Punicalagin Suppresses Acute Inflammatory Response Via Promoting Mouse Macrophages M2Polarization

Punicalagin (PUN) is a bioactive ellagitannin isolated from pomegranate, which is widely used for the treatment of inflammatory bowel disease, diarrhea and ulcers in Chinese traditional medicine. In this study, we tried to determine the anti-inflammation and anti-oxidative stress effects of punicalagin on macrophages. Furthermore, we tried to investigate the underlying mechanisms of M2macrophages polarization induced by punicalagin, which contributes to understanding the anti-inflammation and anti-oxidative stress effects of punicalagin. Experiment1In this study, we detected the anti-inflammation potentials of PUN in lipopolysaccharide (LPS)-induced macrophages and tried to uncover the underlying mechanism. Results demonstrated that PUN (25,50, or100μM) treatment could significantly decrease the LPS-induced production of nitric oxide (NO), prostaglandin E2(PGE2), interleukin (IL)-1β, IL-6and tumor necrosis factor (TNF)-a in RAW264.7cells. Molecular research showed that PUN inhibited the activation of upstream mediator nuclear factor-κB (NF-κB) by suppressing the phosphorylation of IκBα and p65. Results also indicated that PUN could suppress the phosphorylation of mitogen-activated protein kinase (MAPK), including p38, JNK and ERK. In conclusion, we observed that PUN could inhibit LPS-induced inflammation and it may be a potential choice for the treatment of inflammation diseases. Experiment2Hemeoxygenase-1(HO-1) exhibits antioxidant activity in macrophages. Therefore, we hypothesized that HO-1is a potential target of PUN and tried to reveal its antioxidant mechanism. Here, PUN treatment increased HO-1expression together with its upstream mediator nuclear factor-erythroid2p45-related factor2(Nrf2). However, specific inhibition of Nrf2by brusatol (a specific Nrf2inhibitor) dramatically blocked PUN-induced HO-1expression. Previous research has demonstrated that the PI3K/Akt pathway plays a critical role in modulating Nrf2/HO-1protein expression as an upstream signaling molecular. Here, LY294002, a specific PI3K/Akt inhibitor, suppressed PUN-induced HO-1expression and led to ROS accumulation in macrophages. Furthermore, PUN inhibited LPS-induced oxidative stress in macrophages by reducing ROS, NO generation and increasing superoxide dismutase (SOD)1mRNA expression. These findings provide new perspectives for novel therapeutic approaches using antioxidant medicines and compounds against oxidative stress and excessive inflammatory diseases including tissue damage, sepsis and endotoxemic shock. Experiment3In the present study, we evaluated the inhibitory effect of PUN on LPS-induced macrophages M1polarization via measuring relative cytokines level as well as potential mechanism mediators, including STAT-1, inducible nitric oxide synthase (iNOS) and Arg-1. Additionally, we performed research work on PUN-induced M2macrophages polarization together with its regulatory effect on HO-1mediated IL-4or IL-10expression processes. Results based on western blot demonstrated that PUN could significantly decrease LPS-induced M1macrophage phenotype indicators iNOS, IL-1β, IL-6and TNF-a expression while increase the M2macrophage phenotype indicators IL-10and arg-1expression. Flow cytometry results showed that PUN markedly suppressed the expression of CD11c+while increased CD206expression in RAW264.7macrophages, confirming that PUN could promote M2macrophage polarization. Mechanism research illustrated that PUN notably inhibited the phosphorylation of STAT-1and strengthened the phosphorylation of STAT-3. Furthermore, we found that PUN could significantly increase the HO-1expression together with certain indicators in M2phenotype macrophage, which can be remarkably revised by HO-1inhibitor ZnPP, hinting that HO-1plays a key role in PUN-induced M2macrophage polarization. Experiment4To better understand the M2polarization effect of PUN in vivo, we tested our hypothesis using peritoneal macrophages isolated from mouse. Results showed that, like the mechanisms in vitro, PUN could significantly decrease the expression of iNOS, IL-1β,IL-6and TNF-α, while increase the IL-10and arg-1expression. Flow cytometry results showed that PUN markedly suppressed the expression of CD11c+while increased CD206expression, confirming that PUN could promote M2macrophage polarization in mouse peritoneal macrophages. Furthermore, we demonstrated that PUN-induced HO-1is essential to the activation of STAT-3/IL-10axis and thus lead to M2macrophage polarization in mouse peritoneal macrophage. These findings approved that PUN promotes M2macrophage polarization via HO-1induced STAT-3/IL-10axis activation both in vivo and in vitro.