DL-3-n-butylphthalide Alleviate Oxidative Stress In Diabetic Cataract And The Mechanisms

Objective:Reactive oxygen species(ROS) play an important role in the pathogenesis of diabetic cataract(DC). DL-3-n-butylphthalide(NBP) is an antioxidant that is now a therapeutic drug for ischemic stroke. In this study, we aimed to investigate the effect of oral administration of NBP on the development of diabetic cataract in rat diabetes model and the relationship to endogenous transcriptional Activator NF-E2-related factor 2(Nrf2) and its downstream antioxidant enzymes.Methods:1 Male Sprague-Dawley(SD) rats of 6 weeks old(200 ± 15g) were randomly divided into three groups:(1)Normal control group(C group):15 rats(30 eyes);(2)Diabetic group(DM group):15 rats(30 eyes);(3)DM+NBP group:15 rats(30 eyes).2 The rat of DM and DM+NBP groups received a single intraperitoneal injection of STZ at a dose of 60mg/kg. For the control, the rats received the injection with sodium citrate buffer only. The model of diabetes was considered to be successful when the blood glucose was≥16.7mmol/L and the glucose in urine was+++~++++after 72 hours of the injection.3 NBP was dissolved in vegetable oil and administered by oral gavage 5 d/week at a dose of 80mg/kg body weight. Control and DM group received oral gavage in the same manner with vegetable oil only.4 Fasting blood glucose level and the body weight of each rat were measured every 2 weeks and those who do not meet the criteria were discarded. Eyes were examined every week using a slit lamp biomicroscope on dilated pupils and scored every three weeks Initiation. After 12 weeks treatment, animals were sacrificed and the eyeballs were removed for biochemical analysis. The lens of the left eye was fixed in 4% parafo- rmaldehyde in 0.01 mol/L PBS for histological and immunohistochemisty examinations. The lens from the right eye was stored at-80℃ for biochemical measurements analysis. Blood samples were collected from the femoral vein after removal of the eyeballs. Serum was extracted from EDTA-treated whole blood samples for the levels of blood glucose and 8-Hydroxydeov-exyguanosine(8-OHd G) examinations. The oxidative stress parameters such as 4-hydroxynonenal(4-HNE) and MDA were also determined in the lenses by Western blot and Thiobarbituric acid analyses. The expression of NF-E2-related factor 2(Nrf2) and its downstream antioxidant enzyme, catalase and nuclear accumulation of Nrf2 were determined by Western blot and immunohistochemisty analyses.5 Statistical analysis:SPSS13.0 software was used to analyze the data. All data were expressed as mean ± SD and analyzed by one-way ANOVA. A p value less than 0.05 was considered statistically significant.Results:1 The general condition of rats: The blood glucose levels of DM group were significantly higher than the control group(P < 0.05). After the administration of NBP, the glucose levels were markedly reduced compared to non-NBP treated DM group(P<0.05).Futhermore,DM+NBP group showed a better vitality, body weight.2 Serum 8-OHd G content:Compared with normal control group, the serum 8-OHd G of the DM group were significantly higher(P <0.05).Compared with DM group,the serum 8-OHd G of the NBP group were lower(P<0.05)3 Lens and pathological morphology: we have averaged the stages at the given time(3thweek; 6th week;9th week;12thweek) in order to see the onset and progression of cataract in all the groups. All the lenses in the control group appeared to be clear and normal during the experimental period.4 Lens MDA、oxidative damage 、biomaker 4-HNE、oxidation protein levels: The results showed that levels of MDA 、oxidation protein and 4-HNE in diabetic groups were obviously elevated compared to the control group(P < 0.05). Following treatment with NBP, the levels of MDA 、 oxidation protein and 4-HNE were significantly lower than that of non-treated diabetic rats(P<0.05).5 Nrf2 and it’s downstream antioxidant protein catalase levels: The expresses of nuclear Nrf2, and cytoplasmic Nrf2 were hardly detected in diabetic lens compared to the control group by Western blot(P < 0.05); however, a higher level of Nrf2 expression in both nucleus and in cytoplasm were detected in the lens of NBP treated DM group(P<0.05). To further test the expression of Nrf2 localization, we performed immuno-histochemistry. Nrf2 was mainly expressed in the nucleus and cytoplasm of the epithelial and fiber cells in the control lens; while in the lens of diabetics, few expression of Nrf2 was detected in the epithelial and fiber cells. Nrf2 expression increased in DM + NBP group compared diabetic group.However, in concordance with Nrf2, the expression of catalase were significantly decreased in DM group while significantly increased following NBP treatment by Western blot(P<0.05).Conclusions:1 Oxidative stress is closely related to the pathogenesis of diabetic cataract.2 NBP treatment to some extent inhibit systemic and oxidative stress in diabetic rats.3 Treatment with NBP delayed the formation and progression of diabetic-induced cataract.4 NBP mitigated the protein and lipid peroxidation in the lens of diabetic rats.5 Nrf2 and its downstream antioxidants catalase play an important role in the protective action of NBP on diabetic cataract.