Effects of Puerariae Radix Extract on Endotoxin Receptors and TNF-α Expression Induced by Gut-Derived Endotoxin in Chronic Alcoholic Liver Injury

Abstract

Kudzu (Pueraria lobata) is one of the earliest medicinal plants used to treat alcohol abuse in traditional Chinese medicine for more than a millennium. However, little is known about its effects on chronic alcoholic liver injury. Therefore, the present study observed the effects of puerariae radix extract (RPE) on chronic alcoholic liver injury as well as Kupffer cells (KCs) activation to release tumor necrosis factor alpha (TNF-α) induced by gut-derived endotoxin in rats and macrophage cell line. RPE was observed to alleviate the pathological changes and lipids deposition in liver tissues as well as the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and hepatic gamma-glutamyl transpeptidase (GGT) activity. Meanwhile, RPE inhibited KCs activation and subsequent hepatic TNF-α expression and downregulated the protein expression of endotoxin receptors, lipopolysaccharide binding protein (LBP), CD14, Toll-like receptor (TLR) 2, and TLR4 in chronic alcohol intake rats. Furthermore, an in vitro study showed that RPE inhibited the expression of TNF-α and endotoxin receptors, CD14 and TLR4, induced by LPS in RAW264.7 cells. In summary, this study demonstrated that RPE mitigated liver damage and lipid deposition induced by chronic alcohol intake in rats, as well as TNF-α release, protein expression of endotoxin receptors in vivo or in vitro.

Figure 1

Mechanism of Kupffer cells activation releasing TNF-α to promote liver injury induced by gut-derived endotoxin in alcoholic liver disease.

Figure 2

HPLC chromatograms of the RPE and the standard compounds: (a) RPE, (b) puerarin standard (16.988 min), (c) Daidzin standard (21.488 min), and (d) daidzein (32.314 min).

Figure 3

Effects of RPE on liver injury and lipid deposition induced by Lieber-DeCarli diet. (a) Histological observation on the H.E. sections (original magnification, ×200). (b) Hepatic lipid droplets observation on red O staining sections (original magnification, ×200). (c) Serum ALT, AST, hepatic GGT, and TG variation. Values represent the mean ± SD of 10 rats. *P < 0.05, versus control; ^# P < 0.05, versus ethanol; **P < 0.01, versus control, ^## P < 0.01, versus ethanol.

Figure 4

Effects of RPE on endotoxin in the portal vein and hepatic TNF-α concentration. (a) Endotoxin level in portal vein, (b) TNF-α concentration in liver tissue. Values represent the mean ± SD of 10 rats. **P < 0.01, versus control, ^# P < 0.05, versus ethanol.

Figure 5

Effects of RPE on Kupffer cells activation in liver. (a) Hepatic CD68 expression detected with immunohistology (original magnification, ×400), (b) Hepatic CD68 expression detected with western-blot. Values represent the mean ± SD of three independent experiments. *P < 0.05, versus control; ^# P < 0.05, versus ethanol.

Figure 6

Effects of RPE on protein expression of endotoxin receptors in liver. RPE inhibited protein of endotoxin receptors, LBP, CD14, TLR4, and TLR2 (western-blot). Values represent the mean ± SD of three independent experiments. *P < 0.05, versus control; **P < 0.01, versus control; ^# P < 0.05, versus ethanol.

Figure 7

Effect of RPE on TNF-α secretion in RAW264.7 cell culture supernatant induced by LPS. RPE inhibited TNF-α secretion induced by LPS in vitro. Values represent the mean ± SD of three independent experiments. **P < 0.01, versus control; ^# P < 0.05, versus LPS.

Figure 8

Effect of RPE on endotoxin receptors protein expression induced by LPS in vitro. RPE inhibited CD14 and TLR4 protein expression significantly in vitro (western-blot). Values represent the mean ± SD of three independent experiments. *P < 0.05, versus control; ^# P < 0.05, versus LPS.