Unconjugated
Increased number of eosinophils in the circulation and sputum is associated with the severity of asthma. The respiratory epithelium produces chemokine (C-C motif) ligands (CCL) which recruits and activates eosinophils. A chemically synthesized monoacetyl-diglyceride, PLAG (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol) is a major constituent in the antlers of Sika deer (Cervus nippon Temminck) which has been used in oriental medicine. This study was aimed to investigate the molecular mechanism of PLAG effect on the alleviation of asthma phenotypes. A549, a human alveolar basal epithelial cell, and HaCaT, a human keratinocyte, were activated by the treatment of interleukin-4 (IL-4), and the expression of chemokines, known to be effective on the induction of eosinophil migration was analyzed by RT-PCR. The expression of IL-4 induced genes was modulated by the co-treatment of PLAG. Especially, CCL26 expression from the stimulated epithelial cells was significantly blocked by PLAG, which was confirmed by ELISA. The transcriptional activity of signal transducer and activator of transcription 6 (STAT6), activated by IL-4 mediated phosphorylation and nuclear translocation, was down-regulated by PLAG in a concentration-dependent manner. In ovalbumin-induced mouse model, the infiltration of immune cells into the respiratory tract was decreased by PLAG administration. Cytological analysis of the isolated bronchoalveolar lavage fluid (BALF) cells proved the infiltration of eosinophils was significantly reduced by PLAG. In addition, PLAG inhibited the migration of murine bone marrow-derived eosinophils, and human eosinophil cell line, EoL-1, which was induced by the addition of A549 culture medium.
Hepatocellular carcinoma (HCC) is a highly malignant tumor with an extremely poor prognosis. Our preliminary study indicated that bufalin could restrain the proliferation of human hepatoma BEL-7402 cells in a time- and dose-dependent manner. In the present study, the colony formation assay, the Transwell invasion assay, the western blot analysis and the immunofluorescence method were respectively used to investigate the effect and mechanism of bufalin against HCC cell invasion and metastasis. We found that: i) bufalin had significant inhibitory effect on the cell proliferation of BEL-7402 cells; ii) bufalin markedly inhibited the migration and invasion of BEL-7402 cells; iii) bufalin could suppress the phosphorylation of GSK-3β Ser9 site in BEL-7402 cells, decrease the expression of β-catenin, cyclin D1, metalloproteinases-7 (MMP-7) and cyclooxygenase-2 (COX-2) in the cytoplasm, and increase the expression of E-cadherin and β-catenin on the cell membrane; and iv) the expression of α-fetoprotein significantly decreased and the expression of albumin increased in BEL-7402 cells after bufalin was used. Our results indicate that: i) bufalin can regulate the expression of associated factors in Wnt/β-catenin signaling pathway of BEL-7402 cells through suppressing the phosphorylation of GSK-3β Ser9 site; ii) bufalin can strengthen intercellular E-cadherin/β-catenin complex to control epithelial-mesenchymal transition; and iii) bufalin can reverse the malignant phenotype and promote the differentiation and maturation by regulating the AFP and ALB expression in BEL-7402 cells. These are very important mechanisms of bufalin on the inhibition of the invasion and metastasis of HCC cells.