| Obesity is a pro-inflammatory state and as such promotes many of the chronic non infectious diseases.In order to treat obesity-related disorders,it is important that reduction of chronic low-grade inflammation state associated with obesity.Casein glycomacropeptide(GMP)is a glycopeptide of 64 amino acid residues derived from ?-casein,which can reduce plasma total cholesterol and slightly ameliorate the inflammatory state in obese rats.In this study,alcalase,trypsin,pepsin and papain were used for hydrolysation.The anti-inflammatory effects and underlying molecular mechanisms of GMP and its hydrolysates were investigated.The present study aims to prepare enzymatic hydrolysates from GMP under different enzymatic conditions and to determine the inhibitory effects of GMP-derived peptides on the inflammatory response in LPS-stimulated RAW264.7 macrophages.Results showed that GMP hydrolysate produced by papain at 1 h hydrolysis(casein glycopeptide,GHP)exhibited the highest inhibitory effects on LPS-stimulated NO production and TNF-a mRNA expression in RAW264.7 cells.Treatment with GHP suppressed the gene expression of inflammatory mediators such as TNF-?,IL-1?,IL-6,IL-8 and iNOS.Furthermore,the antioxidant potential of GHP was determined using hydrogen peroxide(H2O2)-induced oxidative stress model.Treatment with GHP significantly blocked ROS generation,lipid peroxidation and protein oxidation,as well as cell death via upregulaton of cellular antioxidant enzymes activities of SOD,CAT and GSH-Px in H2O2-damaged RAW264.7 cells.In order to further identify the molecular mechanisms underlying anti-inflammatory activity of casein glycopeptide in macrophages,the inhibitory effects of GHP on the activation of inflammatory signaling pathways were determined.Results showed that treatment with GHP significantly blocked the binding of LPS to TLR4 as well as downregulated TLR4 expression,and subsequently inhibited the activation of IKK?/I?B?/NF-?B signaling pathway.Furthermore,GHP also suppressed intracellular H2O2 accumulation,glutathione oxidation and NOX2 expresson in LPS-stimulated RAW264.7cells.Moreover,GHP inhibited NF-?B activation in macrophages treated with H2O2.These results indicated that GHP suppressed LPS-induced ROS-NF-?B signaling by attenuating intracellular redox state imbalance.To determine whether Nrf2/HO-1 signaling is directly involved in the anti-inflammatory activity of GHP,RAW 264.7 macrophages were treated with LPS and GHP in the presence of ZnPPIX(a selective inhibitor of HO-1.The addition of ZnPPIX reversed the inhibitory effects of GHP on inflammatory cytokines(TNF-a,IL-?,IL-6,iNOS)and NOX2 gene expression,ROS production and NF-?B activation via blocking HO-1 activity.These results showed that Nrf2/HO-1 signaling played important roles in the anti-inflammatory effects of GHP.GHP induced HO-1 expression via induction of nuclear Nrf2 accumulation in a ROS dependent manner.Furthermore,downregulation of P13K,ERK and p38 with selective inhibitor LY29400,PD98059 and SB203580,respectively,attenuated the regulatory effect of GHP.Activation of P13K,ERK and p38 was detected in GHP-treated RAW264.7 cells,which indicated that P13K,ERK and p38 possibly involved in GHP-induced HO-1 expression.Finally,to clarify the potential inhibitory effects of GHP on inflammatory responses in adipose tissue,the medium change experiments were conduct.The addition of macropahges conditioned medium(M?-CM)significantly increased the mRNA levels of TNF-?,IL-1?,iNOS and MCP-1 in 3T3-L1 cells.However,GHP addition significantly suppressed these elevations.Additionally,the elevated mRNA levels of TNF-?,IL-1?,iNOS and MCP-1 in 3T3-L1 adipocyte-conditioned medium(3T3-CM)-stimulated macrophages were also inhibited by GHP.Furthermore,the high TLR4 expression in both RAW264.7 macrophages and 3T3-L1 adipocytes was related to the inflammatory response.GHP ameliorated inflammatory response in RAW264.7 macrophages and 3T3-L1 adipocyte via suppression of TLR4 expression. |
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