Relationship Between Serum Klotho Protein Level And Retinopathy In Patients With Type 2 Diabetes Mellitus And Its Underlying Mechanisms

PART 1 CHANGES OF SERUM KLOTHO LEVEL IN PATIENTS WITH TYPE 2 DIABETES AND ITS RELATIONSHIP WITH RETINOPATHY AND OXIDATIVE STRESS INDICATORSObjective: To observe the changes of serum Klotho(KL)level in patients with type 2 diabetes and its relationship with retinopathy and oxidative stress indicators.Methods: Seventeen healthy people(NC group)and sixty patients with type 2 diabetes(DM group)were included.According to the results from fundus fluorescein angiography,the diabetic patients were divided into three groups: without DR(NDR group),non-proliferative DR(NPDR group)and proliferative DR(PDR group).In all subjects,the anthropometric and biochemical indexes were collected.The body mass index(BMI)was calculated.Venous blood samples were collected and the serum was isolated.Serum KL protein level was measured by enzyme-linked immunosorbent assay(ELISA).Malondialdehyde(MDA)level was detected by the thiobarbituric acid method,and superoxide dismutase(SOD)activity was measured by the xanthine oxidase method.The serum KL level in each group was compared,and the correlations between KL and DR,SOD,MDA were analyzed.Resultes:(1)Comparison of serum KL protein levels: The serum KL level was lower in DM group than that in NC group.Furthermore,subgroup analysis showed that the serum KL level in NDR,NPDR and PDR group,was gradually decreased.(2)Correlation analysis between serum KL level and DR: A Spearman analysis indicated an obvious relationship between the serum KL level and DR.Furthermore,a logistic regression analysis was performed and showed that the decreased serum KL level was an independent risk factor for DR.(3)Comparison of oxidative stress indicators: Compared with the NC group,the serum SOD activity was significantly decreased,while the MDA level was increased in DM group.In NDR,NPDR and PDR subgroups,SOD activity was gradually decreased,and the MDA level was gradually increased.(4)Correlation analysis between serum KL level and SOD,MDA and clinical indicators.Spearman analysis was performed in all diabetic patients.Results showed that the serum KL level was positively correlated with SOD activity,and negatively correlated with MDA.Furthermore,a multiple linear stepwise regression analysis was performed and showed that the SOD was independently related with serum KL protein.Conclusion: Serum KL level was significantly decreased in diabetic subjects,and the decreased serum KL level was an independent risk factor for DR.In diabetic patients,the oxidative stress level was increased,and the serum KL protein was closely related with SOD.PART2 EFFECTS OF KLOTHO ON APOPTOSIS AND REACTIVE OXYGEN SPECIES GENERATION INDUCED BY PALMITIC ACID IN HUMAN RETINAL ENDOTHELIAL CELLS AND ITS POTENTIAL MECHANISMSObjective: To examine the effects of recombinant Klotho protein(KL)on palmitic acid(PA)-induced apoptosis and reactive oxygen species production(ROS)in human retinal endothelial cells(HRECs)and to explore the potential mechanisms.Methods: HRECs were cultured in vitro using the medium with glucose concentrations of 15 mmol/L to simulate the high glucose environments in the diabetic state,and were randomly divided into control(Ctr)group,PA group,PA + KL group,PA + KL + LY294002 group.The apoptosis rate was detected by flow cytometry assay.The protein expression of Bax?Bcl-2 and Cleaved-caspase 3 was detected by W intracellular ROS was detected by 2'7'-(DCFH-DA)method.Results: Compared with the Ctr group,the apoptotic rate in PA group was significantly increased(P < 0.05).Western blot showed that the expression level of anti-apoptotic protein Bcl-2 was lower and the pro-apoptotic protein Bax and Cleaved-caspase 3 were higher in PA group that in Ctr group,and the Bcl-2/Bax was decreased in PA group(P < 0.05).Additionally,PA significantly increased the intracellular ROS level.This suggested that PA could induce apoptosis and ROS generation in HRECs.After pretreatment with KL,the increased apoptosis rate induced by PA was significantly decreased,and the Bcl-2 expression was up-regulated and Bax and Cleaved-caspase 3 expressions were down-regulated.Besides,the Bcl-2/Bax was increasd and the ROS generation was attenuated by KL protein.This suggested that KL could inhibit the apoptosis and ROS production induced by PA in HRECs.Furthermore,the results showed that the protein expression of p-Akt was significantly decreased after treatment with PA(P < 0.05),while KL protein could significantly recover the decreased expression level of p-Akt induced by PA(P < 0.05).Then the PI3K/Akt signaling pathway inhibitor LY294002 was added,and the results showed that it could obviously attenuate the anti-apoptotic and antioxidant effects of KL protein.Conclusions: KL protein could ameliorate the apoptosis and ROS production induced by PA in HRECs.The mechanism was involved in the activation of PI3K/Akt signaling pathway.