group,indicating that DR rats induced RGC necrosis,while JQ1 could improve the condition.Further immunofluorescence staining of BRN3 A and counterstaining of DAPI were applied,and the results revealed that the RGC in the STZ DR group was significantly less than that in the blank control group,while it increased in the JQ1 group,and the difference was significant(P<0.05).These results indicated that RGC was significantly reduced in DR rats,while RGC loss was alleviated after application of JQ1.The changes in RANTES,TNF-α,and caspase3 expression in each group were analyzed using Western blot method,and the results revealed that the trends of RANTES,TNF-α,and caspase3 were consistent.Compared with the control group,the levels of RANTES,TNF-α,and caspase3 were significantly increased in the diabetic rats,and after JQ1 treatment,the above parameters were decreased,with TNF-α decreasing the most.Moreover,RANTES,TNF-α,and caspase3 expression levels slightly decreased with time.RT-PCR was used to detect the changes of NRN1,SNCG,and Thy1 mRNA expression levels in each group of SD rats on days 1,3,and 7.The results showed that there was no significant difference in NRN1,SNCG,and Thy1 changes in each group with time.The m RNA expressions of NRN1,SNCG,and Thy1 were significantly decreased in the diabetic retina group,followed by the JQ1 group,and were highest in the blank control group,and the differences between groups were significant in statistics(P<0.05).DR rats can up-regulate NRN1,SNCG,and Thy1 gene expression in RGC cells after injection of JQ1,which in turn alleviates RGC damage triggered by DR.2 The results of bioinformatics analysis indicated that NTRK2 was significantly highly expressed in diabetic patients,and the pathway effects of JQ1 on diabetic patients were further analyzed based on the KEGG database,mainly enriched in the PI3K/AKT pathway.Morphologically,the number of GCL cells in the diabetic group was significantly increased compared with the control group,and among them,the number of retinal cell death was reduced after JQ1 treatment.After cell count analysis,the number of GCL cells in the control group did not change much over time,and decreased significantly in the diabetic group,while the cellular number of GCL decreased less in the JQ1 treatment group than in the STZ diabetic group,and increased at 7d(P<0.01).The PI3K phosphorylation level was very low in the control group and significantly increased in the diabetic group,which could be reduced to some extent in the JQ1 treatment group,and the number of green positive cells was significantly reduced.Western blotting method was adopt to measured the expression levels of PI3K/AKT and MMP2 and MMP9-related proteins in the retinal tissues of rats in each group,and the results showed that the expression levels of p-PI3K/PI3K,p-AKT/AKT,MMP2 and MMP9 in the diabetic JQ1 treatment group showed an increasing trend compared with the control group(P<0.01),while the expression levels of the above proteins in the retinal tissues of the JQ1 treatment group were lower in statistics than those in the diabetic group(P<0.01)3 The scratch assay and transwell results indicated that the scratch distance gradually decreased,the cell migration distance increased,and the invasion ability increased with culture time.Compared with the control group,the cell migration distance and invasion were significantly increased in the high glucose and JQ1 groups(P<0.01),and after JQ1 treatment,the cell migration distance and invasion ability were significantly decreased compared with the high glucose group(P<0.01).Western blotting results revealed that BV2 cells activated the PI3K/AKT pathway after high glucose stimulation,and the protein expression levels of p-PI3K,PI3K,phosphorylation of AKT and AKT were increased,and the JQ1 treatment group could reduce the PI3K/AKT pathway activation to some extent,and the above protein expression showed a decreasing trend compared with the high glucose group.The PI3K pathway activity was directly increased in each group of cells after treatment with the PI3K pathway activator 740Y-P,and the results showed that the protective effect of JQ-1 on BV2 cells disappeared,and the expression levels of each protein of the phosphatidylinositol-4,5-bisphosphate 3-kinase/AKT cellular signal transduction pathway were not significantly different from those in the high glucose group.The results of immunofluorescence staining showed that the PI3K phosphorylation level was significantly increased in the cells stimulated with high glucose,and the relative fluorescence intensity was increased by about 3-fold compared with the control group(P<0.01),and post 48 h of JQ1 co-culture of the BV2 cells,the number of fluorescence positive cells were decreased,and the relative fluorescence intensity was decreased compared with the high glucose DMEM cultrue group(P<0.01).With the passage of time,the number of retinal ganglion cell layer cells in the blank control group did not change significantly,which showed that the number of retinal GCL cells in the STZ diabetic group was significantly reduced compared with the normal SD rat group,while the number of retinal ganglion cell layer cells did not change significantly on the first day and the third day after JQ1 intravitreal injection,but slightly increased on the 7 days as time went on.The number of ganglion cells in JQ1 injection group was lower than that in diabetes group,and the number of ganglion cells in JQ1 injection group increased after 7 days(P<0.01).To further verify the protective effect of JQ1 on the retina,the PI3K phosphorylation level of retina was observed by immunofluorescence staining.The level of PI3K phosphorylation was very low in the retina of the blank control group,significantly increased in the diabetes mellitus group,and could be reduced to some extent in the JQ1 treatment group,with a significant decrease in the number of green positive cells.Western blot method was adopt to measure the expression levels of PI3K/protein kinase B and MMP2 and MMP9 related proteins in the retinal tissues of each group,and the results showed that the expressions of p-PI3K/PI3K,p-AKT/AKT,MMP2 as well as MMP9 in the diabetic and JQ1 treated groups all showed an increasing trend(P<0.01),whereas the expression levels of the above proteins in the retinal tissues of the JQ1 treated group were significantly decreased(P<0.01)compared with the control group.3.The BV2 microglia high glucose model was established in vitro.The scratch assay and Transwell results indicated that with prolonged culture time,the scratch distance gradually decreased,the cell migration distance increased,and the invasion ability increased.Cell migration distance and invasion were increased by statistics meature in the high glucose and JQ1 groups compared with the blank control BV2 microglia group(P<0.01),and after JQ1 treatment,cell migration distance and invasion were significantly decreased compared with the high glucose group(P<0.01).Western blotting revealed that BV2 microglial cells activated the PI3K/AKT pathway after culture in high glucose DMEM,and the protein expression levels of p-PI3K,total PI3K,and Phosphorylated protein kinase B,as well as AKT,were increased,and PI3K/protein kinase B pathway activation was reduced to some extent in the JQ1 treated group,and the expression levels of the above proteins showed a decreasing trend compared with the high glucose group.And the effects of JQ1 on cell migration and invasion were analyzed using scratch assay and Transwell.Western blotting was used to analyze the PI3K/ protein kinase B pathway protein expression level in different groups of cells,and immunofluorescence staining was used to analyze the PI3K phosphorylation level in each group of cells to deeply explore the molecular signaling pathway by which JQ1 exerts its efficacy.Conclusion: 1.Diabetic retinopathy intravitreal injection of jq1 in rats ameliorates retinal ganglion cell loss and apoptosis,and has therapeutic effects to protect retinal ganglion cells,suggesting that JQ1 has a certain alleviating effect on diabetic retinopathy.2.JQ1 can reduce the levels in statistics of inflammatory factors such as TNF-α,etc in the retina of rats with diabetic retinopathy,possibly by reducing the migratory and invasive capacity of microglia and inhibiting microglial activation.3.JQ1 could downregulate PI3K/AKT pathway gene expression and decrease PI3K phosphorylation levels induced by high glucose stimulation in diabetic model animals and in cells.It suggests a therapeutic approach targeting BET as a therapeutic target for intervention,with a promising future for the treatment of diabetic retinopathy. |
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