Homeopathic Rhus toxicodendron Induces Cell Adhesions in the Mouse Pre-osteoblast Cell Line MC3T3-e1.

BACKGROUND: Rhus toxicodendron (R. tox) has been used as a homeopathic remedy for the treatment of inflammatory conditions. Previously, we reported that R. tox modulated inflammation in the mouse chondrocyte and pre-osteoblastic MC3T3-e1 cell line. During the inflammatory process, cells adhere to the extracellular matrix (ECM) and then migrate to the inflammation site. We examine here the process of cell adhesion in MC3T3-e1 cells after their stimulation with homeopathic R. tox. METHODS: For the cell-substrate adhesion assay, the cultured MC3T3-e1 cells were trypsinized, starved for 1 h in serum-free media, and plated onto culture plates coated with fibronectin (FN), 30c R. tox or gelatin, respectively. The cells were allowed to adhere for 20 min incubation and unattached cells were washed out. Adherent cells were measured using the water-soluble tetrazolium salt-8 assay. The intracellular signals after stimulation of R. tox were examined by analyzing the tyrosine phosphorylation of focal adhesion kinase (FAK), Src kinase, and Paxillin using immunoblot assay. Formation of focal adhesion (FA, an integrin-containing multi-protein structure that forms between intracellular actin bundles and the ECM) was analyzed by immunocytochemistry using NIH ImageJ software. RESULTS: Cell adhesion increased after stimulation with R. tox (FN, 20.50%; R. tox, 44.80%; and gelatin, 17.11% vs. uncoated cells [control]). Tyrosine phosphorylation of FAK, Paxillin, and Src increased compared with that of gelatin when stimulated with R. tox. Additionally, R. tox-stimulated cells formed many FAs (number of FAs per cell, 35.82 ± 7.68) compared with gelatin-stimulated cells (number of FAs per cell, 19.80 ± 7.18) and exhibited extensive formation of actin stress fibers anchored by FAs formed at the cell periphery. CONCLUSION: Homeopathic R. tox promotes the formation of cell adhesions in vitro.

Cereblon Deletion Ameliorates Lipopolysaccharide-induced Proinflammatory Cytokines through 5'-Adenosine Monophosphate-Activated Protein Kinase/Heme Oxygenase-1 Activation in ARPE-19 Cells.

Cereblon (CRBN), a negative modulator of AMP-activated protein kinase (AMPK), is highly expressed in the retina. We confirmed the expression of CRBN in ARPE-19 human retinal cells by Western blotting. We also demonstrated that CRBN knock-down (KD) could effectively downregulate IL-6 and MCP-1 protein and gene expression in LPS-stimulated ARPE-19 cells. Additionally, CRBN KD increased the phosphorylation of AMPK/acetyl-coenzyme A carboxylase (ACC) and the expression of heme oxygenase-1 (HO-1) in ARPE-19 cells. Furthermore, CRBN KD significantly reduced LPS-induced nuclear translocation of NF-κB p65 and activation of NF-κB promoter activity. However, these processes could be inactivated by compound C (inhibitor of AMPK) and zinc protoporphyrin-1 (ZnPP-1; inhibitor of HO-1). In conclusion, compound C and ZnPP-1 can rescue LPS-induced levels of proinflammatory cytokines (IL-6 and MCP-1) in CRBN KD ARPE-19 cells. Our data demonstrate that CRBN deficiency negatively regulates proinflammatory cytokines via the activation of AMPK/HO-1 in the retina.