The Establishment Of Sugar Cataract Related Models, Mechanism Research And Investigation Of Anti Sugar Cataract Agents

Diabetes has aleady been the global epidemic disease.Cataract,opacification of the lens,is closely associated with diabetes as one of the major late complications.Diabetic cataract has seriously affected the quality of patients' life.Different from the normal age-related cataract,diabetic cataract is mainly caused by high concentration of glucose in body fluid and tissue,rather than oxidative stress in lens.Diabetic cataract and galactosemic cataract are together called "sugar cataract".Lens osmotic expansion is a relatively irreversible pathological period in the early stage of sugar cataract.It triggers the occurrence of oxidative stress and the post-translational modification of lens proteins,accelerates the formation of frank cataract,but the precise pathological mechanism of each stage is not clear.Because of the uncertainty of sugar cataract mechanisms and the specificity of ophthalmic pharmacology,the investigations of sugar cataract and anti-sugar cataract drugs develop relatively slowly.The relationship of polyol pathway and diabetic cataract has engendered extensive investigations over the last several decades.Many drugs with varying AR-inhibiting efficacy(e.g., sorbinil,tolrestat and zopolrestat) have been synthesized and tested.Initial trials in animal models showed significant protection against diabetic complication.ARI,in addition to preventing diabetic or galactosemic cataract,ameliorate some of the features of diabetic nephropathy and neropathy. However,clinical trials with AR inhibitors have yielded uncertain results,in part due to the high nonspecific toxicity of this class of drugs.And now only AR inhibitor,Epalrestat,appears on the Japanese market,which is mainly used to anti-diabetic nephropathy.Since there is no effective drugs to treat diabetic cataract,most patients rely on surgery to remove the suffering.In this paper,focus on lens osmotic hypothesis occuring in the early stage of sugar cataract.First of all,optimize and set up a series of sugar cataract associated research methods and models. According to the characters of enzyme reaction,comparing the efficiency of heating,cooling,freezing, acidification and alkalization termination methods to improve the screening methods of ARI.On cellular level,try to establish the primary cultivation methods of lens epithelial cells,which are located below the anterior capsule of lens.On organ level,remove the intact lenses of rat,rabbit and dog with different methods,and establish the organ cultivation method in vitro.On the basis of lens culture,using galactose and sodium selenite/hydrogen peroxide to induce the formation of lens osmotic expansion and oxidative turbidity respectively.Establish economic galactosemic cataract model by continuously adjusting the time and concentration of galactose solution(10-12.5%).In order to rule out the gender interference and facilitate the administration of eye drops,gradually increasing the age of newborn rats and regulating the concentration of sodium selenite to improve the selenic cataract model in vivo.At the same time,study the formation of STZ-induced diabetic cataract in vivo.In the investigation of lens osmotic expansion,morphological changes and biochemical results showed that high concentration of galactose/glucose could induce the osmotic swelling of rat lenses. And also demonstrated the up or down regulations of AR(polyol pathway associated protein),AQP0, AQP1(water metabolism associated proteins),Clcn3 and P-gp(ion metabolism associated proteins) occured during the formation of osmotic expansion.Besides,180 non-orient synthetic compounds and 44 natural extracts are screened using AR inhibitor high-throughput screening system in vitro,and then investigate the anti sugar cataract of two active samples,one is Chinese traditional medicine recipe,ZM and another is a natural compound, DG.They both have good dose-response relationship,the IC₅₀ values of ZM and DG to AR are calculated 27.68μg/ml and 4.59μM respectively.In the lens culture test,ZM and DG can partly prevent the lens osmotic expansion induced by 50 mM galactose.In animal experiments,ZM has no effects to delay or inhibit the progression of galactosemic or diabetic cataract either as eye drops or given orally.As eye drops,DG also has no effect to obviously attenuate the progression of galactosemic cataract.When given orally,DG can partly delay the formation of galactosemic and diabetic cataract.The present study can be mainly concluded that:1.Using tissue explant technique,we has established the primary culture methods of LECs from rat,rabbit,dog and human.2.The intact lenses of rat,rabbit and dog can be removed gently by post-capsule,anterior-capsule or side-capsule and cultivated in vitro.3.Galactose and sodium selenite/hydrogen peroxide can induce rat lenses to form osmotic expansion or oxidative turbidity respectively.4.Galactosemic cataract can be induced on 21-day old rats by drinking 10-12.5%galactose solution.5.Selenic cataract can be induced on 16-day old rats by one-time subcutaneous injection of sodium selenite solution(20μmol/kg).6.Lens osmotic swelling occurs during the formation of galactosemic cataract and diabetic cataract in vivo.7.With lens osmotic expansion,AR,AQP0,AQP1,P-gp and Clcn3 have been up-regulated or down-regulated in mRNA levels.8.The ZM has strong AR inhibitory activity,and can partly prevent the lens osmotic expansion in vitro;but it can't delay the progression of galactosemic and diabetic cataract in vivo.9.A natural compound,DG possesses a significant AR inhibitory activity,it can partly prevent lens osmotic expansion induced by galactose in vitro;and also obviously delay the onset and slow the progression of galactosemic and diabetic cataract when given orally in vivo.