CAJ | 학술논문

目的体外实验研究法舒地尔(fasudil)通过抑制Rho/ROCK信号通路对高糖培养下的人肾小球系膜细胞(HMCs)炎症反应及其纤维化的影响.方法传代培养的HMCs同步化后分组:(1)正常糖浓度对照组(NG,含葡萄糖5.5 mmol/L);(2)高糖组(HG,含葡萄糖30.0 mmol/L);(3)甘露醇渗透压对照组(Man,含5.5 mmol/L葡萄糖+24.5 mmol/L甘露醇);(4)高糖+法舒地尔处理组(HG+F组,法舒地尔浓度分别为25、50、100 μmol/L).培养12、24、36、48、72 h收集上清及细胞,用实时PCR检测细胞中小G蛋白(RhoA)、小G蛋白激酶-Ⅰ(ROCK-Ⅰ)、纤维结缔组织生长因子(CTGF)mRNA浓度的变化,用ELISA方法检测上清中纤维连接蛋白(FN)、CTGF、TNFα的蛋白含量.结果 (1)高糖培养下的HMCs中RhoA、ROCK-Ⅰ、CTGF mRNA的表达较NG组明显升高(P＜0.05),并有一定的时间依赖性,Man组与NG组相比差异无统计学意义(P＞0.05).(2)正常培养的HMCs经不同浓度法舒地尔预处理后,高糖继续培养24 h或48 h,HG+F组与HG组对比,RhoA、ROCK-Ⅰ、CTGF mRNA的表达明显下降(P＜0.05),并有一定的浓度依赖性.(3)高糖呈时间依赖方式增加HMCs对FN、CTGF、TNFα蛋白的分泌(P＜0.05),而NG组和Man组无此作用(P＞0.05).(4)经不同浓度法舒地尔预处理后,高糖继续培养12、24、36、48、72 h后FN、CTGF、TNFα蛋白的分泌较HG组明显降低(P＜0.05).结论法舒地尔通过抑制高糖激活的HMCs的Rho/ROCK信号转导通路,从而降低下游的炎性因子和细胞因子的分泌,减少HMCs的炎症反应及纤维化,为糖尿病肾病的治疗提供新的依据.
목적 체외실험연구법서지이(fasudil)통과억제Rho/ROCK신호통로대고당배양하적인신소구계막세포(HMCs)염증반응급기섬유화적영향.방법 전대배양적HMCs동보화후분조:(1)정상당농도대조조(NG,함포도당5.5 mmol/L);(2)고당조(HG,함포도당30.0 mmol/L);(3)감로순삼투압대조조(Man,함5.5 mmol/L포도당+24.5 mmol/L감로순);(4)고당+법서지이처리조(HG+F조,법서지이농도분별위25、50、100 μmol/L).배양12、24、36、48、72 h수집상청급세포,용실시PCR검측세포중소G단백(RhoA)、소G단백격매-Ⅰ(ROCK-Ⅰ)、섬유결체조직생장인자(CTGF)mRNA농도적변화,용ELISA방법검측상청중섬유련접단백(FN)、CTGF、TNFα적단백함량.결과 (1)고당배양하적HMCs중RhoA、ROCK-Ⅰ、CTGF mRNA적표체교NG조명현승고(P＜0.05),병유일정적시간의뢰성,Man조여NG조상비차이무통계학의의(P＞0.05).(2)정상배양적HMCs경불동농도법서지이예처리후,고당계속배양24 h혹48 h,HG+F조여HG조대비,RhoA、ROCK-Ⅰ、CTGF mRNA적표체명현하강(P＜0.05),병유일정적농도의뢰성.(3)고당정시간의뢰방식증가HMCs대FN、CTGF、TNFα단백적분비(P＜0.05),이NG조화Man조무차작용(P＞0.05).(4)경불동농도법서지이예처리후,고당계속배양12、24、36、48、72 h후FN、CTGF、TNFα단백적분비교HG조명현강저(P＜0.05).결론 법서지이통과억제고당격활적HMCs적Rho/ROCK신호전도통로,종이강저하유적염성인자화세포인자적분비,감소HMCs적염증반응급섬유화,위당뇨병신병적치료제공신적의거.
Objective To study the effect of fasudil on inhibiting the Rho/ROCK signaling pathway under high glucose in human mesangial cells (HMCs) inflammation and fibrosis. Methods Synchronized HMCs were divided into following groups: (1) Normal glucose control group ( NG, 5. 5 mmol/L glucose) ;(2) High glucose group (HG, 30 mmol/L glucose) ; (3) Mannitol group (Man, 5.5 mmol/L glucose + 24. 5 mmol/L mannitol) ; (4) High glucose + fasudil group ( HG + F, the concentrations of fasudil were 25 ,50 and 100 μmol/L, respectively). Collect the supernatant and cells at 0, 12, 24, 36, 48 and 72 h respectively, and determine the concentration changes of the RhoA, ROCK- Ⅰ, connective tissue growth factor (CTGF)mRNA with real-time PCR method in the cells, then used the ELISA method to check the protein content of the fibronectin ( FN) , CTGF, TNFα in the supernatant. Results ( 1) RhoA, ROCK- Ⅰand CTGF mRNA of the HMCs cultured under the high glucose expressed significantly higher than those in the normal group, and there was certain time-dependence. Besides, there was no statistic significance by comparing Man and NG. (2) Under the high glucose situation, after the fasudil pretreatment with different concentrations and 24 h or 48 h culture with high glucose, RhoA, ROCK- Ⅰ , CTGF mRNA expression was significantly decreased in HG + F, compared with HG, and there was certain concentration-dependence. (3) High glucose increased the FN, CTGF, TNFα protein secretion of HMCs in a time-dependent manner, but normal glucose and mannitol had no such effect. (4) After the fasudil pretreatment with different concentrations and culture with high glucose for 12, 24, 36, 48, 72 h, the FN, CTGF, TNFα protein secretion was significantly reduced compared with HG. Conclusion Fasudil can reduce the secretion of downstream inflammatory factors and cytokines by inhibiting high glucose-activated HMCs Rho/ROCK signaling pathway, and reduce the inflammation and fibrosis of HMCs. This provides a new basis for the therapeutic target in the treatment of diabetic nephropathy.