Effects Of Acylated Ghrelin On Adiponectins And MCP-1Parasecretion Of Adipocytes Induced By TNF-α

Objective:Main hazard of obesity is not adipose tissue itself, but the adipose tissue inflammation. Recent research shows that acylated ghrelin has anti-inflammatory effect. Here we aimed to investigate whether acylated ghrelin can protect3T3-L1mouse adipocytes from inflammatory injury mediated by inflammatoral factor TNF-α.Methods:Four experimental groups were set up:control group, TNF-α treated group, acylated ghrelin treated group and a group pretreated by acylated ghrelin followed by TNF-α treatment. In TNF-α treated group,3T3-L1mouse adipocytes were cultured for24hours by TNF-α at the concentration of100ng/ml. In Acylated ghrelin treated group,3T3-L1adipocytes were treated for28hours by acylated ghrelin at different doses (1.5pmol/L,15pmol/L,150pmol/L,1500pmol/L). In the group pretreated by acylated ghrelin followed by TNF-α treatment Pretreatment of the3T3-L1adipocytes with acylated ghrelin for4hours at different doses (1.5pmol/L,15pmol/L,150pmol/L,1500pmol/L) followed by incubating with TNF-α (100ng/ml) for24hours. After the completion of the intervention, the mRNA and protein levels of TLR4in3T3-L1adipocytes were detected by RT-PCR and Western blotting, respectively. NF-κB p65protein phosphorylation level in the3T3-L1adipocytes was detected by Western blotting. In addition, the concentration of monocyte chemotactic protein1(MCP-1) and adiponectin in the supernatant were detected by ELISA.Results:①Compared with control group, the mRNA and protein expression level of TLR4were increased by TNF-α intervention, as well as level of NF-κB p65protein phosphorylation and MCP-1protein in the murine3T3-L1adipocytes (P<0.05). On the contrary, the adiponectin protein was decreased(P<0.05).②Compared with control group, the level of TLR4mRNA and protein expression, as well as NF-κB p65protein phosphorylation were significantly decreased in acylated ghrelin treated group (P<0.05). The adiponectin level in the cell supernatant was decreased (from15pM, P<0.05), while level of the MCP-1went down without statistically significant difference (P>0.05).③Compared with TNF-α (100ng/mL) treated group, the incubation of acylated ghrelin could antagonise TNF-α-induced activation of TLR4/NF-κB pathway in mouse3T3-L1adipocytes, mRNA and protein expression levels of TLR4, the level of NF-κB p65protein phosphorylation were decreased (P<0.05), and in a dose-dependent manner. The secretion of MCP-1and adiponectin did not change significantly in3T3-L1adipocytes (P>0.05).Conclusions:①TNF-α can activate TLR4/NF-λB inflammatory pathways and increase secretion of pro-inflammatory cytokines (MCP-1), while decrease the secretion of anti-inflammatory cytokines (adiponectin) in3T3-L1adipocytes.②Acylated ghrelin can antagonise TNF-α-induced activation of TLR4/NF-κB pathway in a dose-dependent manner in mouse3T3-L1adipocytes, but can not completely improve the secretion disorder of MCP-1and adiponectin.