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. 2022 Oct 13:9:1032722.
doi: 10.3389/fnut.2022.1032722. eCollection 2022.

Triptolide increases resistance to bile duct ligation-induced liver injury and fibrosis in mice by inhibiting RELB

Zihang Yuan  1 , Jie Wang  1 , Haoran Zhang  1 , Yingying Miao  1 , Qianhui Tang  1 , Ziqiao Yuan  1 , Cheng Nong  1 , Zhicheng Duan  1 , Luyong Zhang  1   2 , Zhenzhou Jiang  1   3 , Qinwei Yu  1
Affiliations

Triptolide increases resistance to bile duct ligation-induced liver injury and fibrosis in mice by inhibiting RELB

Zihang Yuan et al. Front Nutr. .

Abstract

Cholestasis is a common, chronic liver disease that may cause fibrosis and cirrhosis. Tripterygium wilfordii Hook.f (TWHF) is a species in the Euonymus family that is commonly used as a source of medicine and food in Eastern and Southern China. Triptolide (TP) is an epoxy diterpene lactone of TWHF, as well as the main active ingredient in TWHF. Here, we used a mouse model of common bile duct ligation (BDL) cholestasis, along with cultured human intrahepatic biliary epithelial cells, to explore whether TP can relieve cholestasis. Compared with the control treatment, TP at a dose of 70 or 140 μg/kg reduced the serum levels of the liver enzymes alanine transaminase, aspartate aminotransferase, and alkaline phosphatase in mice; hematoxylin and eosin staining also showed that TP reduced necrosis in tissues. Both in vitro and in vivo analyses revealed that TP inhibited cholangiocyte proliferation by reducing the expression of RelB. Immunohistochemical staining of CK19 and Ki67, as well as measurement of Ck19 mRNA levels in hepatic tissue, revealed that TP inhibited the BDL-induced ductular reaction. Masson 3 and Sirius Red staining for hepatic hydroxyproline showed that TP alleviated BDL-induced hepatic fibrosis. Additionally, TP substantially inhibited BDL-induced hepatic inflammation. In summary, TP inhibited the BDL-induced ductular reaction by reducing the expression of RelB in cholangiocytes, thereby alleviating liver injury, fibrosis, and inflammation.

Keywords: RelB; TNFSF14; bile duct ligation (BDL); cholangiocyte; triptolide.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Triptolide alleviates liver injury induced by bile duct ligation. Male C57BL/6 mice were sacrificed at seven days after BDL or sham surgery. (A) The diagrammatic experimental procedures. (B) Body weight of mice (each group n = 6). (C) Serum levels of ALT, AST and ALP in mice sacrificed at 7 days after BDL or sham surgery. (D) Representative images of H&E (The black dotted line indicates the necrotic area) from liver tissues. Necrosis area statistics of H&E. Scale Bar: 100μm. Data are shown as the mean ± SD. Data represent at least 6 independent experiments with triplicate measurements. Analysis of variance (one-way ANOVA) was used. p values represents significance different from BDL group.
FIGURE 2
FIGURE 2
Triptolide inhibits proliferation and RelB expression in HiBEC. (A) The growth curve of HiBEC after transfection of siRNA-Relb. (B) The protein level of RelB in HiBEC after TP treatment. (C) The growth curve of HiBEC after TP treatment. (D) Double immunofluorescence staining for CK19 (green) and Ki67 (red) from HiBEC after TP treatment. Nuclei were counter-stained with DAPI (blue). (E) Relb, Tnfsf14 and Ltβ mRNA was measured in HiBEC after TP treatment. Data are shown as the mean ± SD. Data represent at least 3 independent experiments with triplicate measurements. Analysis of variance (one-way ANOVA) was used. p values represents significance different from control group.
FIGURE 3
FIGURE 3
Triptolide inhibits BDL induced RelB and its downstream gene expression. (A) Western blot analysis of RelB in liver. (B) The serum TNFSF14 levels (ELISA data) of mice. (C) The mRNA level of Relb, Tnfsf14 and Ltβ in liver tissues. Data are shown as the mean ± SD. Data represent at least 6 independent experiments with triplicate measurements. Analysis of variance (one-way ANOVA) was used. p values represents significance different from BDL group.
FIGURE 4
FIGURE 4
Triptolide relieves BDL-induced bile duct hyperplasia. (A) Representative images of IHC for CK19 and Ki67 (The black arrows indicate the regenerated cholangiocytes) from liver tissues. Scale Bar: 100 μm. (B) Statistical analysis of immunohistochemically positive regions of (A). (C) The mRNA level of Ck19 in liver tissues. Data are shown as the mean ± SD. Data represent at least 6 independent experiments with triplicate measurements. Analysis of variance (one-way ANOVA) was used. p values represents significance different from BDL group.
FIGURE 5
FIGURE 5
Triptolide relieves BDL-induced liver fibrosis. (A) Representative images of Masson 3, Sirius Red, and IHC for α-SMA from liver tissues. Scale Bar: 100 μm. (B) Collagen positive area statistics of Masson3 and Sirius Red. (C) Hydroxyproline assay of liver tissues. (D) The mRNA levels of Acta2, Col1a1, Ccn2, and Tgf-β1 from liver tissues. Data represent at least 6 independent experiments with triplicate measurements. Analysis of variance (one-way ANOVA) was used. p values represents significance different from BDL group.
FIGURE 6
FIGURE 6
Triptolide relieves BDL-induced liver inflammation. (A) Representative images of IHC of F4/80 and LY6G from liver tissues. Scale Bar: 100 μm. (B) The liver mRNA levels of F4/80, IL-1β and Tnf-α from liver tissues. Data represent at least 6 independent experiments with triplicate measurements. Analysis of variance (one-way ANOVA) was used. p values represents significance different from BDL group.
FIGURE 7
FIGURE 7
TP inhibited the BDL-induced ductular reaction by reducing the expression of RelB in cholangiocytes, thereby alleviating liver injury, fibrosis, and inflammation. Created with BioRender.com.
See this image and copyright information in PMC

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