In our previous study immortalized mouse stellate cell lines that were TLR4 wild type (JS1) and TLR4 knockout (-/-) (JS2) were generated (Guo, et al. Hepatology, 2008). The aim of the present study was to investigate the differential gene expression in these cell lines with or without the stimulation by lipopolysacchirde (LPS), the exogenous TLR4 ligand, and high mobility group box 1 (HMGB1), a potential endogenous TLR4 ligand

and damage pattern molecule that signals the presence of necrosis (Zhang, et al, Lif Sci, 2012). Methods: JS1 LY2157299 clinical trial and JS2 cells that were sub-cultured to 80% confluence were stimulated with normal saline vehicle (control), or 100 ng/ml LPS, or 100 ng/ml HMGB1 for 24 Neratinib nmr hours. The cells were collected with Trizol reagent for RNA extraction. The RNA extracts from the control, LPS and HMGB1 groups were hybridized on a 4644 K Agilent whole mouse genome oligo microarray for the gene expression analysis. Functional analysis of the microarray data was performed using KEGG analyses. Gene interaction network and co-expression network were built on the base of ontology and pathway analysis to which the differentially

expressed genes attributed. Selected genes were validated by real-time polymerase chain reaction (RT-PCR), ELISA and/or Western Blot. Results: The gene expression profiles are different between JS1 and JS2 cells under basal condition and after stimulated with TLR4 ligands. The differentially expressed genes encode extracellular matrix and matrix remodeling proteins,

growth factors and receptors, chemokines and receptors, inflammatory and immune related proteins, as well as transcriptional factors and important signaling molecules. In JS1 cells LPS upregulates genes that belong to the signaling pathways of Toll-like receptors, neurotrophic factor, immune, the spliceosome and nucleotide excision repair and downregulates PPAR signaling, with a variety of MHC molecules, MAPKs, Pik3r3, Prkca, Ikbkb as central regulatory factors. Under HMGB1 stimulation, MAPKs, TRAF6, IGF1R, Gstps appeared to be core regulatory Tangeritin factors in JS1 cells. The gene interaction and co-expression network in JS2 cells under LPS or HMGB1 stimulation are different from JS1 cells, which are simple and lack of core regulatory factors. Conclusion: There were complex gene expression alterations subsequent to the lacking of TLR4 in HSCs. These included key inflammatory, fibrogenic, growth and metabolism related signals in HSCs. These finding emphasizes the complex pathways downstream of TLR4 in this important fibrogenic cell type and the significant consequence of TLR4 signaling on HSC biology and function. Key Word(s): 1. stellate cells; 2. Toll like receptor 4; 3. ligands; 4.