Emodin ameliorates lipopolysaccharides-induced corneal inflammation in rats.

AIM: To investigate the effect of emodin on pseudomonas aeruginosa lipopolysaccharides (LPS)-induced corneal inflammation in rats. METHODS: Corneal infection was induced by pseudomonas aeruginosa LPS in Wistar rats. The inflammation induced by LPS were examined by slit lamp microscope and cytological checkup of aqueous humor. Corneal tissue structure was observed by hematoxylin and eosin (HE) staining. The activation of nuclear factor kappaB (NF-κB) was determined by Western blot. Messenger ribonucleic acid (mRNA) of tumor necrosis factor-α (TNF-α) and intercellular adhesion molecule-1 (ICAM-1) in LPS-challenged rat corneas were measured with reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Typical manifestations of acute corneal inflammation were observed in LPS-induce rat model, and the corneal inflammatory response and structure were improved in rats pretreated with emodin. Treatment with emodin could improve corneal structure, reduce corneal injure by reducing corneal inflammatory response. Emodin could inhibit the decreasing lever of inhibitor of kappaB alpha (IкBα) express, and the mRNA expression of TNF-α and ICAM-1 in corneal tissues was also inhibited by emodin. The differences were statistically significant between groups treated with emodin and those without treatment (P<0.01). CONCLUSION: Emodin could ameliorate LPS-induced corneal inflammation, which might via inhibiting the activation of NF-κB.

Differentiation of lymphatic endothelial cells from bone marrow mesenchymal stem cells with VEGFs.

Although there have been many experimental studies demonstrating that bone marrow-derived mesenchymal stem cells (MSCs) have the potential to differentiate into mesenchymal tissues such as osteocytes, chondrocytes, and adipocytes in vivo and in vitro, little information is available regarding their potential to differentiate into lymphatic endothelial cells. Therefore, we chose to investigate differentiation of MSCs into lymphatic endothelial cells using stimulation with members of the vascular endothelial growth factor (VEGFs) family. Rat MSCs were isolated from bone marrow aspirate of Sprague-Dawley rats as previously described and characterized with flow cytometry for surface markers CD14, CD34, CD29, and CD90. Purified MSCs were plated and cultured in the presence of VEGF-A, VEGF-C, or the combination of both for 10 days. We examined the cells for Prox-1 and LYVE-1 by immunocytochemistry, RT-PCR, and Western blot analysis. Results demonstrated that compared to controls, cell differentiated with VEGF-A, VEGF-C and VEGF-A+VEGF-C expressed Prox-1 and LYVE-1. Our results indicate that MSCs induced by VEGFs are capable of differentiating into lymphatic endothelial-like cells in vitro, and this response has the potential to make them attractive candidates for the development of autologous tissue grafts for future therapy.

[Effects of emodin on expression of cytokines induced by lipopolysaccharide in corneal fibroblasts].

OBJECTIVE: To investigate the effects of emodin on expression of cytokines induced by lipopolysaccharide (LPS) in cultured human corneal fibroblasts in vitro. METHODS: Primary human corneal fibroblasts of passages 4 were used in this research. Cells were treated with 10 microg/L LPS for 1, 2, 4, or 8 hours, which were pretreated with or without emodin for 30 minutes before LPS challenge. The degeneration of inhibitor of kappaB-alpha (I kappaB-alpha) and the effect of emodin on it were analyzed by Western blot analysis with a specific antibody. The cellular abundance of the mRNA of interleukin (IL)-6 and IL-8 from corneal fibroblasts under different conditions was determined by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Compared with cells without LPS treatment, I kappaB-alpha level significantly decreased in every time point after LPS challenge (P < 0.01). Emodin inhibited the LPS-induced degeneration of I kappaB-alpha by corneal fibroblasts in a dose-dependent manner (P < 0.05). Compared with cells without LPS treatment, the expressions of IL-6 and IL-8 mRNA significantly increased in every time point after LPS challenge (P < 0.01). At the same time, the expressions of the mRNA of IL-6 and IL-8 induced by LPS in corneal fibroblasts were also inhibited by emodin in a dose-dependent manner (P < 0.05). CONCLUSION: Emodin can inhibit the expressions of IL-6 and IL-8 mRNA induced by LPS in corneal fibroblasts, which maybe via inhibiting the degeneration of I kappaB-alpha and suppressing the activation of nuclear factor-kappaB.

RNA interference (RNAi)-mediated vascular endothelial growth factor-C (VEGF-C) reduction interferes with lymphangiogenesis and enhances epirubicin sensitivity of breast cancer cells.

It has been reported that over-expression of vascular endothelial growth factor-C (VEGF-C) in tumors leads to increased lymphangiogenesis and resistance to chemotherapy. Therefore, we hypothesized that VEGF-C would be a good molecular target for cancer gene therapy. In this study, we silenced the expression of VEGF-C with the highly specific post-transcriptional suppression of RNA interference (RNAi) in human breast cancer MCF-7 cell line. The expression of VEGF-C was examined by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), and the effect of plasmid on human lymphatic endothelial cells (HLECs) in vitro was analyzed by migration and 3-(4, 5-dimethylt-hiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The sensitivity to anticancer agents was evaluated by MTT and apoptosis assay, and apoptosis-related genes bcl-2/bax ratio was determined by Western Blotting. Results showed that of three siRNA-expressing vectors, P-1/siRNA most significantly suppressed the expression of VEGF-C mRNA and protein (38.1% of control and 117.8 +/- 24.2 pg/ml, respectively) and interfered with proliferation and migration of HLECs in vitro. Moreover, transfection of VEGF-C/siRNA combined with Epirubicin markedly decreased breast cancer cells viability, reaching up to 38.5%, and increased apoptosis rate from 13.1% to 38.9%, as determined by decrease of bcl-2/bax ratio. In summary, VEGF-C would be a good molecular target, and a combination of Epirubicin and RNAi targeting VEGF-C could be an effective means for suppressing lymphatic metastasis and enhancing chemosensitivity of human breast cancer cells.