Trilobatin reduced the inflammatory response in LPS-treated 3T3-L1 adipocytes.
As shown in Fig. 1a, after differentiated 3T3-L1 adipocytes were treated with 0.1, 1.0 and 10 µM trilobatin for 24 hours, the cell viability was 101.6 ± 4.5, 98.6 ± 5.3 and 97.7 ± 3.9% respectively, compared to the mean of 0.01% DMSO group (control = 100%), suggested that trilobatin at concentrations ranging from 0.1 µM to 10 µM had no significant cytotoxicity on differentiated 3T3-L1 adipocytes. Meanwhile, we also evaluated the effect of trilobatin on the cell viability in LPS-induced 3T3-L1 adipocytes, the results showed that preincubation with 0.1, 1.0 and 10 µM trilobatin increased the cell viability about 4.5, 12.4 and 21.3% respectively (Fig. 1b).
To evaluate the anti-inflammatory activity, we determined the effect of trilobatin on the TNFα and IL-6 mRNA levels in LPS-treated differentiated 3T3-L1 adipocytes. Data indicated that, incubation with 1 µg/mL induced a remarkable increase in TNFα mRNA expression by 2.1-fold and IL-6 mRNA by 2.7-fold of the control group, whereas trilobatin (0.1–10 µM) decreased the mRNA levels of TNFα and IL-6 in a dose-dependent manner (Fig. 1c and d). Consistent with the results of mRNA expression, trilobatin treatment (0.1–10 µM) concentration-dependently inhibited the secretion of TNFα and IL-6, and the inhibitory rates of 10 µM trilobatin treatment were 42.5% for TNFα and 47.3% for IL-6 in LPS-treated 3T3-L1 adipocytes (Fig. 1e and f).
FABP4 played a critical role in the anti-inflammatory activity of trilobatin.
The results on qRT-PCR and western blot indicated that treatment with trilobatin decreased the expression of FABP4 induced by LPS in a dose-dependent manner in differentiated 3T3-L1 adipocytes, and incubation with 10 µM trilobatin for 24 h decreased the mRNA and protein levels by 2.5- and 3.5-folds respectively in LPS-treated 3T3-L1 adipocytes (Fig. 2a, b).
To assess the role of FABP4 in trilobatin against inflammatory response induced by LPS, we used siRNA transfection to interrupt the expression of FABP4 in differentiated 3T3-L1 adipocytes. Our data indicated that treatment with 50 nM siRNA for 24 h decreased the mRNA and protein levels of FABP4 to 30% and 20% respectively (Fig. 2c, d). Furthermore, we also found that, in FABP4 gene interrupted 3T3-L1 adipocytes, trilobatin had no significant effect on mRNA and protein levels of TNFα and IL-6 induced by LPS (Fig. 2e-h).
Trilobatin reduced the expression of FABP4 and inhibited the inflammatory response eWAT of ob/ob mice.
Several publications had reported that circulating FABP4 might be linked to obesity, inflammation and metabolic syndrome [5, 12], but the expression of FABP4 in eWAT and its role in inflammation of ob/ob mice remain unknown. In the current study, we determined the mRNA and protein levels of FABP4 in eWAT of ob/ob mice. The data from qRT-PCR, western blot and immunofluorescent staining assay revealed that, compared to control group (saline solution), the mRNA and protein levels of FABP4 in eWAT of ob/ob mice were increased 7.6-fold and 1.5-fold, and administration of 10 mg/kg trilobatin for 4 weeks respectively decreased the mRNA and protein levels of FABP4 by about 39% and 36% in the eWAT of ob/ob mice (Fig. 3a-c). Furthermore, data from qRT-PCR assay showed that, compared to C57BL/6 mice, the mRNA levels of TNFα and IL-6 in ob/ob mice increased by 4.5-fold and 5.6-fold, respectively. Administration of 10 mg/kg trilobatin for 4 weeks dramatically decreased the mRNA levels of TNFα (Fig. 3d) and IL-6 (Fig. 3e). Using ELISA assay, we confirmed that administration of trilobatin also significantly decreased the protein levels of TNFα (Fig. 3f) and IL-6 (Fig. 3g) in serum of ob/ob mice.
miR-375 was involved in trilobatin regulating the expression of FABP4.
A growing body of evidence indicates that obesity-induced chronic inflammation plays a fundamental role in the pathogenesis of metabolic syndrome, and miRNAs are largely dysregulated in obesity . To elucidate the molecular mechanisms associated with trilobatin regulating the expression of FABP4, we screened the miRNAs which could bind with the 3'-UTR sequence or coding sequence of FABP4 using http://mirwalk.umm.uni-heidelberg.de/ and http://www.targetscan.org/ and other resources online. Results showed that there were twelve miRNAs including miR-129, miR-338, miR-375, miR-205, miR-347, miR-350, miR-126a, miR-103, miR-503, miR-330, miR-236 and miR-206 possibly interacted with FABP4 gene (Fig. 4a). And then, we used qRT-PCR to confirm the mRNA expression and determine the effect of trilobatin on these mRNAs in LPS-treated differentiated 3T3-L1 adipocytes and the eWAT of ob/ob mice. Data showed that only pre-miR129 (pmiR-129), pre-miR-338 (pmiR-338) and pre-miR-375 (pmiR-375) were decreased in the cellular and animal inflammatory models, most important, trilobatin significantly increased the mRNA levels of pmiR-129, pmiR-338 and pmiR-375 (Fig. 4b, c). Furthermore, we found that miR-375 inhibitor, but not the miR-129 and miR-338 inhibitors prevented the action of trilobatin on mRNA expression of FABP4 induced by LPS in differentiated 3T3-L1 adipocytes (Fig. 4d).