# Immunomodulatory Effects of Pentoxifylline: Profiling Data Based on RAW 264.7 Cellular Signaling
## 3 Results
A slight increase in AMPK (106.6%) is found in samples treated for 24 h. The expression of LC3 (90.4%) and SOD-1 (89.2%) were decreased in the samples treated for 12 h and increased with time to 105.6% and 99.3% of baseline, respectively. GST (89.5%) was down-regulated by PTX treatment. However, the expression of NOS1 decreased to 88.0% in the sample treated for 48 h. These results suggest that PTX inhibits NO synthesis and the action of other antioxidant enzymes (Figure 6b). **Expression of angiogenesis-related proteins**. Increased expression of angiogenesis-related proteins such as HIF (108.6%), angiogenin (107.3%), and VEGF-C (107.4%) was found in the 48-hour-treated sample. MMP-2 (109.2%) was up-regulated in the 24-hour-treated sample; however, it was down-regulated in the 48-hour-treated sample. The expression levels of VEGF-A, vWF, ET-1, CD31, MMP-10, and VCAM showed a minimal change within 5%. CMG2 (85.8%) decreased significantly over time. These findings reveal that PTX had weak angiogenic properties (Figure 6c). **Expression of oncogenic proteins**. A slight reduction in the expression level of TERT (93.7%) and 14-3-3 (94.3%) was observed. The expression of YAP (97.4%) and mTOR (94.0-105.7%) did not show significant changes. PTX treatment did not upregulate tumorigenic protein expression in RAW264.7 cells (Figure 7). **Expression of osteogenesis-related proteins**. A slightly increased expression of OPG (108.4%), osteopontin (115.8%), and osterix (110.5%) is shown compared with untreated controls. The expression level of RANKL was obviously increased to 117.1% after PTX treatment. The expression level of cathepsin K was down-regulated to 92.7%, and the expression of HSP-90 was reduced to 93.2% in the 24-hour-treated sample. The expression levels of BMP-2, osteonectin, and ALP demonstrated a small change similar to the control proteins. PTX slightly increased osteogenesis-related proteins in RAW 264.7 cells compared with the untreated control. However, RAW 264.7 cells are a macrophage cell line, which have the potential to become osteoclasts; after PTX treatment, the cells showed slight osteogenic properties compared with the untreated control. Thus, RAW264.7 cells treated with PTX may have an effect on bone formation (Figure 8). Figure 7: Changes to oncogenic proteins after PTX treatment in RAW 264.7 cells. Left-sided graph shows changes to protein expressions according to the application time of PTX, blue bar; control group, red bar; 12 h treatment of PTX, green bar; 24 h treatment of PTX, purple bar; 48 h treatment of PTX. Right-sided polygonal graph shows protein expression profiles after 12 h treatment of PTX. (JPG 342 kb). **Effects of PTX on the global protein expressions**. The results of this in vitro study are summarized in Figure 9. PTX was shown to inhibit cellular proliferation-, apoptosis-, and inflammation-related proteins in RAW 264.7 cells. Conversely, angiogenesis and antioxidant activities were not significantly affected. Although the origin of RAW 264.7 cells is a murine macrophage, PTX was expected to have an osteogenesis promoting effect (Table 2). Figure 8: Changes to osteogenesis-related proteins after PTX treatment in RAW 264.7 cells. Left-sided graph shows changes to protein expressions according to the application time of PTX, blue bar; control group, red bar; 12 h treatment of PTX, green bar; 24 h treatment of PTX, purple bar; 48 h treatment of PTX. Right-sided polygonal graph shows protein expression profiles after 12 h treatment of PTX. Figure 1:0 Global protein expression diagrams showing the effects of PTX on RAW 264.7 cells after 12 h treatment. Updated (red dots) and down-regulated (blue dots) proteins are displayed in this diagram. PTX down-regulated the proliferation-, apoptosis-, inflammation-, oncogenic-related proteins and up-regulated the osteogenesis-related proteins.