科研之友
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摘要
Background and PurposeHere, we have studied the effects of the dinucleotide P-1, P-4-Di (adenosine-5) tetraphosphate (Ap(4)A) on corneal barrier function conferred by the tight junction (TJ) proteins and its possible involvement in ocular drug delivery and therapeutic efficiency. Experimental ApproachExperiments in vitro were performed using human corneal epithelial cells (HCLEs) treated with Ap(4)A (100M) for 5min. Western blot analysis and transepithelial electrical resistance (TEER) were performed to study the TJ protein levels and barrier function respectively. Intracellular pathways involved were determined using an ERK inhibitor and P2Y(2) receptor siRNAs. In in vivo assays with New Zealand rabbits, TJ integrity was examined by zonula occludens-1 (ZO-1) staining. The hypotensive compound 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT) was used to assess improved delivery, measuring its levels by HPLC and measuring intraocular pressure using 5-MCA-NAT, P2Y receptor antagonists and P2Y(2) siRNAs. Key ResultsTwo hours after Ap(4)A pretreatment, TJ protein levels in HCLE cells were reduced around 40% compared with control. TEER values were significantly reduced at 2 and 4h (68 and 52% respectively). TJ reduction and ERK activation were blocked by the ERK inhibitor U012 and P2Y(2) siRNAs. In vivo, topical application of Ap(4)A disrupted ZO-1 membrane distribution. 5-MCA-NAT levels in the aqueous humour were higher when Ap(4)A was previously instilled and its hypotensive effect was also increased. This action was reversed by P2Y receptor antagonists and P2Y(2) siRNA. Conclusions and ImplicationsAp(4)A increased corneal epithelial barrier permeability. Its application could improve ocular drug delivery and consequently therapeutic efficiency.
- 出版日期2015-2
