TMAO is closely related to obesity in clinical samples
To understand the correlation between TMAO and obesity, 30 volunteers in different body mass index (BMI) were collected to test the TMAO concentration. According to WHO standard, BMI was classified into normal (20.0-24.9 kg/m2), overweight (25.0-29.9 kg/m2), mild obesity (30.0-34.9 kg/m2) and moderate obesity (35.0-39.9 kg/m2). We observed that TMAO was increased significantly with the development of obesity in comparison to normal group, especially in moderate obesity (Fig. 1). It was indicated that increasing of TMAO concentration is related to the severity of obesity.
TMAO inhibited the proliferation of LO2 cells
In order to further investigate whether different concentration of TMAO affects the proliferation and migration of LO2 cells, we used cck-8 assay and wound healing assay to estimate the following indicators. LO2 cells were treated with TMAO in 0, 50, 100, 200, 400 and 800μM. Wound healing analysis had shown that TMAO in 50, 100μM had complete cells morphology but TMAO destroyed the cell shapes over 200μM (Fig. 2A-B). Also, TMAO in 50 and 100μM did not destroy the migration of LO2 cells, but it did destroy in 200, 400, 800μM in comparison to control group. Furthermore, 50μM TMAO group had better wound healing ability than that in other groups. The toxicity of TMAO was examined by CCK-8 assay. As illustrated (Fig. 2C), we found that 205μM was the half-maximal inhibition concentration (IC50) of TMAO-treated LO2 cells after 24 hours. In particular, compared with control group, 50μM TMAO promoted cells proliferation, while other groups, such as 100, 200, 400 and 800μM, had significant inhibitory with the increase of TMAO concentration. At the same time, the inhibition of cell proliferation in high concentration groups, 200, 400 and 800μM TMAO, was significantly higher than that in low concentration groups, 50 and 100μM TMAO, which was in comparison to control group. These results indicated that TMAO in 50μM significantly ameliorated LO2 cells proliferation and in 200, 400 and 800μM could destroy it.
TMAO inhibited LO2 cell cycle and promoted apoptosis, and inflammatory cytokines expression
To further verify the ability of TMAO to induce apoptosis and block cycle cycle, the flow cytometry assay was used. Apoptosis analysis of TMAO treated LO2 cells were stained with Annexin V-FITC and PI. TMAO had significant increase in 100, 200, 400 and 800μM concentrations of total apoptosis rates (including early apoptosis and late apoptosis), compared with the control group (Fig. 3A). Furthermore, the cell cycle analysis also had significant increase in 100, 200, 400 and 800μM concentrations in total cell cycle G0/G1 distribution (Fig. 3B). Meanwhile, IL-1β, IL-6, TNF-α, AST and ALT were detected. After exposure with TMAO, the levels of IL-1β, IL-6, TNF-α, AST and ALT was significant increased, especially in 400 and 800μM (Fig. 3C-D).
TMAO inhibited LO2 cells steatosis generation
According to the above, TMAO 100μM was lower damage than higher concentration groups so that we used 100μM TMAO and 40μM oleic acid to testify TMAO function in liver. Oleic acid, as a free fatty acid used into steatosis model, obviously promoted the apoptosis and increased in ALT and AST (Fig. 4A-B). It was found that compared with OA group, TMAO could inhibit the apoptosis in TMAO and OA-treated group. We investigated the effect of TMAO on OA-induced steatosis using the analysis of ALT and AST levels. The result has revealed a significant inhibition in steatosis compared with OA group. Next, we determined the levels of IL-1β, IL-6 and TNF-α. The result has showed that OA promoted the level of IL-1β, IL-6, TNF-α. Respectively, when TMAO was added, IL-1β was increased, but IL-6 was no significant change and TNF-α was decreased.
To further confirm whether TMAO on hepatitis steatosis were associated with autophagy, we detected the protein expression level of IκBα, NF-κB p65, ABCG5, SERBP1 and PPAR gamma through western blotting (Fig. 5). Compared with control group, both the protein expressions of IκBα, NF-κB p65, ABCG5, SERBP1 and PPAR gamma were significantly increased in TMAO and OA group. However, the protein expressions of NF-κB p65, ABCG5, PPAR gamma and SERBP1 were decreased when TMAO and OA group was added with SN50, an NF-κB p65 inhibitor, which could block the NF-κB signaling pathway.
TMAO reduced hepatitis steatosis and inflammation response through NF-κB signaling pathway
To further confirm TMAO decreased steatosis through NF-κB signaling pathway, we used the SN50 to stimulated LO2 cells. Compared with control or TMAO groups, SN50 added with TMAO and OA was marked increased lipid accumulation in LO2 cells (Fig. 6A). Moreover, we also found that TMAO could significantly promote cells apoptosis in steatosis while SN50 was added (Fig. 6B). Expectantly, NF-κB signaling pathway was closed related to TMAO-stimulated in liver cells, especially in hepatic steatosis.