Combined Use of Bicyclol and Berberine Alleviates Mouse Nonalcoholic Fatty Liver Disease

Abstract

Nonalcoholic fatty liver disease (NAFLD), ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), is a liver disease worldwide without approved therapeutic drugs. Anti-inflammatory and hepatoprotective drug bicyclol and multi-pharmacological active drug berberine, respectively, have shown beneficial effects on NAFLD in murine nutritional models and patients, though the therapeutic mechanisms remain to be illustrated. Here, we investigated the combined effects of bicyclol and berberine on mouse steatosis induced by Western diet (WD), and NASH induced by WD/CCl₄. The combined use of these was rather safe and better reduced the levels of transaminase in serum and triglycerides and cholesterol in the liver than their respective monotherapy, accompanied with more significantly attenuating hepatic inflammation, steatosis, and ballooning in mice with steatosis and NASH. The combined therapy also significantly inhibited fibrogenesis, characterized by the decreased hepatic collagen deposition and fibrotic surface. As per mechanism, bicyclol enhanced lipolysis and β-oxidation through restoring the p62-Nrf2-CES2 signaling axis and p62-Nrf2-PPARα signaling axis, respectively, while berberine suppressed de novo lipogenesis through downregulating the expression of acetyl-CoA carboxylase and fatty acid synthetase, along with enrichment of lipid metabolism-related Bacteroidaceae (family) and Bacteroides (genus). Of note, the combined use of bicyclol and berberine did not influence each other but enhanced the overall therapeutic role in the amelioration of NAFLD. Conclusion: Combined use of bicyclol and berberine might be a new available strategy to treat NAFLD.

Keywords: berberine; bicyclol; combination; gut microbiota; lipid metabolism; nonalcoholic fatty liver disease.

FIGURE 1

Combination of bicyclol (Bic) and berberine (BBR) exerts better preventive effects in mice with NAFL induced by Western diet. Male C57BL/6J mice were treated by free feeding with Western diet (WD) or WD mingled with bicyclol or/and berberine (A). The average food intake during the whole experiment (B) and body weight at week 16 (C) were recorded. At week 16, ALT (D) and AST (E) in serum were detected, and TG (F) and CHO (G) in the liver were measured. Liver histopathology was evaluated using H&E and ORO staining (H) and quantified with NAS score criteria (I). Results were presented as mean ± SD. n = 5 for each group, ^*p < 0.05 and ^**p < 0.01, WD-induced model group vs. the control group; ^#p < 0.05 and ^##p < 0.01 vs. the WD-induced model group or monotherapy group.

FIGURE 2

Combination of bicyclol and berberine demonstrates better therapeutic effects on NASH in mice induced by WD/CCl₄. Male C57BL/6J mice were freely fed with Western diet plus intraperitoneal injection of 0.2 ml/kg CCl₄ (WD/CCl₄) once per week for 4 weeks (A). Liver steatosis was performed with H&E staining at week 4 (B). Then the mice were continuously treated with WD/CCl₄ or WD/CCl₄ plus drug for 8 weeks. The body weight (C) was recorded. ALT (D) and AST (E) in serum and TG (F) and CHO (G) in the liver were measured. Liver histopathology was evaluated using H&E, ORO, and Masson’s trichrome staining (H) and quantified with NAS score criteria (I) and fibrotic surface (J). Results were presented as mean ± SD. n = 5–9 for each group, *p < 0.05 and **p < 0.01, WD/CCl₄-induced model group vs. the control group; ^#p < 0.05 and ^##p < 0.01 vs. the WD/CCl₄-induced model group or monotherapy group.

FIGURE 3

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.

FIGURE 4

Berberine with or without bicyclol suppresses de novo lipogenesis by inhibiting the protein expression of ACC and FAS. Male C57BL/6J mice were freely fed with WD or WD plus bicyclol and/or berberine for 16 weeks (A), or induced with WD/CCl₄ for 4 weeks and then treated with drugs for 8 weeks (B). The protein expressions of ACC and FAS were detected and quantified. 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. ACC, acetyl-CoA carboxylase; FAS, fatty acid synthase.

FIGURE 5

Berberine but not bicyclol modulates the composition of gut microbiota in WD/CCl₄-induced NASH mice. A total of 25 male C57BL/6J mice fecal samples with five in each representative group were collected and analyzed using 16S rRNA gene sequencing. (A) The alpha diversity of each group was obtained using the Shannon index. (B–C) The beta diversity among samples was obtained using the unweighted UniFrac diversity distance (B) and PCoA (C) of gut microbiota. (D–E) The relative abundance of bacteria at family (D) and genus (E) level. (F) Representative histogram of the most abundant gut microbiota at the family and genus level. (G) Prediction of lipid metabolism-related microbiome function based on KEGG database. *p < 0.05, **p < 0.01 vs. the control group; ^#p < 0.05, ^##p < 0.01 vs. the WD/CCl₄ group. N, Control; M, WD/CCl₄; B, WD/CCl₄+BBR(200 mg/kg); D, WD/CCl₄+Bic(200 mg/kg); G, WD/CCl₄+BBR(200 mg/kg)+Bic(200 mg/kg).

FIGURE 6

Combined use of bicyclol and berberine decreases lipid accumulation via regulating lipid metabolism-related gene expression in FFA-induced HepG2 cells. HepG2 cells were induced by 0.1 mM FFA and simultaneously treated for 24 h with 2 μM berberine, 2 μM bicyclol, or their combinations. The cells were assayed with an MTT method to show the cytotoxicity (A), or carried out Nile Red staining to show LDs (B) and the level of LDs in cells were quantified using Image-Pro Plus 6.0 (C). Total proteins were extracted and detected with Western blot (D). n ≥ 3, **p < 0.01, the model group vs. control group; ^#p < 0.05 and ^##p < 0.01 vs. the model group or the monotherapy group. FFA, free fatty acid; LDs, lipid droplets; PPARα, proliferator-activated receptor α; CES2, carboxylesterase 2; ACC, acetyl-CoA carboxylase; FAS, fatty acid synthase.

FIGURE 7

Schematic diagram of the therapeutic mechanism of combined use of bicyclol and berberine against NAFLD. Bicyclol enhanced lipolysis through p62-Nrf2-CES2 signaling axis and improved β-oxidation through p62-Nrf2-PPARα signaling axis, while berberine suppressed the de novo lipogenesis by inhibiting protein expressions of ACC and FAS, along with the regulation of lipid metabolisms via the gut microbiota, and thus co-facilitating to ameliorate the occurrence and progress of NAFLD. The green arrow shows a stimulatory effect, the red flathead shows an inhibitory effect.