| Part one Effect of curcumin on PI3K/Akt/mTOR signal pathway in retina of DR ratsObjective: To detect the expression of PI3K/Akt/mTOR signaling pathway related cytokines in the retina of DR rats.Methods:1.Establish DR rat model by streptozocin(60mg/kg)intraperitoneal injection and intravitreous vascular endothelial growth factor(0.05 μg).Divide the rats into control group,DR group and different curcumin concentration groups(0.25 μg/μL,0.5 μg/μL,0.75 μg/μL).Observe the retinal pathological changes in each group by HE staining.2.Detect PI3 K,Akt and mTOR mRNA in the retina of each group by fluorescent quantitative PCR.3.Detect the expression of PI3 K,Akt,mTOR in the retina by western bolt.Results:1.In the control group,the retina structure was intact,and the granule cell morphology and intercellular space were normal and the cells were arranged closely and orderly.The inner and outer plexiform layers were arranged in a network.In the DR group,the structure of the retina showed different degrees of edema.The structural integrity was disrupted and loose.The inner and outer granular layer cells were swollen and disordered,and the cell gap was enlarged.Capillary dilatation and some congestion were observed.Visible swelling in the inner and outer plexiform layers,and scattered angiogenesis in the outer plexiform layer were shown.The structural changes of the retina in the 0.25 μg/μL curcumin concentration group were similar to those in the DR group.The retinal structure of rats in 0.5 μg/μL group was almost complete,and the edema of each layer was less than that of 0.25 μg/μL group.The structure of inner and outer granular layer was relatively complete,the arrangement was relatively regular,and the swelling of inner and outer plexiform layers was reduced.Significant telangiectasia,congestion,and neovascularization were not found.The retinal structure of the rats in the 0.75 μg/μL group was almost complete,and no edema was observed in each layer.The inner and outer granule cells were arranged regularly,and the swelling of the inner and outer plexiform layer was significantly improved compared with the DR group.No telangiectasia,congestion or neovascularization was observed.2.Compared with the control group,PI3 K,Akt and mTOR mRNA in the DR group were significantly increased.Compared with the DR group,PI3 K,Akt and mTOR mRNA in the curcumin group were significantly decreased,and they declined as the curcumin concentration increased.When the curcumin concentration was 0.75 μg/μL,the levels of PI3 K,Akt and mTOR mRNA were the lowest.(P <0.05).3.Compared with the control group,the expression of PI3 K,Akt and mTOR in the DR group was significantly increased.Compared with the DR group,the PI3 K,Akt and mTOR in the curcumin group were significantly decreased,and they declined as the curcumin concentration increased.When the curcumin concentration was 0.75 μg/μL,the levels of PI3 K,Akt and mTOR were the lowest.(P <0.05).Part two Curcumin inhibits high glucose-induced inflammatory injury in human retinal pigment epithelial cells through the ROS-PI3K/Akt/mTOR signaling pathwayObjective: To investigate the role of curcumin in alleviating high glucose-induced RPE inflammatory factor secretion,ROS production,PI3K/Akt and mTOR activation.Methods:1.ARPE-19 cells were randomly divided into 5 groups,control group,high glucose treatment group(HG),HG+ different concentrations of curcumin group(CUR1μmol/L,5μmol/L,10μmol/L),RPE viability was measured by CCK-8 assay.2.ARPE-19 cells were randomly divided into(1)control group,HG group,mannitol group(Mannitol);(2)control group,HG group,HG+N-acetylcysteine group(HG+NAC),HG+LY294002 group,HG+Rapamycin group.The contents of IL-1β,IL-6 and TNF-α in the cell culture supernatants of group(1)and group(2)were detected by ELISA.Intracellular reactive oxygen species(ROS)levels were measured by laser scanning confocal microscope in group(1).The expression of Akt、p-Akt、mTOR、p-mTOR in the retina were assessed by western bolt in group(2).3.ARPE-19 cells were randomly divided into HG group,HG+NAC group,NAC group.The expression of Akt、p-Akt、mTOR、p-mTOR in the retina were assessed by western bolt.4.ARPE-19 cells were randomly divided into control group,HG group,HG+CUR 10 μmol/L group,CUR 10 μmol/L group.The contents of IL-1β,IL-6 and TNF-α in the cell culture supernatants were detected by ELISA.ROS levels were measured by laser scanning confocal microscope.Akt、p-Akt、mTOR、p-mTOR in the retina were assessed by western bolt.Results:1.High glucose reduced RPE viability,increased TNF-α,IL-6 and IL-1β secretion,increased ROS formation,and promoted phosphorylation of Akt and mTOR.2.The antioxidant N-acetylcysteine(NAC),PI3K/Akt inhibitor LY294002 and mTOR inhibitor rapamycin ameliorated the effect of high glucose.3.Pretreatment with 10 μmol/L CUR reduced secretion of TNF-α,IL-6 and L-1β,ROS formation,and phosphorylation of Akt and mTOR.Part three Expression and significance of PI3K/Akt/mTOR signaling pathway related factors in vitreous of DR ratsObjective: To observe the retinal pathological changes of DR rats and detect the expression of PI3K/Akt/mTOR signaling pathway related cytokines in the vitreous humor.Methods:1.Establish diabetic rat model by streptozocin(60mg/kg)intraperitoneal injection.Establish diabetic retinopathy rat model by intravitreous vascular endothelial growth factor(0.05 μg).The rats were divided into control group,IVE 2 weeks group(2w),4 weeks(4w)and 8 weeks(8w).2.Observe the retinal pathological changes in each group by HE staining.3.Detect the expression of PI3 K,Akt,mTOR in the vitreous humor by ELISA.Results:1.Fasting blood glucose in diabetic rats was more than 16.7mmol/L,model establishment rate was100%.2.In the control group,the retina structure was intact,and the granule cell morphology and intercellular space were normal and the cells were arranged closely and orderly.The inner and outer plexiform layers were arranged in a network.In the 2W group,the cells in the inner and outer layers of the retina were sparse,slightly disordered,and vacuoles were seen in the inner and outer plexiform layers.In the 4W group,the inner and outer granule cells were further sparse and arranged disorderly.Visible vacuolar-like changes,vacuolization changes in the inner and outer plexiform layers were shown.In the 8W group,the structure of the retina showed different degrees of edema.The structural integrity was disrupted and loose.The inner and outer granular layer cells were swollen and disordered,and the cell gap was enlarged.Capillary dilatation and some congestion were observed.Visible swelling in the inner and outer plexiform layers,and scattered angiogenesis in the outer plexiform layer were shown.3.Compared with the control group,the expression levels of PI3 K,Akt,and mTOR in the vitreous humor of 2W group,4W group and 8W group were significantly increased.In each group of diabetes,they were significantly higher in 8W group than that in the other two groups,and they were significantly lower than that in group 4W(P <0.05).Conclusions:1.The PI3K/Akt/mTOR pathway-related cytokines in the retina of DR rats were significantly increased;the corresponding mRNA of each cytokine increased at the same time;curcumin had protective effect on the retina of DR rats;its mechanism may be related to PI3K/Akt/mTOR pathway.2.In high glucose environment,the expression of TNF-α,IL-1β,IL-6 inflammatory factors in RPE increased,ROS increased,phosphorylation levels of Akt and mTOR increased;NAC,PI3K/Akt inhibitor,mTOR inhibitor blocked the expression of inflammatory factors;curcumin has a similar function as the ROS/PI3K/Akt /mTOR signaling pathway inhibitor and can interfere with ROS-PI3K/Akt /mTOR signaling in ARPE-19.3.A satisfactory animal model of diabetes can be obtained by intraperitoneal injection of STZ.The ideal DR animal model can be obtained by intravitreal injection of VEGF.The retinopathy gradually increases with the prolongation of diabetes duration.The PI3 K,Akt and mTOR cytokines in the vitreous increase with the severity of the disease. |
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