[The effect of puerarin on prevention of oxidative damage of cultured lens capsule epithelial cells].

OBJECTIVE: To investigate the peroxynitrite damage to the lens epithelial cells (LEC) and the prevention of this damage by puerarin in vitro. METHODS: This paper was experimental study. Rabbit LEC were isolated and cultured and the third or forth passage LEC were used in this experiment The experiment groups included: (1) CONTROL GROUP: Heat-pathogen free saline (NS) 200 microl was added to the medium; (2) ONOO- group: ONOO- 200 microl was added to obtain the terminal concentration at 0. 5 mmol/L; (3) Puerarin group: 5 microg/ml ONOO- and 10 microg/ml puerarin were added simultaneously. Then, the cells were cultured and collected after 6,12 or 24 hours. The nitrotyrosine (NT), a symbol of the ONOO-, was tested with immunofluorescence technique. The expression of NT protein was examined with Western blot method. The cell morphology was observed with light microscope. Cell apoptosis was examined via DNA ladder, flow cytometry and Fas/FasL immunohistochemical staining. These datas were analyzed by one-way-ANOVA and q test. RESULTS: During the 6 to 24 hours of experiment period, green color could be observed in the cell nucleus and cytoplasm of control group. Staining ranged from yellow to brown-yellow, then to brown color were observed in STZ group. Staining ranged from faint green to yellow green or faint green color were observed in puerarin group. Slight expression of nitrotyrosine (NT) could be seen in the control group. A moderate to strong expression of NT was observed at different stages in the STZ group (A = 77.22 +/- 2.44, 145.00 +/- 3.94, 235. 8 +/- 5.97). At 6 hours, a slight expression of NT could be seen in the control group (A = 72.78 +/- 2.64), this increased at 12 hours (A =89. 94 +/- 3.01) and decreased at 24 hours (A = 74. 44 +/- 3.00). With computer photo-analysis, there were significant differences between the control, STZ and puerarin groups at different period during the experiment (q = 78.12, 82.76, 69.98, P <0. 01). In the control group, cell morphology and gene DNA ladder were normal, minor apoptosis could be observed but no expression of Fas/FasL in the membrane and cytoplasm of the cells. Distinctive cell morphology changes and the typical "ladder bands" as well as the expression of Fas/FasL could be observed in STZ group. All of these aspects were comparatively normal in puerarin group. CONCLUSIONS: The LEC apoptosis induced by ONOO- in vitro could be alleviated by puerarin. Fas/FasL cell signal transduction pathway may affect and strengthen the apoptosis process mediated by ONOO-.

Puerarin decreases lens epithelium cell apoptosis induced partly by peroxynitrite in diabetic rats.

The present study was designed to observe if puerarin decreases lens epithelium cell (LEC) apoptosis induced partly by peroxynitrite (ONOO(-)). One hundred and eight rats were randomly divided into control group (n=36), streptozotocin (STZ) group (n=36) and STZ + puerarin group (n=36). The rats in the control group intraperitoneally (i.p.) received 0.5 ml of saline. The rats in STZ group and STZ + puerarin group received intraperitoneal injection of STZ (45 mg/kg). Three days later, the rats in STZ + puerarin group were given puerarin (140 mg/kg per day, i.p.). On days 20, 40 and 60 of the experiment, morphologic changes of lenses were observed with slit lamp. Then the animals were sacrificed for further analysis. The amount and percentage of apoptotic LECs were determined by flow cytometry. Nitrotyrosine (NT, the foot print of ONOO(-)) was examined by immunohistochemistry. Apoptosis-related genes (iNOS, etc.) were analyzed by gene array. The results showed that in the control group, all the lenses were clear. In STZ group, gradually severe opacity of the lens was observed on days 20, 40 and 60. But in STZ + puerarin group, mild opacity of the lens was observed on day 20 and more severe on day 40, but markedly decreased on day 60. In the control group, mild apoptosis of LECs was observed. In STZ group, time-dependent increase in apoptosis of LECs was observed. In STZ + puerarin group, mild apoptosis of LECs was observed on day 20, significantly increased on day 40, but markedly decreased on day 60. There was no expression of NT in the lens in the control group, but an increased expression of NT in STZ group. In STZ + puerarin group, mild expression of NT was observed on day 20, significantly increased on day 40, but markedly decreased on day 60. There was no expression of iNOS in the lens in the control group, but continuous up-regulation of iNOS expression in STZ group. In STZ + puerarin group, mild expression of iNOS was observed on day 20, significantly increased on day 40, but markedly decreased on day 60. Except the changes of iNOS related to NO production, the other apoptosis-related genes, including BCL-2 and SOD were down-regulated, while NF-kappaB and TNFR1-FADD-caspase signal transduction way were up-regulated in STZ group. The results were opposite in STZ + puerarin group and the control group. These findings show that NT is expressed in diabetic rat lens, which proves that LEC apoptosis in diabetic lens is partly induced by ONOO(-) which may be a new oxidative damage way to form cataract. Puerarin partly decreases LEC apoptosis induced by ONOO(-) and is a potential medicine for therapy of diabetic cataract. The mechanism of puerarin dealing with diabetic cataract may be related to its direct inhibition of LEC apoptosis and antagonism of ONOO(-) in diabetic rats.