Polygoni Multiflori Radix interferes with bile acid metabolism homeostasis by inhibiting Fxr transcription, leading to cholestasis

Abstract

Objective: To explore the possible mechanisms of cholestasis induced by Polygoni Multiflori Radix (PM). Methods: Low and high doses of water extract of PM were given to mice by gavage for 8 weeks. The serum biochemical indexes of aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutamyltransferase (GGT) alkaline phosphatase (ALP) and so on were detected in the second, fourth, sixth, and eighth weeks after administration. At the end of the eighth week of administration, the bile acid metabolic profiles of liver and bile were screened by high-performance liquid chromatography tandem triple quadrupole mass spectrometry (HPLC-QQQ-MS/MS). Liver pathological changes were observed by hematoxylin and eosin staining. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the mRNA transcription of the target genes and Western blotting (WB) was used to the detect target protein expression. Results: Biochemical tests results showed the values of ALP and GGT were two and three times greater than the normal values respectively, and the value of R was less than 2. Histopathology also showed that PM caused lymphocyte infiltration, a small amount of hepatocyte necrosis and nuclear fragmentation in mouse liver. The proliferation of bile duct epithelial cells was observed in the high group. These results indicated that PM may lead to cholestatic liver injury. HPLC-QQQ-MS/MS analysis with the multivariate statistical analysis revealed significant alterations of individual bile acids in liver and gallbladder as compared to those of the control group. RT-qPCR showed that the transcription of Fxr, Shp, Bsep, Bacs, Mdr2, and Ugt1a1 were downregulated and that of Cyp7a1, Mrp3, and Cyp3a11 was significantly upregulated in the treatment group. WB demonstrated that PM also markedly downregulated the protein expression of FXR, BSEP, and MDR2, and upregulated CYP7A1. Conclusion: PM inhibited the expression of FXR, which reduced the expression of MDR2 and BSEP, leading to the obstruction of bile acids outflow, and increased the expression of CYP7A1, resulting in an increase of intrahepatic bile acid synthesis, which can lead to cholestasis.

Keywords: Radix Polygoni Multiflori; bile acid metabolism; bile salt output pump; cholestasis; cholesterol-7α-hydroxylase; farnesoid X receptor.

FIGURE 1

The structure of bile acids.

FIGURE 2

Contents of AST (A), ALT (B), AST/ALT ratio (C), ALP (D), fold change of ALP (E) and R values (F) in Control, PM-L and PM-H group at the end of second, fourth, sixth and 8 week after administration. (mean ± SEM, n = 8, *p < 0.05, **p < 0.01).

FIGURE 3

Contents of GGT (A), TBA (C), TBIL (D), DBIL (E), IBIL (F) and change of GGT (B) in control, PM-L and PM-H group at the end of second, fourth, sixth, and eighth week after administration. (mean ± SEM, n = 8, *p < 0.05, **p < 0.01).

FIGURE 4

Liver histology (H&E stain, 200x, Scale bar = 50 µm) of mice; Hepatocyte necrosis and dissolution, replaced by hyperplastic connective tissue (yellow arrow), Inflammatory cell aggregation (black arrow), Peribiliary injury (red arrow).

FIGURE 5

Score chart of PCA analysis of 19 bile acids in liver (A); score chart and VIP values of OPLS-DA analysis of 19 bile acids in liver (B–E); The screened important differential bile acids and their contents (F). (X± SEM, n = 8, *p < 0.05, **p < 0.01).

FIGURE 6

Score chart of PCA analysis of 19 bile acids in gallbladder (A); core chart and VIP values of OPLS-DA analysis of 19 bile acids in liver (B–E); The screened important differential bile acids and their contents (F). (X± SEM, n = 8, *p < 0.05, **p < 0.01).

FIGURE 7

Relative expression of transcription factor (A), metabolic enzyme (B, C) and transporters (D) genes in mice after 8 weeks administration (X ± SEM, n = 6, *p < 0.0, **p < 0.01).

FIGURE 8

Effect of the protein expression of FXR (A, E), CYP7A1 (B, F), BSEP (C, G) and MDR2 (D, H) in mice after PM-L and PM-H treatment for 8 weeks. Quantification of the protein expression was performed by densitometric analysis of the blots following normalization to GAPDH expression. (mean ± SEM, n = 3, *p < 0.05, **p < 0.01).

FIGURE 9

The possible mechanism of cholestatic liver injury by PM.