Investigation Of Pharmacological Activities Of Pinocembrin Against Staphylococcus Aureus Infection And Inflammation

Pinocembrin or5,7-dihydroxyflavanone is a flavanone, a type of flavonoid that hasbeen isolated from propolis. Strongly adhesive, this resin is collected from the cracks inthe bark of trees. The aims of this study were to investigate1)-the anti-inflammatory effectof pinocembrin in LPS-stimulated RAW264.7macrophage cells in vitro;2)-the activity ofof pinocembrin on LPS-induced endotoxemia mouse model;3)-the properties ofpinocembrin on LPS-induced acute lung injury in mice and4)-to ascertain theantibacterial effect of pinocembrin against S. aureus pneumonia in a murine model, and itsinfluence on the production of S. aureus α-hemolysin.In the study, we first assessed the anti-inflammatory effects of pinocembrin in RAWmacrophage cells; based on the positive results of in vitro activity of pinocembrin, wedesigned and performed in vivo study with pinocembrin.In first study, RAW264.7cells were pretreated with pinocembrin prior to treatmentwith1μg/ml of LPS and cytokine concentrations in the supernatants of RAW264.7cellculture were determined using an enzyme-linked immunosorbent assays (ELISA) kit;additionally we examined the effect of pinocembrin on the lipopolysaccharide(LPS)-induced phosphorylation of ERK1/2, JNK, p38MAPK and IκBα in the cytoplasmby Western blotting analysis. We observed that in vitro pretreatment with pinocembrinremarkably regulated the production of TNF-α, IL-1β, IL-6and IL-10via inhibiting thephosphorylation of IκBα, ERK1/2, JNK and p38MAPK.Next, we examined the effects of pinocembrin on LPS-induced endotoxemia toinvestigate whether protection of mice from death could be an effect of pinocembrin.Cytokine concentrations and responses were assessed by measuring concentrations in serum separated from clotted blood at0,2,4,6,8and24h following LPS administration.Our results showed lower production of TNF-α, IL-6and IL-1β in serum ofLPS-challenged mice supplemented with pinocembrin. In addition, pretreatment of themouse with pinocembrin improved animal survival rate during LPS-induced lethalendotoxemia.In the murine model of LPS-induced acute lung injury (ALI), we examined inhibitoryactivity of pinocembrin on production of pro-inflammatory cytokines such as TNF-α, Il-1βand Il-6. The molecular mechanisms underlying the anti-inflammatory property ofpinocembrin in mouse model of acute lung injury were also examined. Six hours after LPSadministration, the levels of the cytokines TNF-α, IL-1β and IL-6in lung homogenatewere dramatically increased, while IL-10was only slightly increased compared withcontrol group. Pretreatment with pinocembrin (20or50mg/kg) up-regulated the IL-10leveland down-regulated TNF-α, IL-1β and IL-6levels in dose-dependent manner. Furthermore,pinocembrin (20or50mg/kg, i.p.) attenuated the development of pulmonary edema,histological alterations, as well as neutrophil, lymphocyte and macrophage cell numbers,which were significantly increased following LPS administration. The results of themolecular mechanisms let us suggest that pinocembrin attenuated LPS-induced ALIthrough suppression of constitutive activation of IκBα, JNK and p38MAPK.Staphylococcus aureus remains one of the most important bacteria that cause alarmamong health care. Among the infections caused by this pathogen, pneumonia is one themost prominent. In order to ascertain the combined antibacterial activity of pinocembrin,we aimed to elucidate possible antibacterial acitivity of pinocembrin (PNCB) againstα-hemolysin production and S. aureus pneumonia in a murine model. The in vitroactivities of PNCB on α-hemolysin production were determined using hemolysis, westernblot, and real-time RT-PCR assays. The viability and cytotoxicity assays were performedto evaluate the influence of PNCB on α-toxin-mediated injury of human alveolar epithelialcells. Moreover, through histopathologic analysis, we further determined the in vivoeffects of PNCB on S. aureus pneumonia in a mouse model. In vitro, PNCB at lowconcentrations exhibited inhibitory activity against α-hemolysin production and attenuatedα-haemolysin-mediated cell injury. Taken together, these findings suggest that pinocembrin may represent a new targetfor novel therapeutics.