# Immunomodulatory Effects of Pentoxifylline: Profiling Data Based on RAW 264.7 Cellular Signaling
## 4 Discussion
In this study, immune-related markers were increased, and matrix inflammation-associated markers were decreased. The level of cytokines and T-lymphocytes in COVID-19 patients was studied, and it was suggested that a decrease in T cells in COVID-19 patients may be associated with a high serum concentration of TNF-\(\alpha\), IL-6 negatively regulating T cell survival or proliferation. [45] The severity of a cytokine storm and decreased T cells are associated with exacerbation of diseases such as pulmonary damage and respiratory distress [46; 47]. The expression of CD4 is slightly increased by PTX treatment, in which it can be presumed that PTX contributes to macrophage immune function. PTX downregulated the expression of p53, MDM2, and PARP. However, BAX expression was increased in the 12 and 24 h PTX treatment group and decreased slightly in the 48 h PTX treatment group to 98.8% of baseline. The expression of BAK was decreased in the 48 h sample, APAF-1 increased at 24 h and decreased at 48 h. The expression of caspase 9 decreased with time; however, the changes were not significant. PTX seems to down-regulate p53, but downstream proteins associated with the intrinsic apoptotic pathway require further study. PTX also down-regulated FAS-mediated apoptosis-related proteins, FASL, FAS, FADD, PARP, and caspase 8 were down-regulated by PTX treatment. PTX inhibited the extrinsic apoptotic pathway, which is in contrast to a previous study [48]. In this in vitro study, it was found that PTX suppressed the proliferation of RAW 264.7 cells and also suppressed the expression of proteins related to cellular apoptosis. However, we did not perform the cellular proliferation and apoptosis assay. In a previous animal study, an experiment irradiated the mandible of rats with radiation. It was shown that PTX had an effect of inhibiting cellular apoptosis [20]. Therefore, protein expressions related to proliferation and apoptosis were analyzed through the IP-HPLC in this study. Referring to the antioxidant-related proteins, NOS1, SOD-1, GST, and LC3 were downregulated by PTX in contrast to the control group. This suggests that the antioxidant effect of PTX in RAW 264.7 cells is unclear. [PERSON] et al. [49] studied the protective effects of Pentoxifline on acute liver injury; the levels of SOD and glutathione (GSH) in liver tissue were elevated by PTX treatment compared to that of the control group. In a study of methotrexate-induced damage in the livers and kidneys of rats, immunostaining of iNOS and TNF-\(\alpha\) was decreased in the PTX administered group, while SOD levels decreased in liver tissue. These results are similar to the result of our in vitro study [50]. The oncogenic protein expressions, TERT, and 14-3-3 were down-regulated; PTX did not elevate tumorigenic protein expression but actually showed some anti-cancer effects by up-regulating the expression of MBD4, which has some function in DNA repair. PTX not only has anti-tumor activity, but it also increases the susceptibility of cancer cells to radiation therapy [51]. Mild increases in cell protection-related proteins, such as HSP-70 (110.2%), AP-1 (111.3%), SP-1 (113.4%), and PKC (105.1%) also occurred. Most proteins associated with cellular protection increased in cells treated with PTX for 12 h and decreased for the next 24, and 48 h back to baseline, with the exception of the transcription factor. This means that the PTX has no deleterious effect on RAW 264.7 cells. The expressions of OPG, osteeopontin, and osterix, were slightly increased, and RANKL expression was markedly up-regulated by PTX treatment, which was not surprising since RAW 264.7 cells originate from murine macrophages and are likely to differentiate into osteoclasts. The advantage of IP-HPLC research is that it can investigate the expression of multiple proteins in one experiment. Therefore, when investigating osteogenesis, we tried to observe as many related proteins as possible. Very few studies have been published on PTX and osteogenesisis; [PERSON] et al.