Immunochemical detection of glycated β- andγ-crystallins in lens and their circulating autoantibodies (IgG) instreptozocin induced diabetic rat
[摘要] Purpose: This study used an immunochemical approach aimed to detectthe glycated crystallins (β- and γ-crystallin) in rat lens andtheir circulating specific autoantibodies in serum during the course ofcataractogenesis.Methods: Streptozocin (STZ; 55 mg/kg body mass) induced diabeticmale Wistar/NIN rats (2-3 months old) and control nondiabetic rats wereused for this study. Plasma glucose, glycated hemoglobin and body weightwere evaluated on day zero, and at the interval of every two weeks up tothe eighth week of post-injection in both the groups. Other biochemicalparameters, such as the levels of nonprotein sulfhydryl (-SH) groups andthe activity of γ-glutamyl transpeptidase (γ-GT) in lensproteins were also estimated. Cataract progress was monitored bymeasuring the advanced glycation end product (AGE)-like fluorophores inboth intact lens as well as in lens homogenate employing digital basedimage analysis and spectrofluorimetric methods. Similarly, thepolyclonal antibodies specific to β-glycated-, γ-glycated-,β-, and γ-crystallins were used to determine the concentrationof respective immunogens in lens by noncompetitive ELISA and theirrespective circulating antibodies by antibody capture assay. The profileof glycated lens protein (soluble and insoluble fractions) during thecourse of cataractogenesis was assessed by the western blot technique.Results: STZ induced diabetic rats showed typical signs of diabetes(hyperglycemia, increased water and food intake with no increase in bodyweight). Biochemical analysis of total lens protein showed a significant(p=<0.001) decrease in the levels of nonprotein -SH groups. Theactivity of lenticular γ-GT in diabetic rats was found to beunaltered as compared to the control group. Digital analysis of intactlens illustrated a positive correlation (r2=0.888) with theformation of AGE-like fluorophores during the course ofcataractogenesis. A similar trend was also observed in the levels ofAGE-like fluorophores in the total lens homogenate of diabetic animalsduring the course of cataractogenesis. The concentration of β- andγ-glycated-crystallins in the rat lens (soluble and insolublefractions) was analyzed by non-competitive ELISA. The concentration ofβ- and γ-glycated-crystallins were found to be enhanced by theend of week eight, as compared to the control group. Concomitantly,crystallin-specific (β- and γ-glycated-crystallin)autoantibodies were also detected in the serum of the diabetic rats fromweek two onwards. Western blot analysis indicated the formation ofenhanced glycated lens crystallins (β- and γ-crystallin) in theinsoluble fraction.Conclusions: The following was observed during the course ofcataractogenesis: (1) there was an enhanced formation of AGEs-likefluorophores in intact lens; (2) β- and γ-glycated-crystallinlevels increased in the rat lens (insoluble fraction) by the end of weekeight; and (3) release of these glycated lens proteins into peripheralcirculation resulted in the production of autoantibodies to β- andγ-glycated-crystallins that could be detected as early as week two,after induction of diabetic status in experimental rats.
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[效力级别] [学科分类] 生物化学/生物物理
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