Abstract: ObjectiveTo study the effects of proliferation inhibitionof C.butyricum culture supernatant (C.b.cs) on colon cancer SW-480 cells,and to explore the possible active ingredient and related molecular mechanism. MethodsCCK-8 and FCM wereused to test the cell proliferation and apoptosis.Furthermore, high efficiency liquid chromatography (HPLC) was used to analyze the concentrations of the main organic acid in C.b.cs. After the stimulation of lipopolysaccharide (LPS), the expression of TLR4 was detected by Western blot and qRT-PCR after the treatment of C.b.cs andbutyrate.ResultsC.b.cs inhibits the proliferation of SW-480cells in dose-dependent manner, which can also induce apoptosis and G0/G1 phase arrest. HPLC analysis showed that the concentrations of acetic acid and butyric acid were 9.27g/L and 4.53g/Lin C.b.cs respectively. The expression of TLR4 was inhibited after the treatment of C.b.cs and butyrate（p<0.01）,as the downstream gene of TLR4, the expression of NF-κBwas also inhibited（p<0.01）.Furtherly, the expression of TLR4 was regulated by LPS（p<0.01）, and C.b.cs and butyrateattenuatedthe upregulation（p<0.05）. ConclusionsC.b.cs and butyrate inhibit SW-480 cell proliferation by downregulation the expression of TLR4/NF-κB pathway.

Key words: C. butyricum, colon cancer, apoptosis, Toll-like receptor 4, butyrate