| Objective: To investigate the correlation between the development of diabetic retinopathy and changes in serum Klotho protein levels with the prolongation of the diabetes mellitus course and changes in laboratory indicators.At the same time,cellular level studies were conducted to observe the effect of recombinant Klotho protein on the function of diabetic retinal pigment epithelial cells,and to comprehensively reveal the molecular basis of Klotho protein in the treatment of diabetic retinopathy from a holistic perspective.Method:(1)Eighty patients with DR and 30 patients with DN who were hospitalized in the ophthalmology department and endocrinology department of the People’s Hospital of Inner Mongolia Autonomous Region from January 2021 to August 2021 were selected as the experimental group.And 33 patients with ophthalmic cataracts and retinal fissures unrelated to metabolic diseases were also selected as the control group.The enzyme linked immunosorbent assay technique was used to detect Klotho protein in peripheral serum,analyze the significance of the changes in Klotho protein content in patients of the DR group,DM group and NC group,and the experimental results were analyzed as data to determine the diagnostic significance of klotho protein for diabetic retinopathy.(2)The pigment epithelial cell model of diabetic retinopathy was constructed and cell passages were performed.After the cells were resuscitated,they were grouped and HRP tracing was performed to detect the phagocytosis of RPE cells under the conditions of low and high glucose,and to detect the change of phagocytosis of RPE cells after the addition of recombinant Klotho protein under the conditions of high glucose.The cells were also grouped and the effect of Klotho on VEGF secretion by RPE cells was detected by ELISA.And the changes of IGF-1Rβ and VEGFR2 expression in RPE cells after WB assay treatment were analyzed.Result:(1)Statistical results of biochemical indexes and serum ELISA tests of patients showed that serum creatinine,blood urea,blood glucose and glycosylated hemoglobin were different among the three groups(P<0.001),and Klotho expression could be influenced by various physiological factors and was negatively correlated with serum creatinine.The results of correlation between changes in serum Klotho protein levels of patients and metabolic indicators of patients pointed out that compared to controls,serum Klotho protein was reduced in patients with type 2 diabetes and significantly lower than that in diabetic retinopathy,with a progressive decrease with increasing degree of DR(p<0.001).Single factor correlation analysis yielded a negative correlation between Klotho protein levels and DR,and logistic regression analysis indicated that each unit increase in Klotho protein was associated with a 0.420-fold decrease in the risk of disease.The ROC curve analysis showed that Klotho can be used for the risk assessment of DR conditions.(2)In vitro culture of ARPE-19 cells and observation revealed that treatment of primary human RPE cells with recombinant Klotho protein(10 ng/ml)for 30 minutes or 16 hours examined the phagocytosis of RPE cells.The incubation of RPE cells with Klotho for 30 minutes reduced the phagocytosis of RPE cells(p < 0.01),while long-term incubation for 16 hours significantly elevated the phagocytosis of RPE cells(p < 0.01).At the same time,to test the ability of Klotho protein to inhibit VEGF secretion from human RPE,VEGF secretion from the upper and lower chambers of Transwell was significantly reduced after treatment of polarized RPE cells with recombinant human Klotho.The analysis of the results of Western blot assay showed reduced protein content of Klotho protein to IGF-1R tyrosine phosphorylation and that Klotho concentrations at 20 ng/ml and 40 ng/ml inhibited IGF-1R tyrosine phosphorylation(p<0.001),indicating that Klotho can inhibit VEGF secretion by inhibiting IGF-1 signaling in human RPE.The Klotho assay for VEGFR2 phosphorylation showed a decrease in p-VEGFR2 with increasing Klotho protein and a concomitant decrease in VEGFR2 content.Conclusion: Lower levels of the circulating vasoprotective hormone Klotho are associated with an increased risk of diabetic retinopathy development and progression in patients with type 2 diabetes.It was found that Klotho promotes phagocytosis of the RPE,inhibits VEGF secretion,and attenuates the development of retinopathy.Based on these findings,we proposed that Klotho has a protective function in DR and is an important regulator of RPE physiology.Thus Klotho may be a novel biomarker for diabetic retinopathy and a potential therapeutic target for diabetes and diseases. |
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