Melatonin inhibits high glucose-induced cell proliferation and expressions of inflammatory factor via Toll-like receptor 4 signaling pathway in mouse mesangial cells / 中华肾脏病杂志

ABSTRACT

Objective To investigate effects of melatonin (MT) on high glucose-induced cell proliferation,Toll-like receptor 4 (TLR4) signaling pathway and expressions of inflammatory factor in mouse mesangial cells (SV40).Methods SV40 cells were divided into mannitol control group (30 mmol/L mannitol),normal control group (5 mmol/L glucose),control (5 mmol/L glucose)+ 1000 μmol/LMT group,high glucose group (25 mmogL glucose),high glucose +10,100,1000 μmol/L MT group and high glucose + TLR4 inhibitor (TAK242) group.(1) The cell viability was measured by CCK-8 cytotoxicity kits,and cell proliferation was measured by EdU kits.The expression of TLR4 and the nuclear translocation of nuclear factor-κB (NF-κB p65) were observed by immunofluorescence.(2) Realtime quantitative PCR was used to detect TLR4 mRNA expression.Real-time quantitative PCR and ELISA were used to determine the mRNA and protein secretion levels of the downstream inflammatory factors,such as monocyte chemoattractant-1 (MCP-1),interleukin-1β (IL-1β) and tumor necrosis factor of α (TNF-α);Western blotting was used to detect TLR4 pathway proteins,such as TLR4,myeloid differentiation factor 88 (MyD88),β interferon TIR domain adaptor (Trif),phosphorylated interferon regulatory factor 3 (p-IRF3) and phosphorylated NF-κB inhibitory protein (p-IκB).Results High glucose stimulated mesangial cell proliferation,promoted TLR4 expression and NF-κB p65 transcription activity.Both MT and TAK242 inhibited the above reactions,and the effects of MT was concentration-dependent.Compared with the normal control group,high glucose group had up-regulated expressions of TLR4,MCP-1,IL-1β and TNF-α mRNA (all P ＜ 0.05),but also significantly increased the protein expressions of MyD88,Trif,p-IRF3 and p-IκB (all P ＜ 0.05).Compared with those in the high glucose group,the expression of TLR4 was down-regulated in the high glucose+ 10,100,1000 μmol/L MT group and the high glucose+TAK242 group (all P ＜ 0.05),while the expressions of MyD88,Trif,p-IRF3,p-IκB,MCP-1,IL-1β and TNF-α decreased (all P ＜ 0.05).The effects of MT was concentration-dependent.Conclusions High glucose stimulates the proliferation of SV40,and MT can inhibit the proliferation of mesangial cells and the expressions of inflammatory factors through TLR4 signaling pathway.