Abstract:

Objective To observe the treatment effect of Zhuang medicine Herba abri formula (HaF) on hepatic fibrosis induced by Echinococcus multilocularis infection in vivo and in vitro. Methods The HaF solution was prepared from Zhuang medicine Herba abri capsule commercial available. Human hepatic stellate cell-LX2 (HSC-LX2) were cultured with 25, 50, 100 or 200 µg/ml HaF solution for 72, 96 and 120 h, then the relative expression of α-smooth muscle actin (α-SMA) and collagen Ⅰ in HSC-LX2 was detected with Western blotting, respectively. Separately, HSC-LX2 cells were stimulated with 0.1 mg/ml E. multilocularis crude antigen for 24 h, followed by addition of 100 μg/ml HaF for 24, 48 and 72 h, subsequently, the relative expression of α-smooth muscle actin (α-SMA) and collagen Ⅰ in HSC-LX2 were detected by Western blotting. Thirty-six female Kunming mice were randomly assigned into the following groups: control group (4 mice), infection group (4 mice), HaF treatment groups at doses of 0.5 g/(kg•d) (5 mice), 1.0 g/(kg•d) (5 mice), and 2.0 g/(kg•d) (6 mice), albendazole (ABZ) treatment group (6 mice), and ABZ + HaF combined treatment group (6 mice). The infection group and each treatment group received intraperitoneal injection of 2 000 protoscolices, while the control group was injected with an equal volume of normal saline. After a period of 60 days post-infection, the mice in treatment groups were treated daily by gavage with the corresponding medication for 60 days, and then the mice liver and spleen tissues were collected and weighed to calculate hepatic index and splenic indix. The content of hydroxyproline (HYP) in liver tissue was determined by the alkaline hydrolysis method, and the level of serum aspartate aminotransferase (AST) was measured by the colorimetric method. Western blotting was performed to detect the relative expression of α-SMA and collagen Ⅰ in the liver, while hematoxylin-eosin staining (HE) and Masson staining were used to observe pathological changes in liver and spleen tissues. The comparison between two groups of data was conducted using an independent-samples t-test. Results The relative expression of α-SMA protein and collagen Ⅰ was 0.401 ± 0.218 and 0.352 ± 0.058, respectively, in HSC-LX2 cells treated with 100 µg/ml HaF solution at 96 h, which were significantly lower than those in the control group (1.435 ± 0.297, 1.340 ± 0.416) (t = 2.755, 11.120; both P < 0.05). The control group did not exhibit any statistically significant differences compared to the other concentrations and durations of treatment. Compared to the control group (0.895 ± 0.417, 1.009 ± 0.378), the relative expression levels of α-SMA and collagen Ⅰ in HSC-LX2 treated with HaF for 48 h after E. multilocularis crude antigen stimulation significantly decreased to (0.326 ± 0.106) and (0.315 ± 0.076), respectively (t = 6.359, 9.059; both P < 0.05). In vivo experiments showed that the hepatic indices, splenic indices, serum AST levels and liver HYP contents of infection group were (4.366 ± 0.284) %, (5.129 ± 1.114) mg/g, (22.194 ± 1.509) U/L, and (21.743 ± 2.503) × 10^-2 μg/mg respectively, which were higher than (3.389 ± 0.045) %, (2.031 ± 0.165) mg/g, (17.355 ± 0.574) U/L and (9.330 ± 1.519) × 10^-2 μg/mg in the control group (t = 3.393, 2.752, 2.616, 4.549; all P < 0.05). The serum AST levels of mice in the HaF treatment group at doses of 0.5, 1.0, and 2.0 g/(kg·d), the ABZ treatment group, and the combined ABZ + HaF treatment group were (17.375 ± 0.746), (15.411 ± 1.338), (17.057 ± 1.066), (16.190 ± 1.559), (14.637 ± 1.888) U/L, respectively, lower than those in infection group (t = 2.863, 3.362, 2.838, 2.717, 3.002; all P < 0.05). Among the treatment groups, the HaF treatment group at a dosage of 1.0 g/(kg•d) showed the most significant reduction in serum AST levels. There were no significant differences in hepatic/splenic indices and HYP content in liver tissue between the five treatment groups and the infection group (all P > 0.05). Western blotting results showed that the relative expression level of α-SMA protein in the liver tissue of mice in the 1.0 g/(kg•d) HaF treatment group was 0.818 ± 0.195, and the relative expression level of collagen Ⅰ in the liver tissue of mice in the combined treatment group of ABZ and HaF was 0.799 ± 0.167, both lower than those in the infected group (1.668 ± 0.131, 1.341 ± 0.141) (t = 3.619, 2.486; P < 0.05). The results of HE staining and Masson staining demonstrated that the fibrotic tissue area in the ABZ + HaF combined treatment group was (6.184 ± 1.951)%, which was significantly reduced compared to (19.916 ± 2.046)% in the infection group (t = 4.703, P < 0.01), indicating a notable improvement in liver fibrosis. Conclusion Zhuang medicine HaF demonstrates the ability to attenuate hepatic fibrosis induced by E. multilocularis infection both in vitro and in vivo. It also provides an experimental basis for HaF as a novel therapeutic agent for treatment of alveolar echinococcosis.

Key words: Echinococcus multilocularis infection, Zhuang medicine Herba abri, Hepatic fibrosis