Skip to main content
. 2022 Aug 5;26:372–386. doi: 10.1016/j.omto.2022.08.001

Figure 4.

Figure 4

BBR inhibited LPA-induced p38 phosphorylation and leptin transcription

(A) BBR reduced the leptin expression induced by LPA. HepG2 cells were subjected to 20 μM LPA combined with BBR (0.1 μM, 0.5 μM, 2.5 μM) and Met (250 μM) respectively for 18 h, after being starved overnight and pretreatment with BBR or Met for 4 h. Cellular total proteins were extracted for western blot analysis, which were normalized to that of ACTB. (B) Leptin mRNA expression was detected by real-time RT-PCR, normalized to ACTB, and presented as fold of the DMSO group without LPA treatment. (C) BBR and Met significantly decreased the abnormal activity of leptin promoter induced by LPA in HepG2 cells transfected with the pGL4.17-LP1 plasmid. After transfection, cells were screened using 0.4 mg/mL G418. Stable transfected cells were then treated as above. The promoter activity was detected as in section “methods.” (D) The promoter region (247 bp) was sufficient for LPA to induce the transcription of leptin gene. HepG2 cells were co-transfected with pGL4.74 and four pGL4.17-LP plasmids containing full-length and truncated 5′ regulatory region of the leptin gene respectively and treated as above. (E) BBR inhibited LPA-induced p38 phosphorylation at Thr180/Tyr182. HepG2 cells were treated as above. Western blot of P-p38 and p38 was conducted as described in section “methods,” and normalized to that of ACTB. (F) BBR inhibited p38 activation in tumor and liver tissues from male mice 8 months after DEN injection. The ratio of P-p38/ACTB was set as 1 in sham-treated group. (G and H) The LPA-induced activity of leptin promoter was reduced by the inhibitors of p38 (SB 203580 and SB 202190) and HIF-1α (KC7F2 and BAY 87-2243). Transfected cells were subjected to LPA (20 μM) combined with BBR (2.5 μM), Met (250 μM), SB 202190 (5 μM), SB 203580 (5 μM), KC7F2 (40 μM), and BAY 87-2243 (10 μM) as above. The luminescence was set as 1 in the vehicle group treated with DMSO. (I) BBR decreased the mRNA expression of CEBPA and HIF1A. HepG2 cells were treated as above. Results were normalized to ACTB and presented as fold of the DMSO group without LPA treatment. Values were the mean ± SEM of three independent experiments. Data are expressed as mean ± SEM, ∗p < 0.05, ∗∗p < 0.01 compared with the LPA- or S1P-stimulated group.