【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 homologousgene-Rho-associatedcoiled-coilcontainingkinases,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2μl/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,q RT-PCR)检测Rho A和ROCK2m RNA的相对表达量,采用Western blot检测Rho A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Rho A和ROCK2m RNA相对表达量均显著低于模型组、竹沥组和丹参酚酸B组(Rho A m RNA:1.05±0.18 vs 2.61±0.37 vs 1.62±0.21 vs 1.50±0.14,ROCK2 m RNA:1.04±0.17vs2.32±0.29vs1.46±0.08vs1.45±0.09;P均<0.05),模型组Rho A和ROCK2m RNA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Rho A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(Rho A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Rho A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。更多还原

【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(q RT-PCR) was used to detect the m RNA relative expression of Rho A and ROCK2. Western blot was used to detect the protein relative expression of Rho A 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 Rho A and ROCK2 m RNA relative expression levels of mice in blank group were significantly lower than those in model group, Zhuli group and salvianolic acid B group(Rho A m RNA: 1.05 ± 0.18 vs 2.61 ± 0.37 vs 1.62 ± 0.21 vs 1.50 ± 0.14, ROCK2 m RNA: 1.04 ± 0.17 vs 2.32 ± 0.29 vs 1.46 ± 0.08 vs 1.45 ± 0.09; all P < 0.05), the Rho A and ROCK2 m RNA 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 Rho A and ROCK2 m RNA relative expression levels between mice in Zhuli group and salvianolic acid B group(all P > 0.05). The Rho A 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(Rho A 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 Rho A 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 Rho A 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.更多还原