A polysaccharide from Pueraria lobata ameliorates hepatic fibrosis via gut microbiota-dependent suppression of ferroptosis.

PLP2 is a homogeneous, water-soluble polysaccharide with a molecular weight of 142.9 kDa, primarily composed of (1→4)-α-D-Glc and (1→4)-α-D-GalA units.

• PLP2 was purified from Pueraria lobata Radix using standard polysaccharide extraction and purification methods.
• The major structural components are (1→4)-α-D-Glc and (1→4)-α-D-GalA units.
• A minor presence of →4,6)-α-D-Glc-(1→ residues was also identified.
• Molecular weight was determined to be 142.9 kDa.

PLP2 treatment effectively ameliorated liver injury, histopathological damage, and inflammatory responses in a CCl4-induced murine model of hepatic fibrosis.

• A CCl4-induced murine model was used to evaluate hepatoprotective activity.
• PLP2 treatment reduced markers of liver injury and inflammation in vivo.
• Histopathological analysis confirmed reduced tissue damage in PLP2-treated animals.
• These protective effects were observed in the context of established hepatic fibrosis.

PLP2 treatment restored mitochondrial ultrastructure and hepatic ATP levels in CCl4-treated mice.

• Mitochondrial ultrastructure was assessed and found to be restored following PLP2 treatment.
• Hepatic ATP levels were recovered by PLP2 treatment, indicating improved mitochondrial function.
• Mitochondrial dysfunction is a known contributor to ferroptosis, linking this finding to the mechanistic pathway studied.
• These effects were observed in the in vivo CCl4 fibrosis model.

PLP2 suppressed hepatic ferroptosis through activation of the Nrf2/HO-1/GPX4 axis.

• Ferroptosis, a form of iron-dependent cell death driven by oxidative stress, was measured as a mechanistic endpoint.
• PLP2 treatment activated the Nrf2/HO-1/GPX4 signaling axis in hepatic tissue.
• The indispensable role of Nrf2 was validated using the specific inhibitor ML385, which abolished PLP2's hepatoprotective effects.
• Suppression of ferroptosis was linked to restoration of mitochondrial function and ATP levels.

The hepatoprotective effects of PLP2 were predominantly dependent on gut microbiota integrity, as direct PLP2 treatment failed to protect hepatocytes in vitro.

• In vitro treatment of hepatocytes with PLP2 did not confer protection, indicating PLP2 does not act directly on liver cells.
• Antibiotic treatment to deplete gut microbiota abolished the hepatoprotective effects of PLP2 in vivo.
• Fecal microbiota transplantation (FMT) from PLP2-treated animals transferred the hepatoprotective benefits to recipient animals.
• These three lines of evidence collectively demonstrate that gut microbiota integrity is required for PLP2's anti-fibrotic activity.

The anti-fibrotic mechanism of PLP2 operates through a gut microbiota-dependent pathway leading to suppression of ferroptosis via the Nrf2/HO-1/GPX4 axis.

• PLP2 requires an intact gut microbiome to exert its hepatoprotective effects.
• The gut microbiota mediates downstream activation of the Nrf2/HO-1/GPX4 ferroptosis-suppression pathway in the liver.
• This mechanism links prebiotic-like polysaccharide activity in the gut to hepatic oxidative stress regulation.
• The findings provide mechanistic support for the traditional use of Pueraria lobata Radix as a hepatoprotective herb.