Effects of novel Fufang Biejia Ruangan Tablets with sheep placenta as substitute for Hominis Placenta on CCl4-induced liver fibrosis

Abstract

Objective: Fufang Biejia Ruangan Tablet (FBRT) is widely used for the treatment of liver fibrosis. However, Hominis Placenta (HP), as an important adjuvant of FBRT, has been restricted for medicinal using due to the limited availability, ethical controversy and safety issues. The present study aimed to investigate the therapeutic effects of novel FBRT (N-FBRT) with sheep placenta (SP) as substitute for HP on liver fibrosis and explore its possible mechanisms. Different dosages of SP in N-FBRT were also evaluated.

Methods: Rats were subcutaneously injected with CCl₄ to induce liver fibrosis and then treated with N-FBRT and FBRT. The anti-hepatic fibrosis effect was determined based on biomarkers analysis of liver function and hepatic fibrosis, and the liver pathology was visualized by H&E staining and Masson staining. The oxidative stress and inflammatory cytokines were also detected. Immunohistochemical staining of α-SMA, real time PCR and Western blotting were performed to evaluate hepatic stellate cells (HSCs) activation and TGF-β1/Smad signaling pathway.

Results: N-FBRT and FBRT could ameliorate CCl₄-induced liver fibrosis and improve liver function, as evidenced by lowering serum biomarkers levels of liver function and hepatic fibrosis, and decreasing hepatic Hyp content and collagen deposition, and improving the hepatic morphology and architecture changes. Moreover, the anti-liver fibrosis effect was better when the dosage of SP used in N-FBRT was 1/2 of HP in FBRT. Administration of N-FBRT markedly alleviated oxidative stress and inflammatory cytokines, and inhibited α-SMA expression. Furthermore, the mRNA expression of Col I, Col III, α-SMA and TGF-β1, and proteins expression of α-SMA, TGF-β1, Smad2/3 and p-Smad2/3 were significantly down-regulated by N-FBRT treatment.

Conclusion: SP can be used as substitute for HP to prepare N-FBRT for the treatment of liver fibrosis and the anti-liver fibrosis effect of N-FBRT is achieved by eliminating oxidative stress and inflammation, and inhibiting HSCs activation and ECM production by blocking TGF-β1/Smad signaling pathway.

Keywords: Fufang Biejia Ruangan Tablet; Hominis Placenta; TGF-β1/Smad signaling pathway; liver fibrosis; sheep placenta; substitute.

Fig. 1

Effects of N-FBRT and FBRT on hepatic histopathological changes of rats with liver fibrosis (magnification, 200×). (A) H&E staining; (B) Masson staining; (C) α-SMA immunohistochemical staining; (D) Relative fibrosis area; (E) α-SMA positive area. Data are expressed as mean ± SD (n = 8). ^**P < 0.01 vs control group; ^##P < 0.01 vs CCl₄ model group; ^ΔP < 0.05, ^ΔΔP < 0.01 vs FBRT group.

Fig. 2

Effects of N-FBRT and FBRT on mRNA expression levels of Col-I and Col-III (A), α-SMA and TGF-β1 (B) in liver of rats with liver fibrosis. Data are expressed as mean ± SD (n = 4). ^**P < 0.01 vs control group; ^#P < 0.05, ^##P < 0.01 vs CCl₄ model group.

Fig. 3

Effects of N-FBRT and FBRT on proteins expression of α-SMA and TGF-β1(A), Smad2/3 and p-Smad2/3 (B) in liver of rats with liver fibrosis. Bands 1–8 of western-blot images represent the control, CCl₄ model, N-FBRT1, N-FBRT2, N-FBRT3, N-FBRT4, FBRT and colchicine groups, respectively. Data expressed as mean ± SD (n = 3). *P < 0.05, ^**P < 0.01 vs control group; ^#P < 0.05, ^##P < 0.01 vs CCl₄ model group; ^ΔP < 0.05, ^ΔΔP < 0.01 vs FBRT group.