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摘要：
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摘要：目的 探究竹沥对肝纤维化小鼠Ras同源基因-Rho相关螺旋卷曲蛋白激酶（Ras
homologous gene-Rho-associated coiled-coil containing kinases，Rho-ROCK）信号转导通
路的作用，分析竹沥抗肝纤维化的分子机制。方法 将40只KM小鼠随机分为空白组、
模型组、竹沥组和丹参酚酸B组，每组10只，模型组、竹沥组和丹参酚酸B组小鼠给予
40% CCl4花生油混合液（剂量2 μl/g），空白组给予等量生理盐水，腹腔注射，2次/周，
连续8周。模型构建成功后，竹沥组以竹沥灌胃，每日1次，每次30 ml/100 g；丹参酚
酸B组以丹参酚酸B溶液灌胃，每日1次，每次用量0.25 mg/100 g；其余2组给予等量
生理盐水灌胃，持续4周。末次给药后对小鼠眼球取血并处死，采用HE染色观察肝组
织病理并进行Ishak评分，采用γ放射免疫全自动双探头计数器检测肝纤维化指标，包
括透明质酸、层粘连蛋白、Ⅲ型前胶原和Ⅳ型胶原，采用实时荧光定量聚合酶链式
反应（real-time quantitative polymerase chain reaction，qRT-PCR）检测RhoA和ROCK2
mRNA的相对表达量，采用Western blot检测RhoA和ROCK2蛋白的相对表达量。结果 空
白组、模型组、竹沥组和丹参酚酸B组小鼠Ishak评分分别为（0.40 ± 0.05）分、（4.80 ±
0.84）分、（3.20 ± 0.45）分和（2.80 ± 0.83）分，差异有统计学意义（F = 34.807，P ＜
0.001），其中空白组显著低于模型组、竹沥组和丹参酚酸B组（t = 8.690，P ＜ 0.001；
t = 5.674，P ＜ 0.001；t = 4.768，P = 0.001），模型组显著高于竹沥组和丹参酚酸B
组（t = 3.016，P = 0.017；t = 3.922，P = 0.004），竹沥组和丹参酚酸B组差异无统计
学意义（t = 0.809，P = 0.464）。空白组小鼠肝纤维化指标均显著低于模型组、竹沥
组和丹参酚酸B组 [透明质酸：（26.14 ± 2.69）μg/L vs（53.69 ± 5.87）μg/L vs（37.21 ±
3.04）μg/L vs（32.88 ± 3.60）μg/L，层粘连蛋白：（120.37 ± 15.57）μg/L vs（214.48 ±
21.39）μg/L vs（162.54 ± 19.22）μg/L vs（161.66 ± 14.23）μg/L，Ⅲ型前胶原：（4.04 ±
1.01）μg/L vs（11.70 ± 3.09）μg/L vs（8.12 ± 1.87）μg/L vs（7.80 ± 1.72）μg/L，Ⅳ型胶
原：（12.96 ± 2.82）μg/L vs（31.34 ± 4.44）μg/L vs（23.72 ± 3.69）μg/L vs（22.85 ±
3.00）μg/L；P均＜ 0.05]，模型组小鼠肝纤维化指标均显著高于竹沥组和丹参酚酸B
组（P均＜ 0.05），竹沥组和丹参酚酸B组差异无统计学意义（P均＞ 0.05）。空白组
RhoA和ROCK2 mRNA相对表达量均显著低于模型组、竹沥组和丹参酚酸B组（RhoA
mRNA：1.05 ± 0.18 vs 2.61 ± 0.37 vs 1.62 ± 0.21 vs 1.50 ± 0.14，ROCK2 mRNA：1.04 ±
0.17 vs 2.32 ± 0.29 vs 1.46 ± 0.08 vs 1.45 ± 0.09；P均＜ 0.05），模型组RhoA和ROCK2
mRNA相对表达量均显著高于竹沥组和丹参酚酸B组（P均＜ 0.05），竹沥组和丹参酚
酸B组差异无统计学意义（P均＞ 0.05）。空白组RhoA和ROCK2蛋白相对表达量均显
著低于模型组、竹沥组和丹参酚酸B组（RhoA蛋白：0.14 ± 0.03 vs 0.43 ± 0.05 vs 0.26 ±
0.02 vs 0.30 ± 0.15；ROCK2蛋白：0.28 ± 0.03 vs 0.76 ± 0.09 vs 0.38 ± 0.04 vs 0.49 ± 0.03；
P均＜ 0.05），模型组RhoA和ROCK2蛋白相对表达量均显著高于竹沥组和丹参酚酸B
组（P均＜ 0.05），竹沥组和丹参酚酸B组差异无统计学意义（P均＞ 0.05）。结论 竹
沥可抑制Rho-ROCK信号转导通路的活动，可能是其抗肝纤维化的作用机制。
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Abstract: Objective To investigate the effects of Zhuli on the Ras homologous gene-Rhoassociated coiled-coil containing kinases (Rho-ROCK) signaling transduction pathway
in mice with liver fibrosis and to analyze the molecular mechanism of Zhuli against liver
fibrosis. Methods A total of forty KM mice were divided into blank group, model group,
Zhuli group and salvianolic acid B group, 10 mice in each group. Mice in model group,
Zhuli group and salvianolic acid B group were given 40% CCl4 peanut oil mixture (2 μl/g)
and mice in blank group were given equal amounts of saline by intraperitoneal injection
for 8 weeks. After the successful model construction, mice in Zhuli group were gavaged
with Zhuli once a day (30 ml/100 g), mice in salvianolic acid B group were gavaged with
salvianolic acid B once a day (0.25 mg/100 g) and mice in the other two groups were
gavaged with equal amounts of saline for 4 weeks. After the last administration, blood was
taken from the eyeballs and the mice were sacrificed. Liver histopathology was visualized
by HE staining and Ishak score was calculated. Liver fibrosis indexes were detected by a
fully automated double probe counter, including hyaluronic acid, laminin, procollagen Ⅲ
and Ⅳ collagen. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to
detect the mRNA relative expression of RhoA and ROCK2. Western blot was used to detect
the protein relative expression of RhoA and ROCK2. Results The Ishak score of mice in
blank group, model group, Zhuli group and salvianolic acid B group were (0.40 ± 0.05) points, (4.80 ±
0.84) points, (3.20 ± 0.45) points and (2.80 ± 0.83) points, respectively, the difference was
statistically significant (F = 34.807, P ＜ 0.001), the Ishak score of mice in blank group
was significantly lower than that in model group, Zhuli group and salvianolic acid B group
(t = 8.690, P ＜ 0.001; t = 5.674, P ＜ 0.001; t = 4.768, P = 0.001), the Ishak score of mice
in model group was significantly higher than that in Zhuli group and salvianolic acid B group
(t = 3.016, P = 0.017; t = 3.922, P = 0.004), and there was no significant difference of Ishak
score between mice in Zhuli group and salvianolic acid B group (t = 0.809, P = 0.464).
Liver fibrosis indexes of mice in blank group were significantly lower than those in model
group, Zhuli group and salvianolic acid B group [hyaluronic acid: (26.14 ± 2.69) μg/L vs
(53.69 ± 5.87) μg/L vs (37.21 ± 3.04) μg/L vs (32.88 ± 3.60) μg/L, laminin: (120.37 ± 15.57) μg/L vs
(214.48 ± 21.39) μg/L vs (162.54 ± 19.22) μg/L vs (161.66 ± 14.23) μg/L, procollagen Ⅲ: (4.04 ±
1.01) μg/L vs (11.70 ± 3.09) μg/L vs (8.12 ± 1.87) μg/L vs (7.80 ± 1.72) μg/L, Ⅳ collagen:
(12.96 ± 2.82) μg/L vs (31.34 ± 4.44) μg/L vs (23.72 ± 3.69) μg/L vs (22.85 ± 3.00) μg/L; all
P ＜ 0.05), liver fibrosis indexes of mice in model group were significantly higher than
those in Zhuli group and salvianolic acid B group (all P ＜ 0.05), and there were no
significant differences of liver fibrosis indexes between mice in Zhuli group and salvianolic
acid B group (all P ＞ 0.05). The RhoA and ROCK2 mRNA relative expression levels of
mice in blank group were significantly lower than those in model group, Zhuli group and
salvianolic acid B group (RhoA mRNA: 1.05 ± 0.18 vs 2.61 ± 0.37 vs 1.62 ± 0.21 vs 1.50 ±
0.14, ROCK2 mRNA: 1.04 ± 0.17 vs 2.32 ± 0.29 vs 1.46 ± 0.08 vs 1.45 ± 0.09; all P ＜
0.05), the RhoA and ROCK2 mRNA relative expression levels of mice in model group were
significantly higher than those in Zhuli group and salvianolic acid B group (all P ＜ 0.05),
and there were no significant differences of RhoA and ROCK2 mRNA relative expression
levels between mice in Zhuli group and salvianolic acid B group (all P ＞ 0.05). The RhoA
and ROCK2 protein relative expression levels of mice in blank group were significantly
lower than those in model group, Zhuli group and salvianolic acid B group (RhoA protein: 0.14 ± 0.03
vs 0.43 ± 0.05 vs 0.26 ± 0.02 vs 0.30 ± 0.15; ROCK2 protein: 0.28 ± 0.03 vs 0.76 ± 0.09 vs
0.38 ± 0.04 vs 0.49 ± 0.03; all P ＜ 0.05), the RhoA and ROCK2 protein relative expression
levels of mice in model group were significantly higher than those in Zhuli group and
salvianolic acid B group (all P ＜ 0.05), and there were no significant differences of RhoA
and ROCK2 protein relative expression levels between mice in Zhuli group and salvianolic
acid B group (all P ＞ 0.05). Conclusions Zhuli can inhibit the activity of Rho-ROCK
signaling pathway, which may be its anti-liver fibrosis mechanism.
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