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. 2019 Apr 24:10:389.
doi: 10.3389/fphar.2019.00389. eCollection 2019.

Baishouwu Extract Suppresses the Development of Hepatocellular Carcinoma via TLR4/MyD88/NF-κB Pathway

Yong-Fang Ding  1 , Zi-Xuan Peng  2 , Lan Ding  3 , Yun-Ru Peng  1
Affiliations

Baishouwu Extract Suppresses the Development of Hepatocellular Carcinoma via TLR4/MyD88/NF-κB Pathway

Yong-Fang Ding et al. Front Pharmacol. .

Abstract

Purpose: The root of Cynanchum auriculatum Royle ex Wight, known as Baishouwu, has been widely used for a tonic supplement since ancient times. The current study was performed to explore the effect of Baishouwu extract on the development of experimental hepatocellular carcinoma (HCC) and the potential mechanism involved. Methods: Rats were injected diethylnitrosamine (DEN) to initiate the multistep hepatocarcinogenesis. Animals were treated concurrently with Baishouwu extract given daily by oral gavage for 20 weeks to evaluate its protective effects. Time series sera and organ samples from each group were collected to evaluate the effect of Baishouwu extract on hepatic carcinogenesis. Results: It was found that Baishouwu extract pretreatment successfully attenuated liver injury induced by DEN, as shown by decreased levels of serum biochemical indicators (AST, ALT, ALP, TP, and T-BIL). Administration of Baishouwu extract inhibited the fibrosis-related index in serum and live tissue, respectively from inflammation stage to HCC stage after DEN treatment. It significantly reduced the incidence and multiplicity of DEN-induced HCC development in a dose-dependent manner. Macroscopic and microscopic features suggested that pretreatment with Baishouwu extract for 20 weeks was effective in inhibiting DEN-induced inflammation, liver fibrosis, and HCC. Furthermore, TLR4 overexpression induced by DEN was decreased by Baishouwu extract, leading to the markedly down-regulated levels of MyD88, TRAF6, NF-κB p65, TGF-β1 and α-SMA in hepatitis, cirrhosis, and hepatocarcinoma. Conclusion: In conclusion, Baishouwu extract exhibited potent effect on the development of HCC by altering TLR4/MyD88/ NF-κB signaling pathway in the sequence of hepatic inflammation-fibrosis-cancer, which provided novel insights into the mechanism of Baishouwu extract as a candidate for the pretreatment of HCC in the future.

Keywords: Baishouwu extract; TLR4; hepatocellular carcinoma; inflammation-fibrosis-cancer axis; pretreatment.

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Figures

Figure 1
Figure 1
Total ion chromatograms in the negative mode of the reference compounds and the sample of Baishouwu extract. (A) Reference compounds and (B) Sample of Baishouwu extract. M1, caudatin 2,6-dideoxy-3-O-methy-β-D-cymaroseglycoside; M2, wilfoside C1N; M4, caudatin; M5, wilfoside K1N; M6, wilfoside C1G; M7, cynauricuoside A; M8, cynauricuoside C; M9, auriculoside IV.
Figure 2
Figure 2
Experimental schedule.
Figure 3
Figure 3
Effect of Baishouwu extract on growth curve of rats treated with DEN. Results are expressed as mean ± SD (1st∼6th week n = 24, 7th∼10th week n = 16, 11th∼20th n = 8, respectively). ΔΔP < 0.01 vs. control group, ∗∗P < 0.01 vs. model group.
Figure 4
Figure 4
Effect of Baishouwu extract on serum biochemical indicators induced by DEN. Serum levels of (A) ALT, (B) AST, (C) TP, (D) ALB, (E) ALP, (F) T-BIL, (G) IL-6, and (H) TNF-α. Results are expressed as mean ± SD. (n = 8). ΔΔP < 0.01 vs. control group, ∗P < 0.05, ∗∗P < 0.01 vs. model group.
Figure 5
Figure 5
Effect of Baishouwu extract on fibrosis related indicators induced by DEN. Tissue levels of (A) hydroxyproline, (B) Collagen I, and (C) Collagen III. Results are expressed as mean ± SD (n = 8). ΔΔP < 0.01 vs. control group, ∗P < 0.05, ∗∗P < 0.01 vs. model group.
Figure 6
Figure 6
Effect of Baishouwu extract on macroscopic features of livers in DEN-treated rats. Representative photographs of the livers from each group at the stages of inflammation (6 weeks), fibrosis (10 weeks), and HCC (20 weeks). Arrows indicate the representative tumors.
Figure 7
Figure 7
Effect of Baishouwu extract on the liver histological changes in the DEN-treated rats. Representative photomicrographs of HE (A) and Masson (B) staining of the livers in the DEN-treated rats from each group at the stages of inflammation (6 weeks), fibrosis (10 weeks), and HCC (20 weeks). Arrows indicate the representative tumors. Original magnification: 200×.
Figure 8
Figure 8
Effect of Baishouwu extract on the expression of NF-κB p65 in the livers of the DEN-treated rats. Representative photomicrographs of immunohistochemical analysis of NF-κB p65 in the livers developed in DEN-treated rats. (A) Representative liver tissues immunostained with anti- NF-κB p65 antibody in hepatitis, cirrhosis and hepatocarcinoma control at 6th week, 10th week, and 20th week (magnification: 200×). (B) The percentage of NF-κB p65 positive cells in control and experimental groups. Results are expressed as mean ± SD (n = 3). ΔΔP < 0.01 vs. control group, ∗∗P < 0.01 vs. model group.
Figure 9
Figure 9
Effect of Baishouwu extract on the TLR4/MyD88/NF-κB signaling pathway. The expression levels of TLR4, MyD88, TRAF6, NF-κB p65, TGF-β1, and α-SMA were detected by Real-time PCR (A) and western blot (B): (1) control group, (2) model group, (3) Baishouwu extract-L group, and (4) Baishouwu extract-H group. Results are expressed as mean ± SD (n = 3). ΔΔP < 0.01 vs. control group, ∗P < 0.05, ∗∗P < 0.01 vs. model group.
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