Bupleuri radix total saikosaponins alleviate liver fibrosis by inhibiting hepatic stellate cell activation via SIRT3-SMAD3/HIF-1α -mediated glycolysis.

BACKGROUND: An essential uncontrolled process-liver fibrosis-is vital to cirrhosis and liver cancer onset. There are currently no approved antifibrotic drugs in clinical application. Saponins are a class of major bioactive components of Bupleuri radix (Chaihu) that play important roles against liver fibrosis. Glycolysis inhibition may control the activation of hepatic stellate cells (HSCs), serving as an effective anti-fibrotic strategy. However, the mechanism by which saikosaponins exert anti-liver fibrosis effects by regulating glycolysis through HIF-1α is unclear.

PURPOSE: To explore the mechanism by which Bupleuri radix total saikosaponins (BRTS) inhibit HSC activation and alleviate liver fibrosis.

METHODS: Purified BRTS were obtained by extraction with saturated aqueous n-butanol solution, followed by adsorption and purification with AB-8 macroporous resin. After removing impurities using 40% ethanol, the product was washed with 90% ethanol. LC-MS/MS and HPLC were used to characterize the BRTS components and determine their contents. We used Network pharmacology to predict the possible mechanism underlying BRTS. CCl4 was intraperitoneally injected to create a liver fibrosis rat model, and we added TGF-β1 to LX-2 cells to create a HSC activation model. Haematoxylin and eosin (H&E), Masson, and Sirius Red staining of liver tissue, along with serum and liver biochemical indicator measurement analysed the extent of liver injury and fibrosis. Western blotting, qRT-PCR, immunofluorescence, immunohistochemistry, transcriptomics, and metabolomics practices analysed liver fibrosis- or HSC activation-related protein, gene, and metabolite expression. ELISA kits were used to detect glycolytic activity, SIRT3 deacetylase activity, lactate secretion, and the glucose uptake rate. Moreover, the mechanism by which BRTS improves liver fibrosis was validated using a glycolysis inhibitor, i.e., 2-deoxy-D-glucose (2-DG), 2-methoxyestradiol (2-ME, a kind of HIF-1α inhibitor), dimethyl oxoglutarate (DMOG, a kind of HIF-1α stabilizer), and SIRT3 inhibitor (3-TYP).

RESULTS: The overall yield of BRTS was 6.56 ± 0.41%, and the main components were saikosaponin A (176.1 ± 10.5 mg/g) and saikosaponin D (132.7 ± 7.7 mg/g). Network pharmacology and LC-MS analyses revealed that the core anti-liver fibrosis target was HIF-1α. BRTS may show anti-liver fibrosis properties by regulating the HIF-1 signalling pathway, per the KEGG pathway enrichment analysis. Molecular docking suggested that both saikosaponin A and saikosaponin D can stably bind to SIRT3. In vivo, BRTS can improve the symptoms of CCl4-mediated liver fibrosis, alleviate liver injury, reduce collagen fibre content, and decrease collagen deposition, including significantly reduced serum AST and ALT activity, serum LN, HA, PC III, and COL IV levels, and liver lactate levels. BRTS significantly reversed the increased α-SMA, COL1, HIF-1α, and p-SMAD3 protein expression and the decreased SIRT3 protein expression (p<0.05). In addition, BRTS downregulated GLUT1, PKM2, HK2, and LDHA mRNA expression (all glycolytic genes) than the MOD group (p<0.05). The in vitro experimental results revealed that BRTS significantly inhibited LX-2 cell activation caused by TGF-β1, and reduced glucose uptake rates and level of lactate released (p<0.05). BRTS administration raised SIRT3 protein expression and activation, reduced HIF-1α and p-SMAD3 nuclear translocation, downregulated COL1, HIF-1α, α-SMA, and p-SMAD3 protein expression, and reduced glycolytic gene mRNA expression (p<0.05). In LX-2 cells, 2-DG and 2-ME further promoted the suppressive impact of BRTS on glycolysis and activation. Compared with BRTS treatment, DMOG resulted in significant reversal of the suppressive impact of BRTS on glycolysis as well as activation and COL1 and HIF-1α protein expression. In addition, BRTS promoted the deacetylase activity of SIRT3. In LX-2 activated cells where SIRT3 is inhibited, the inhibitory effect of BRTS on HIF-1α and p-SMAD3 protein expression is eliminated.

CONCLUSIONS: BRTS regulates the SMAD3/HIF-1α signalling axis through SIRT3 to mediate glycolysis in HSCs, which suppresses HSC activation and improves liver fibrosis.