The effect of puerarin against IL-1ß-mediated leukostasis and apoptosis in retinal capillary endothelial cells (TR-iBRB2).

PURPOSE: Blood-retinal barrier (BRB) breakdown, the early hallmark of diabetic retinopathy (DR), is thought to depend on retinal inflammation and cell damage. The proinflammatory factor interleukin-1ß (IL-1ß) was demonstrated to cause inflammation as well as cell apoptosis during the process of BRB breakdown. This study extensively evaluated the protective effect of puerarin, a major active component extracted from the traditional herb Radix puerariae, against IL-1ß-induced cell dysfunction in TR-iBRB2 cells, a retinal capillary endothelial cell line. METHODS: TR-iBRB2 cells were pretreated with IL-1ß (10 ng/ml) for 24 h and then exposed to puerarin (0, 10, 25, and 50 µM) for another 24 h. Leukocyte endothelial adhesion was assessed through a cell-based assay using lymphoblastoid cells. Cell apoptosis was evaluated with flow cytometry, and the expression of adhesion molecules and apoptosis-related molecules was assessed with western blot analysis. RESULTS: Our data showed that puerarin attenuated IL-1ß-mediated leukostasis and cell apoptosis in TR-iBRB2 cells. Furthermore, puerarin strikingly prevented IL-1ß-induced molecular events of the upstream and downstream signaling pathways involved in this cellular process. CONCLUSIONS: These findings may significantly contribute to better understanding of the protective effect of puerarin, in particular for DR, as well as provide novel insights into the potential application of this compound in DR therapy.

Inhibition of Hsp90 attenuates inflammation in endotoxin-induced uveitis.

Heat shock protein (Hsp) 90 inhibitors, such as 17-allylamino-17-demethoxy-geldanamycin (17-AAG), constitute promising novel therapeutic agents. We investigated the anti-inflammatory activity of 17-AAG in endotoxin-induced uveitis (EIU) in rats. After the induction of EIU with a footpad injection of lipopolysaccharide (LPS), female Lewis rats received a single intraperitoneal. (i.p.) injection of 17-AAG or vehicle. Twenty-four hours later, the retinas were extracted and assayed for leukocyte adhesion; blood-retinal barrier breakdown; VEGF, TNF-alpha, IL-1beta, and CD14 protein levels; NF-kappaB and HIF-1alpha activity; hsp90 and 70 levels and expression and phosphorylation of the tight junction proteins ZO-1 and occludin. 17-AAG treatment significantly suppressed the LPS-induced increase in retinal leukocyte adhesion; vascular leakage; NF-kappaB, HIF-1alpha, p38, and PI-3K activity; and VEGF, TNF-alpha, and IL-1beta levels. 17-AAG also suppressed phosphorylation of ZO-1 and occludin by inhibiting their association with p38 and PI-3K. Although 17-AAG treatment did not reduce the LPS-induced increase in total CD14 levels in leukocytes, it significantly decreased membrane CD14 levels. These data suggest that Hsp90 inhibition suppresses several cardinal manifestations of endotoxin-induced uveitis in the rat. 17-AAG has demonstrated a favorable safety profile in clinical trials in cancer patients and represents a promising therapeutic agent for the treatment of inflammatory eye diseases.