Fig 2: Bicyclol with or without berberine enhances the lipolysis and β-oxidation through p62-Nrf2-CES2/PPARα axis. Male C57BL/6J mice were freely fed with WD or WD plus bicyclol and/or berberine for 16 weeks (A,C), or induced with WD/CCl4 for 4 weeks and then treated with drugs for 8 weeks (B,D). The protein levels of PPARα, CES2, Nrf2, p62, CD36, FABP1, ATGL, HSL, ACSL1, and ACSL5 were detected with Western blot and quantified using a Gel-Pro analyzer. Results were presented as mean ± SD, and a representative band was shown. n = 5–9 for each group, *p < 0.05 and **p < 0.01, the model group vs. control group; #p < 0.05 and ##p < 0.01 vs. the model group. PPARα, proliferator-activated receptor α; CES2, carboxylesterase 2; Nrf2, NF-E2-related factor 2; p62, ubiquitin-binding protein p62; CD36, cluster determinant 36; FABP1, fatty acid binding protein 1; ATGL, adipose triglyceride lipase; HSL, hormone-sensitive lipase; ACSL, acyl-CoA synthetase long chain family member.

Fig 3: BSGLWE inhibits osteosarcoma growth in vivo. (A) After 21 days of BSGLWE treatment, luciferase intensity of tumor was measured and calculated by an in vivo imaging system. (B, C) The photographs show tumor formation in two groups. There was a significant difference in tumor sizes and weights between the two groups after 21 days. (D) TUNEL assays were utilized to assess the apoptotic status of tumor tissues. BSGLWE promoted extensive tumor cell necrosis was proved by H&E staining. The levels of BCL-XL, LC3B and P62 were further examined by immunohistochemistry. (E) There were no obvious toxic effects observed in the important organs of the experimental mice between the BSGLWE-treated group and the control group. Error bar = mean ± SEM. Magnification, ×200 (d1–2), ×40 (d3–4,e1-10), ×400 (d5–10). Scale bar, 100 μm (d1–2), 500 μm (d3–4, e1–10), 50 μm (d5–10). Error bar = mean ± SD. *P < 0.05, **P < 0.01.

Fig 4: BSGLWE restricts autophagic flux of OS cells in vitro. (A) Morphological changes induced by BSGLWE were observed by transmission electron microscopy. BSGLWE group cells were treated with BSGLWE (4ng/mL). The yellow arrows mean apoptotic bodies and nuclear condensation. The red arrows mean autophagosomes. Scale bar, 2 μm. (B) The HOS and U2 osteosarcoma cells were treated with different concentrations of BSGLWE for 24 h. The expression of those autophagy-related proteins, which included ATG5, ATG12, LC3B, P62, was measured by a Western blot assay. (C, D) An immunofluorescence assay revealed the level of protein LC3B increased in the BSGLWE (4mg/mL) group compared with the NC group in both HOS and U2 cell lines. (E) HOS and U2 cells were treated with different concentrations of BSGLWE (0,4,8 mg/mL), with or without 3-MA (3mM), (F) or with or without CQ (8uM) for 24 h. The protein expressions of LC3B and p62 were measured by Western blotting. (G) HOS and U2 cells were treated with different concentrations of BSGLWE (0,4,6,8 mg/mL), with or without 3-MA (3mM), CCK-8 assay was used to test cell viability. Each assay was repeated 3 times. Magnification, ×200 (C). Scale bar, 100 μm (C). Error bar = mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 versus control, #P< 0.05 versus BSGLWE treatment.