RhoA/ROCK信号转导通路介导高糖诱导的大鼠肝星状细胞的增殖和胶原合成  

RhoA/ROCK signaling transduction pathway mediates high-glucose-induced proliferation of hepatic stellate cell and synthesis of collagen in rats

作　　者：李贵芝[1] 刘莎 张艳[1,3] 周红 Li Guizhi;Liu Sha;Zhang Yan;Zhou Hong(Department of Endocrinology,the Second Hospital of Hebei Medical University,Shijiazhuang 050000,China;Department of Ultrasonography,the Third Hospital of Hebei Medical University,Shijiazhuang 050000,China;Department of Endocrinology,Hengshui Harrison Hospital,Hengshui 053000,Hebei Province,China)

机构地区：[1]河北医科大学第二医院内分泌科,石家庄050000 [2]河北医科大学第三医院超声科,石家庄050000 [3]衡水市哈励逊医院内分泌科,河北衡水053000

出　　处：《中国肝脏病杂志（电子版）》2023年第2期28-35,共8页Chinese Journal of Liver Diseases：Electronic Version

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(12.5μmol/L、25μmol/L、50μmol/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(25μmol/L、50μmol/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12Objective 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(12.5μmol/L,25μmol/L,50μmol/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 typeⅠandⅢprocollagen mRNA were determined by realtime fluorescence 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 significantly,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 typeⅠ(2.783±0.167 vs 1.004±0.008,P<0.001)and typeⅢ(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(25μmol/L,50μmol/L)decreased significantly,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 significantly and expression of typeⅠ(1.580±0.1

关 键 词：肝星状细胞 RhoA/ROCK信号转导通路 高糖 增殖 胶原合成 

分 类 号：R575.5[医药卫生—消化系统] R587.2[医药卫生—内科学]