ABSTRACT
Objective:To study the role of ethanol extract of Euonymus alatus stems (EAT) and ethanol extract of Euonymus alatus wings (EAW) in anti-hepatic fibrosis induced by carbon tetrachloride in mice, and to explore its preliminary mechanism. Methods:Sixty C57BL/6 mice were selected and randomly divided into healthy control group, carbon tetrachloride model (CTM) group, EAW low dose (EAW-L) group, EAW high dose (EAW-H) group, EAT low dose (EAT-L) group and EAT high dose (EAT-H) group, with 10 mice in each group. Three days before modeling, the mice of EAT-L, EAT-H, EAW-L and EAW-H group were gavaged with EAT or EAW at 2.0 or 8.0 g/kg, respectively, and the mice of healthy control group and CTM group were gavaged with equal volume of pure water, once a day till the 30th day after modeling (total 33 times). Five percent carbon tetrachloride olive oil solution was intraperitoneally injected at 8 mL/kg to establish liver fibrosis model in CTM, EAT-L, EAT-H, EAW-L and EAW-H groups. The mice in the healthy control group were intraperitoneally injected with equal volume of 0.9% sodium chloride solution, twice per week for 30 days, and a total of 9 times of injection. The liver index, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil) and interleukin-6 (IL-6) were detected. Hematoxylin-eosin and Masson staining were used to observe the pathological changes of mouse liver tissue and calculate the collagen volume fraction. The liver inflammatory response and fibrosis degree were evaluated by histological activity index (HAI) and Ishak system score. The level of α-smooth muscle actin(α-SMA)in liver tissue was both detected by immunohistochemistry and Western blotting. The expression of matrix metalloproteinase 2 (MMP2) and extracellular signal-regulated kinase (ERK) 1/2 at protein and mRNA level was detected by Western blotting and fluorescent quantitative polymerase chain reaction. Analysis of variance, Tukey test and Dunn test were used for statistical analysis.Results:The hepatic indexes of EAW-L, EAW-H and EAT-H groups were lower than that of CTM group(0.06±0.01, 0.05±0.01 and 0.05±0.01 vs. 0.07±0.01), and the differences were statistically significant ( q=5.12, 7.70, 7.11; all P<0.01). The serum ALT and AST levels of EAW-L, EAW-H, EAT-L and EAT-H groups were lower than those of CTM group((601.76±141.38), (283.35±42.32), (734.74±116.06) and (391.60±34.33) U/L vs.(982.45±96.04) U/L, (509.49±152.29), (345.41±67.39), (282.30±65.72) and(243.23±45.20) U/L vs.(766.01±114.49) U/L), and the differences were statistically significant ( qALT =9.88, 20.81, 7.65, 17.58, qAST =5.11, 12.52, 14.92, 15.56; all P<0.001). The serum TBil levels of EAW-H and EAT-H groups were lower than that of CTM group((6.81±0.49) and (7.08±1.78) μmol/L vs.(12.68±3.28) μmol/L), and the differences were statistically significant( q=6.31, 6.01; both P<0.01). The serum IL-6 levels of EAW-L, EAW-H, EAT-L and EAT-H groups were lower than that of CTM group((29.26±5.42), (24.28±4.75), (9.05±1.74) and (8.01±1.24) ng/L vs.(53.21±10.05) ng/L); the serum IL-6 level of EAT-L group was lower than that of EAW-L group; the serum IL-6 level of EAT-H group was lower than that of EAW-H group, and the differences were statistically significant( q=12.20, 14.73, 22.48, 22.11, 10.28, 7.96; all P <0.001). The collagen volume fractions of EAW-L, EAW-H, EAT-L and EAT-H groups were lower than that of CTM group (6.15±1.09, 2.91±0.76, 7.07±1.37 and 5.31±0.80 vs. 12.36±1.96); the collagen volume fraction of EAW-H group was lower than that of EAW-L, EAT-L and EAT-H groups, and the differences were statistically significant( q=11.68, 17.78, 9.94, 13.25; 6.10, 7.84, 4.53; all P <0.05). The HAI and Ishak system scores of EAW-H and EAT-H groups were lower than those of CTM group (6.0 (5.5, 7.5) and 7.0 (6.0, 7.5) vs. 13.0 (12.0, 13.0), 1.0 (1.0, 2.0) and 2.0 (1.0, 2.0) vs. 4.0 (3.0, 4.0)), and the differences were statistically significant( ZHAI=3.38, 3.23, Zlshak=3.22, 3.03; all P<0.05). The result of immunohistochemical analysis showed that the expression levels of α-SMA in the mice liver tissues of EAW-L, EAW-H, EAT-L, EAT-H and CTM groups were 4.76±0.36, 2.75±0.29, 3.72±0.34, 5.20±0.79 and 5.98±0.52, respectively. The result of Western blotting showed that the expression levels of α-SMA in the mice liver tissues of CTM, EAW-L, EAW-H, EAT-L and EAT-H groups were 0.96±0.11, 0.67±0.07, 0.22±0.01, 0.78±0.08 and 0.68±0.07, respectively. Two detection methods both showed that the expression levels of α-SMA of EAW-L, EAW-H and EAT-H groups were lower than that of CTM group; the expression level of α-SMA of EAW-H group was lower than that of EAW-L, EAT-L and EAT-H group, and the differences were statistically significant( qimmunohistochemical =6.06, 15.95, 11.18, 9.92, 12.10 and 4.79, qWestern blotting=7.29, 18.34, 6.84, 11.05, 13.97 and 11.49, all P<0.05). The expression levels of MMP2 and ERK1/2 at protein and mRNA levels in the mice liver tissues of EAW-L, EAW-H, EAT-L, EAT-H and CTM groups were 0.18±0.04, 0.16±0.04, 0.28±0.02, 0.21±0.02 and 0.84±0.02, 0.80±0.02, 0.57±0.08, 0.83±0.03, 0.69±0.02 and 0.91±0.04, 18.74±1.90, 10.73±1.24, 24.99±1.84, 7.19±0.48 and 24.68±1.18, 29.44±4.47, 11.96±0.53, 24.75±4.04, 5.30±0.36 and 35.76±0.85, respectively. The expression levels of MMP2 at protein level in EAW-L, EAW-H, EAT-L and EAT-H groups were lower than that in CTM group; the expression levels of ERK1/2 at protein level in EAW-H and EAT-H groups were lower than that in CTM group; the expression level of ERK1/2 at protein level in EAW-H group was lower than that in EAT-H group; the expression levels of MMP2 and ERK1/2 at mRNA level in EAW-H and EAT-H group were lower than those in CTM group; the expression levels of MMP2 and ERK1/2 at mRNA level in EAW-H group were lower than those in EAW-L group; the expression levels of MMP2 and ERK1/2 at mRNA level in EAT-H group were lower than those in EAT-L and EAW-H groups, and the differences were statistically significant( q=22.15, 22.96, 18.87, 21.31; 13.42, 8.53; 4.90; 18.57, 23.29, 16.49, 21.11; 10.66, 12.12; 23.70, 15.38, 13.48, 16.73; all P<0.05). Conclusions:Both EAT and EAW can alleviate carbon tetrachloride-induced liver injury and liver fibrosis in mice, which may be related with inhibiting the expression of ERK1/2 and IL-6 and then affecting the Ras/ERK-MMP2 signaling pathway.