The Analysis Of The Oxidative And Antioxidative Level In The Blood Of The Patients With Eales Disease

Eales disease is an idiopathic retinal vascular inflammatory disease. Itnow seen more commonly in the asian countries such as India and China.The disease most commonly affects healthy young adults, predominantlymale. It has become the primary cause that lead to blindness in youngadults. Since the disease seen more commonly in the Indian subcontinent,little research has been done in the developed countries. Although theincidence rate of the disease is high, the basic research of the disease islittle in our country. Currently, the major research of the disease comefrom India. Untill now, the precise etiology of this disease is stillundetermined. Base on the theory that oxidant damage participate in manypathologic process of the inflammatory diseases, we evaluated theoxidative and antioxidative level in the blood of the patients with Ealesdisease, in order to assess the relationship between the oxidant stress andEales disease.1.Patients and control subjectsTwenty patients with Eales disease were enrolled in this study, andthey were divided into two groups. Ten of them which were untreatedbefore the study served as untreated group and the others which wereclinical cured served as cured group. Ten healthy young blood donors wereselected as the control group. All of the subjects were male and agedbetween 20 and 40. They were all non-obese, non-smoking, non-alcoholic,and none of them has taken antioxidant drugs and steroids in the recentmonths before the blood sample were collected.To ensure the representativeness of the patients, In our study, Ealesdisease were diagnosed by the criteria suggested by Biswas,MS andslightly modified by ourself. The clinical cured group was characterized bythe clear of vitreous hemorrhage, no proliferative vitreouretinopathy,peripheral venous sheathing or slcerosis without retinal edema andhemorrhage and no leakage were seen in FFA.2.Evaluated the biomarker of oxidative damage in patients withEales disease.8-OHdG and TBARS were served as biomarker of oxidative damageto DNA and lipid peroxidation respectively, and they were detected in theblood of the patients with Eales disease.1) Assay the level of 8-OHdG: DNA was extracted from 100ul bloodby the Blood Genome DNA Extraction Kit, and its quantity and purity wasdetermined by spectrophotometer. 5ug DNA was hydrolyzed by nucleaseP1 and alkaline phosphatase, and purified by Wizard DNA Clean-upSystem. The product was derivatized by BSTFA and acetonitrile, and theproduct was analysis by GC/MS. The ions with m/z 368 and 383 whichappeared most intense in Mass Spectrometry were selected as thecharacteric ions. The quantity of 8-OHdG was calculated by the ratio of theamplitude area between the sample and the standard.Compared with the control, the level of 8-OHdG was significantly risein both groups of the patients with Eales disease. And the level of8-OHdG in the untreated group was significantly higher than the clinicalcured group. The result implicated that serve oxidant damage in thepatients and the status can not return to normal even in the clinical curedstage.2) Assay the level of TBARS: 100ul serum were collected and thequantity of MDA were measured by the TBARS assay kit. The result showthat the level of MDA was significantly rise in both groups of the patients.And the level of MDA in the untreated group was significantly higher thanthe clinical cured group. The result implicated that serve lipid peroxidationin the patients and the status can not return to normal even in the clinicalcured stage.3. Determine the gene expression of inducible nitric oxidesynthase (iNOS) in the leucocytes and the capacity of the iNOSin the serum.To evaluate the function of iNOS and NO in the pathologic process ofEales disease by the method of determine the gene expression of iNOS inthe leucocytes and the capacity of the iNOS in the serum.1)The gene expression iNOS in the leucocytes: leucocytes wereseparated from 1ml blood by ficoll hypaque density gradient solution.RNA were extracted from the leucocytes. Its quantity and purity wasdetermined by spectrophotometer. ?-actin was served as referencetemplate and was amplificated with the target gene segment of iNOS bythe method of RT-PCR. The ratio of the target gene to β-actin PCRproduct intensity was indicated the level of the target gene expression. Theresult shown that the level of the expression of target gene in both groupsof the patients was significantly higher than the control. Furthermore, thelevel was significantly higher in the untreated group than the clinical curedgroup.2) The capacity of iNOS in the serum were evaluated by the assay kit.The result shown that the capacity of iNOS in both groups of the patientswas significantly higher than the control. the capacity was significantlyhigher in the untreated group than the clinical cured group.Too much NO can lead to lethal oxidant damage to the cell. SinceiNOS can generate and release amount of NO in the status of inflammatory,the level of NO in the tissue was determined by the level of iNOS. Theresult of our research indicated that the oxidant damage induced by the NOplay an important role in the pathologic process of Eales disease.4. Evaluated the antioxidant capacity in the blood of the patientswith Eales disease.The antioxidant system in the body is consist of enzymatic andnon-enzymatic antioxidant defense system. The enzymatic antioxidantinclude superoxide disutase (SOD), glutathione peroxidase (GPX) andcatalase (CAT). The non-enzymatic antioxidant include vitamine, aminoacid and metal-binding proteins. In our study, we detected the capacity oftotal antioxidant, SOD, GPX and the level of vitamine E in the serum inorder to access the capacity of antioxidant in the serum of the patients withEales disease.All of the subjects were determined by the corresponding assay kits.The result revealed that the antioxidant capacity of the patients significantlylower than the control, and the antioxidant capacity of the clinical curedgroup higher than the untreated group significantly.The result of our research reveal that an imbalance between theoxidant and the antioxidant in the blood of the patients with Eales disease.Furthermore, the severity of the imbalance varied with the stage of thedisease. We suspect that the oxidant stress play a important role in theetiology and the pathology of the Eales disease.This theory may be give a interpretation to the result that smoking may bea risk factor to Eales disease which concluded in our previous epidemicresearch.The retina is considered to be the tissue most susceptible for oxidativestress among other body tissues. Oxidative damage has been proved totake part in the pathologic process of many diseases of the retina.Moreover, antioxidant therapy has been proved effective in many retinadisease. Based on the result of our research, we believe that inhibitingiNOS and supply VitE might be beneficial to patients with Eales disease.