| Objective：To observe the inhibitory effect of osthole on inflammatory cytokinerelease from3T3-L1adipocytes stimulated with lipopolysaccharide (LPS) and toexplore its possible mechanisms.Methods: The3T3-L1cells were treated with the classic“cocktail†method toinduce the transformation of these cells into mature adipocytes, and these adipocyteswere then divided into the control, DMSO, LPS, osthole0.025,0.1and0.4μg/mlgroups, LPS+osthole0.025,0.1and0.4μg/ml groups, positive drugs lipanthyl200μg/ml and rosiglitazone10μg/ml groups. Medicine-treated cells were firstly incubatedwith corresponding drugs for2h, and then stimulated with LPS2μg/ml for6h. Thetumor necrosis factor (TNF)-α and interleukin (IL)-6levels in the cultured supernatantwere determined by ELISA method. The peroxisome proliferator-activated receptor(PPAR) α, PPARγ and nuclear factor (NF)-κB protein expressions in the adipocyteswere determined by Western blot method, and the nuclear translocation of NF-κB wasdetermined by immunocytochemistry method. In order to determine whether theinhibitory effect of osthole on TNF-α and IL-6release from LPS-stimulated adipocyteswas associated with the activation of PPARα/γ, the cultured adipocytes were pretreatedwith4μM PPARα inhibitor MK886or/and10μM PPARγ inhibitor GW9662for2h,then incubated with0.4μg/ml osthole for8h, and finally stimulated with LPS for6h,the changes of TNF-α and IL-6levels in the cultured supernatant, NF-κB proteinexpression in the cells and NF-κB nuclear translocation were examined.Results: After treatment of the mature adipocytes with osthole0.025,0.1and0.4μg/ml for8h, the significant changes of TNF-α and IL-6levels in the culturedsupernatant as well as the PPARγ and NF-κB protein expressions in the adipocytes werenot observed, but the PPARα protein expression in the adipocytes was significantly increased（P<0.05or P<0.01）. After the mature adipocytes were stimulated with LPS2μg/ml for6h, the TNF-α and IL-6levels in the cultured supernatant were inceased(P<0.05), the NF-κB protein expression in the cells was also inceased (P<0.01), butPPARα protein expression in the cells was decreased (P<0.01). After the matureadipocytes were treated with osthole0.025,0.1and0.4μg/ml for2h and thenstimulated with LPS2μg/ml for6h, the TNF-α level in cultured supernatant wasdose-dependently decreased, particularly in osthole0.4μg/ml (P<0.01). The IL-6levelin cultured supernatant in the osthole-treated cells were also decreased, particularly inosthole0.025μg/ml group (P<0.01). Osthole could significantly increase the proteinexpressions of PPARα/γ（P<0.05or P<0.01）, decrease the protein expression of NF-κB(P <0.05or P <0.01) and reduce the nuclear translocation of NF-κB. After pretreatmentwith PPARα inhibitor MK886and/or PPARγ inhibitor GW9662for2h, the inhibitoryeffect of0.4μg/ml osthole on these inflammatory factors, NF-κB protein expression andnuclear translocation were decreased or cancelled.Conclusion：Osthole could inhibit the secretion of inflammatory cytokines TNF-αand IL-6from LPS-stimulated adipocytes, and its mechanisms might be associated withactivation of PPARα/γ and subsequent reduction of NF-κB protein expression andnuclear translocation. |
