title: Toxicity of endogenous peroxynitrite and effects of puerarin on transplanted retinal pigment epithelial sheets in the subretinal space in mice.
abstract: OBJECTIVE
  To evaluate the toxicity of endogeneous peroxynitrite on transplanted retinal pigment epithelial (RPE) sheets and the effect of puerarin on their survival in the C57BL/6 mice after RPE sheets have been transplanted into SD rats' subretinal space .
  
  
  METHODS
  C57BL/6 mice eyes were used to culture RPE cells. Ninety-six SD rats were involved in the experiment. They were divided into control (block control), streptozotocin (STZ, negative control), untransplanted RPE (positive control) and transplanted RPE groups respectively. Diabetes was induced in SD rats by intra-peritoneal STZ injection in the latter three groups. Saline was injected into the subretinal space of 24 SD rats in the untransplanted RPE group and primary RPE sheets were injected into the subretinal space of 24 SD rats in the transplanted RPE group. Puerarin (45mg/kg) was administrated into both untransplanted RPE and transplanted RPE groups of diabetic rats through intra-peritoneal injection route after RPE sheets transplantation. At 20, 40, 60 days after surgery, Western blotting analysis, DNA ladder and RT-PCR were used for determining the differences in expression of nitrotyrosine (NT, the foot print of peroxynitrite ), apoptosis and iNOS mRNA in the control, STZ, untransplanted RPE and transplanted RPE groups respectively. HE staining was used for determining the RPE survival in the subretinal space of the transplanted RPE group.
  
  
  RESULTS
  Apoptosis and expression of NT and iNOS mRNA were observed in STZ, untransplanted RPE and transplanted RPE groups, but were delayed in untransplanted RPE and transplanted RPE groups in a time-dependent manner compared with control and STZ groups (P<0.01). There were no differences between the two groups (P>0.01). NT, DNA ladder, iNOS mRNA were down-regulated, which were associated with the decrease of expression of peroxynitrite. Numerous pigmented cells emerged and increased in number in the subretinal space during the 60-day observation period after transplantation. On day 20, heavily pigmented cells were visible at the transplant site; On day 40, monolayer and multilayered transplant was visible in the subretinal space; On day 60, heavily pigmented monolayer and multilayered transplants with round apical profile were present along Bruch's membrane.
  
  
  CONCLUSIONS
  Puerarin increased the 60-day survival of C57BL/6 mice RPE xenografts in the SD rats' subretinal space, which may be related to its direct inhibition of apoptosis of RPE cells and antagnism of damage of peroxynitrite to RPE cells.
authors.affiliation: Department of Ophthalmology, Hebei Province People's Hospital, Shijiazhuang 050051, Hebei Province, China.
authors.forename: Li-Na
authors.initials: LN
authors.lastname: Hao
authors.forename: Yan-Qing
authors.initials: YQ
authors.lastname: Zhang
authors.forename: Yu-Hua
authors.initials: YH
authors.lastname: Shen
authors.forename: Mei-Qiao
authors.initials: MQ
authors.lastname: Li
authors.forename: Tao
authors.initials: T
authors.lastname: Yang
authors.forename: Zhi-Hua
authors.initials: ZH
authors.lastname: Zhao
authors.forename: Zhi-Yun
authors.initials: ZY
authors.lastname: Wang
authors.forename: Yan-Hua
authors.initials: YH
authors.lastname: Wang
authors.forename: Shou-Zhi
authors.initials: SZ
authors.lastname: He