摘要：目的 探讨RhoA/ROCK信号转导通路在高糖诱导大鼠肝星状细胞（hepatic stellate
cell， HSC）增殖和胶原合成中的作用。 方法 将SD大鼠肝星状细胞株HSC-T6在1640
培养基中培养24 h，实验设置对照组（含5.5 mmol/L葡萄糖）、高糖组（含25 mmol/L
葡萄糖）、高渗透压组（5.5 mmol/L葡萄糖 + 19.5 mmol/L甘露醇）、高糖 + 法舒地
尔（12.5 μmol/L、 25 μmol/L、 50 μmol/L）组。采用MTS法检测细胞增殖率；采用羟
脯氨酸（hydroxyproline， Hyp）试剂盒测定细胞上清中Hyp水平；采用实时荧光定
量聚合酶链式反应（real-time fluorescence quantitative polymerase chain reaction， FQPCR）测定Ⅰ和Ⅲ型前胶原mRNA的相对表达量；采用Western blot检测肌球蛋白磷
酸酶靶亚基1（myosin phosphatase target subunit 1， MYPT1）、细胞外信号调节激酶
（extracellular signal-regulated kinase， ERK）、 c-Jun氨基末端激酶（c-Jun N-terminal
kinases， JNK）和p38MAPK的磷酸化和总体水平。 结果 与对照组相比，高糖组MYPT1
（0.270 ± 0.007 vs 0.090 ± 0.008， P ＜ 0.001）、 ERK（0.851 ± 0.027 vs 0.175 ± 0.038，
P ＜ 0.001）、 JNK（0.869 ± 0.037 vs 0.488 ± 0.022， P ＜ 0.001）和p38MAPK（0.498 ±
0.020 vs 0.144 ± 0.011， P ＜ 0.001）磷酸化水平显著增高， HSC增殖率（A值）（2.372 ±
0.098 vs 1.588 ± 0.087， P ＜ 0.001）和Hyp水平（27.924 ± 1.069 vs 17.643 ± 0.112， P ＜
0.001）显著增高，Ⅰ型前胶原mRNA（2.783 ± 0.167 vs 1.004 ± 0.008， P ＜ 0.001）和
Ⅲ型前胶原mRNA（4.958 ± 0.143 vs 1.098 ± 0.014， P ＜ 0.001）表达显著上调。与高
糖组相比，高糖+法舒地尔（25 μmol/L、 50 μmol/L）组MYPT1（0.110 ± 0.007， P ＜
0.001； 0.101 ± 0.006， P ＜ 0.001）、 ERK（0.473 ± 0.025， P ＜ 0.001； 0.223 ± 0.031，
P ＜ 0.001）、 JNK（0.688 ± 0.024， P = 0.019； 0.576 ± 0.035， P ＜ 0.001）和p38MAPK
（0.350 ± 0.021， P = 0.012； 0.305 ± 0.015， P = 0.019）磷酸化水平显著降低， HSC增
殖率（A值）（1.819 ± 0.104， P ＜ 0.001； 1.613 ± 0.103， P ＜ 0.001）和Hyp水平（21.430 ±
0.714， P ＜ 0.001； 18.574 ± 0.825， P ＜ 0.001）显著降低，Ⅰ型前胶原mRNA（1.580 ±
0.154， P ＜ 0.001； 1.167 ± 0.157， P ＜ 0.001）和Ⅲ型前胶原mRNA（3.166 ± 0.073，
P ＜ 0.001； 2.524 ± 0.085， P ＜ 0.001）表达显著下调。高糖+法舒地尔12.5 μmol/L
组MYPT1、 ERK、 JNK和p38MAPK磷酸化水平及Hcy水平均显著高于高糖+法舒地尔
25 μmol/L组和高糖+法舒地尔50 μmol/L组（P均＜ 0.001），高糖+法舒地尔25 μmol/L
组显著高于高糖+法舒地尔50 μmol/L组（P均＜ 0.001）。 结论 RhoA/ROCK信号转导通
路可能通过激活下游的MAPKs介导了高糖诱导的肝HSC的增殖和胶原合成， ROCK可
能是防治糖尿病肝纤维化的新靶点。

 Abstract: Objective To investigate the role of RhoA/ROCK signaling transduction pathway
on high-glucose-induced proliferation of hepatic stellate cell (HSC) and synthesis of collagen
in rats. Methods HSC-T6 cells of SD rats were cultured in 1640 medium for 24 h and divided
into control group (5.5 mmol/L D-glucose), high-glucose group (25 mmol/L D-glucose), high
osmotic pressure group (5.5 mmol/L D-glucose + 19.5 mmol/L mannose) and high-glucose +
fasudil group (12.5 μmol/L, 25 μmol/L, 50 μmol/L). Proliferation of HSC was measured
by MTS assay. Level of hydroxyproline (Hyp) in the cell supernatant was determined by
Hyp kit. The expression of type Ⅰ and Ⅲ procollagen mRNA were determined by realtime ?uorescence quantitative polymerase chain reaction. Western blot was used to evaluate
the phosphorylation of myosin phosphatase target subunit 1 (MYPT1), extracellular signalregulated kinase (ERK), c-Jun N-terminal kinases (JNK) and p38mitogen-activated protein
kinase (p38MAPK). Results Compared with control group, phosphorylation level of MYPT1
(0.270 ± 0.007 vs 0.090 ± 0.008, P ＜ 0.001), ERK (0.851 ± 0.027 vs 0.175 ± 0.038, P ＜ 0.001),
JNK (0.869 ± 0.037 vs 0.488 ± 0.022, P ＜ 0.001) and p38MAPK (0.498 ± 0.020 vs 0.144 ± 0.011,
P ＜ 0.001) in high-glucose group increased signifcantly, proliferation of HSC (A value) (2.372 ±
0.098 vs 1.588 ± 0.087, P ＜ 0.001) and Hyp level (27.924 ± 1.069 vs 17.643 ± 0.112, P ＜
0.001) increased significantly and expression of type Ⅰ(2.783 ± 0.167 vs 1.004 ± 0.008,
P ＜ 0.001) and type Ⅲ (4.958 ± 0.143 vs 1.098 ± 0.014, P ＜ 0.001) procollagen mRNA
upregulated significantly. Compared with high-glucose group, phosphorylation level of
MYPT1 (0.110 ± 0.007, P ＜ 0.001; 0.101 ± 0.006, P ＜ 0.001), ERK (0.473 ± 0.025, P ＜
0.001; 0.223 ± 0.031, P ＜ 0.001), JNK (0.688 ± 0.024, P = 0.019; 0.576 ± 0.035, P ＜ 0.001)
and p38MAPK (0.350 ± 0.021, P = 0.012; 0.305 ± 0.015, P = 0.019) in high-glucose + fasudil
group (25 μmol/L, 50 μmol/L) decreased signifcantly, proliferation of HSC (A value) (1.819 ±
0.104, P ＜ 0.001; 1.613 ± 0.103, P ＜ 0.001) and Hyp level (21.430 ± 0.714, P ＜ 0.001;
18.574 ± 0.825, P ＜ 0.001) decreased signifcantly and expression of type Ⅰ(1.580 ± 0.154,
P ＜ 0.001; 1.167 ± 0.157, P ＜ 0.001) and type Ⅲ (3.166 ± 0.073, P ＜ 0.001; 2.524 ± 0.085,
P ＜ 0.001) procollagen mRNA downregulated signifcantly. Phosphorylation level of MYPT1
ERK, JNK and p38MAPK and level of Hcy in high-glucose + fasudil group (12.5 μmol/L)
were signifcantly higher than those in high-glucose + fasudil 25 μmol/L group (all P ＜ 0.001),
the above indexes in high-glucose + fasudil 25 μmol/L group were signifcantly higher than
those in high-glucose + fasudil 50 μmol/L group (all P ＜ 0.001). Conclusions The RhoA/
ROCK signaling transduction pathway may mediate the high glucose-induced hepatic HSC
proliferation and collagen synthesis through the activation of downstream MAPKs, and
ROCK may be a novel target for the prevention of liver fbrosis in diabetes.