The Role Of LRG1 In Diabetic Retinal Microangiopathy

PartⅠ: Serum and vitreous concentration of LRG1 in diabetic retinopathyObjective:LRG1(leucine-rich-alpha-2-glycoprotein1)protein,a highly conserved member of the leucine-rich repeat family of proteins(leucine-rich repeat,LRR),is involved in the processes of abnormal angiogenesis and cell apoptosis.In this study,quantitative detection of LRG1 was applied to the serum of the normal population group,diabetic patients without diabetic retinopathy(DR),with non-proliferative diabetic retinopathy(NPDR),proliferative diabetic retinopathy(PDR)and the PDR patients’ vitreous fluid,to explore the possible role of LRG1 in the onset and during the development of DR.Methods:The study included experimental groups and control group of patients at a total of 119,among which are 22 control subjects,22 diabetic patients without DR,20 with NPDR,22 with PDR,plus 33 subjects ready to receive vitrectomy,22 due to PDR and11 due to epiretinal membranes ormacular hole(diabetes excluded).Serum was obtained from blood samples from all subjects by centrifugation and was stored at-80°C until analysis,as well as vitreous samples of last two groups of patients during surgery.Enzyme-linked immunosorbent assay(ELISA)method was used to detect the concentrations of LRG1 protein.The resulting data were presented as means± SD or median(interquartile range).Kolmogorov-Smirnov test was utilized to determine data normality.The differences of characteristics between two groups were compared using two independent samples t test while Chi-square tests,one-way ANOVA,or Kruskal-Wallis test were used between multiple groups.? values less than 0.05 were considered to be statistically significant.Results:No significant difference was found in age or gender between different groups(P> 0.05),so that the experimental groups were comparable.The serum concentration of LRG1 in PDR patients(9025 ± 1994 pg / mL)were markedly increased compared with control group(5975 ± 2069 pg /mL),diabetic patients without DR(6550 ± 2203 pg / mL)and NPDR patients(6771 ± 2120 pg / mL),all with significant difference(P <0.0001,P <0.001,P <0.005).There were no significant differences between the restthree groups.The vitreous LRG1 levels in PDR patients(562.1 ± 273.5 pg /mL)were significantly higher than control group(130.0 ± 102.8 pg / mL).In addition,the serum LRG1 concentration was not correlated with the vitreous.(r = 0.1415,P = 0.5196> 0.05).Conclusions:Serum and vitreous LRG1 levels in PDR patients were markedly increased compared with control group.There’s no correlation between the serum LRG1 concentration and the vitreous one.The practicability of rapid screening PDR by detecting the blood level of LRG1 needs further investigation.Part Ⅱ : The effect of LRG1 on pericyte apoptosis induced by high glucoseObjective:To investigate the LRG1 levels on diabetic rat retina induced by streptozotocin and primary bovine retinal capillary pericytes cultured with high glucose,and to detect the change of pericyte apoptosis influenced by the level of LRG1,the possible intrinsic mechanisms were explored preliminarily,in order to provide new targets for the prevention and treatment of DR.Methods : Diabetic mouse model was established by STZ and the primary cultured bovine retinal pericytes were isolated and cultured.TheLRG1 expression level on mouse retina at 8 weeks was detected by Western blot in the control group and STZ-induced diabetic rats.The immunohistochemistry assay was usd to locate the expression of LRG1 and validate the immunoreactive in retinal.LRG1 protein levels,mRNA levels and cellular immune staining in bovine retinal microvascular pericytes under high glucose were detected by Western blot method,quantitative PCR,immunohistochemistry assay respectively.LRG1 knockout model was established by transfection of siRNA on cellular level.MTS,TUNEL and flow cytometry were applied to test apoptosis ratio of retinal capillary pericytes induced by high glucose.The expression of BCL-2 family protein,PI3K/Akt pathway and MAPK pathway changed with different LRG1 levels,as well as the pericytes’ apoptosis under the high glucose condition were measured by Western blot and the MTS assay.Results:1.The diabetic rat model is successfully established.Compared with the control group,LRG1 protein expression was increased in the retinal blood vessels,the difference was statistically significant(P <0.05),and LRG1 mainly expressed on blood vessels.2.In 30 mmol / L high glucose condition,LRG1 protein and mRNA extracted from bovine retinal pericytes was significant ly higher than control group(P <0.001).And LRG1 mainly expressed in the cytoplasm.3.Transfection of LRG1 siRNA targeted toinhibiting LRG1 expression in normal glucose concentration had no significant effect on cell viability(P > 0.05).Unlike in high glucose conditions,it can significantly reduce the pericytes’ apoptosis(P <0.005).4.Under conditions of high glucose regulation PDGF pathway can be significantly reduced apoptosis of pericytes(P <0.05).5.Under high glucose,pericyte apoptosis-related factors Bax,Bcl-2,p-Akt,p-P38,p-JNK level changes,which can be reduced by the suppression of LRG1 accordingly.6.Under conditions of high glucose,inhibiting PI3 K / Akt pathway can weaken the protective effect of the intervention of LRG1 in pericytes(P <0.05).Conclusions : 1.LRG1 protein was expressed in retinal vascular structure in SD rats,as well as in pericytes’ cytoplasm,and the expression was significantly higher at high glucose;2.The upregulation of PDGF can reduce high glucose-induced pericyte apoptosis;3.Under high glucose conditions,the inhibition of LRG1 attenuated pericytes’ apoptosis;4.PI3 K /Akt pathway,P38 MAPK pathway may be involved in this regulation.