【作者】 王曼；

【导师】 许立；

【作者基本信息】 南京中医药大学 ， 中药学， 2017， 硕士

【摘要】 目的:观察丹酚酸 B(salvianolic acid B,SalB)对单侧输尿管梗阻(unilateral ureteral obstruction,UUO)小鼠肾间质纤维化以及对血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导人肾小管上皮细胞(HK-2)上皮间质转分化(EPithelial-mesenchymal transition,EMT)中HPSE、SCD1及HPSE/SDC1轴的影响,在整体水平和细胞水平上探讨SalB对肾间质纤维化的作用及其可能的机制。方法:1.C57小鼠随机分为7d和14d组,每组又分为:假手术组(Sham组)、单侧输尿管结扎组(UUO 组)、SalB低 6 25mg·kg-1·d-1)、中(12.50mg·kg-i·d-1)、高(25.00mg·kg-1·d-1)剂量组。UUO组与SalB给药组小鼠在无菌条件下进行左侧输尿管结扎。Sham组除不结扎外其余操作均与UUO组相同。术后第1d起给药组分别腹腔注射对应剂量SalB,Sham组和UUO组小鼠腹腔注射等体积的生理盐水,并观察给药期间各小鼠的体重变化。各组小鼠分别在给药第7d和14d时,取血和肾脏。检测血清中肌酐(serum creatinine,Cr)、尿素氮(blood urea nitrogen,BUN)、转化生长因子_β1(transforming growth factor-β1,TGF_β1)、纤维母细胞生长因子-2(fibroblast growth factor-2,FGF-2)、多配体蛋白聚糖-1(Syndecan-1,SDC1)的含量以及乙酰肝素酶(Heparanase,HPSE)的活性。苏木精-伊红染色(HE染色)和胶原染色(Masson染色)检测肾组织病理学变化;免疫组化(Immunohistochemistry,IHC)检测平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、HPSE、SDC1 的表达水平。蛋白免疫印迹法(Western Blotting,WB)法检测肾组织中α-SMA、E-钙粘蛋白(E-cadherin)、TGF-β1、FGF-2、HPSE、SDC1 蛋白表达水平。2.MTT 法检测浓度为(0、10-4、10-3、10-2、10-1、1、101、102μmol·L-1)的AngⅡ作用于人肾小管上皮细胞(HK-2)48h,对细胞的增殖情况的影响,以确定最佳的 AngⅡ浓度;浓度为(0、10-3、10-2、10-1、1、101、102、103、104μmol·L-1)SalB作用于AngⅡ(1μmol·L-1)诱导的HK-2细胞24h时,对细胞增殖情况的影响,以确定合适的SalB浓度。显微镜观察不同浓度SalB对HK-2细胞形态的影响;ELISA试剂盒检测细胞上清液中TGF-β1、FGF-2、SDC1的含量以及HPSE的活性。Western Blotting 法和免疫荧光法(Immunofluorescence,IF)检测 HK-2 细胞中α-SMA、E-cadherin、TGF-βl、FGF-2、HPSE、SDC1 蛋白表达水平。3.合成人HPSE(Homo HPSE)基因重组质粒、对照质粒空载体。通过荧光显微镜观察和 Western Blotting 法检测(0、2、3、4、5pg·mL-1)Homo HPSE 转染 HK-2细胞24h时,细胞中HPSE蛋白的表达水平,以确定合适的Homo HPSE的转染浓度。将体外培养的HK-2细胞随机分为空白对照组(Blank组)、阴性对照组(NC组)、Homo HPSE转染组,分别采用Real-time PCR法和Western Blotting法检测比较Homo HPSE转染的HK-2细胞和正常的HK-2细胞中HPSE基因和蛋白的表达水平,以验证是否成功构建HPSE基因过表达的HK-2细胞。体外培养的HK-2细胞随机分为 NC 组、NC+AngⅡ组、HomoHPSE+AngⅡ组、HomoHPSE+AngⅡ+SalB组,采用Real-time PCR法和WB法检测各组HK-2细胞中HPSE、SCD1基因和蛋白的表达水平,ELISA试剂盒检测细胞上清液中TGF-β1、FGF-2、SDC1的含量以及HPSE的活性。结果:1.小鼠单侧输尿管结扎7d和14d后,相较于Sham组,UUO小鼠的体重先是降低(P<0.05,P<0.001),然后缓慢增长,肾重和肾指数显著增加(P<0.001);血清中Cr、BUN、TGF-β1、FGF-2的含量以及HPSE活性均显著增加(P<0.01,P<0.001),SDC1含量显著下降(P<0.01)。病理结果显示:UUO小鼠左侧肾脏体积明显比右侧变大,里面充满积液,肾实质变薄,肾盂肾盏扩张,肾小管扩张变性坏死,炎性细胞大量浸润,肾脏胶原质蓝色染色明显增多,肾间质纤维化的区域增宽,随着梗阻时间的延长,病理改变更为显著。肾脏中α-SMA、TGF-Jβ1、FGF-2、HPSE蛋白的表达水平显著上升(P<0.01,P<0.001),E-cadherin和SCD1蛋白的表达水平显著下调(P<0.001),其中14d组较7d组的蛋白表达水平的改变更为显著。UUO小鼠腹腔注射不同剂量的SalB后,相较于UUO组,各组小鼠的体重均有所增加,但不显著(P>0.05),肾重和肾指数显著下降(P<0.05,P<0.01,P<0.001);血清中Cr、BUN、TGF-β1、FGF-2 的含量以及 HPSE 活性均显著降低(P<0.05,P<0.01,P<0.001),SCD1的含量显著升高(P<0.05,P<0.001),且与SalB的给药时间和给药剂量呈正相关。病理结果显示:腹腔注射不同剂量的SalB7d和14d对UUO小鼠的上述的肾脏损伤均有明显的改善作用,且呈剂量和时间依赖性。SalB给药组小鼠肾脏中α-SMA、TGF-β1、FGF-2、HPSE 蛋白的表达水平下降(P<0.05,P<0.01,P<0.001),E-cadherin和 SCD1 蛋白的表达水平上升(P<0.05,P<0.01,P<0.001),随着 SalB 给药时间的延长以及剂量的增加,对UUO小鼠肾脏中上述蛋白的表达水平改善作用更为显著。2.MTT结果显示,相较于DMEM组,1μmol·L-1AngⅡ处理HK-2细胞48h,能够明显抑制HK-2细胞的增殖(P<0.05),再给予不同浓度的SalB作用24h,低浓度(0.1μmol·L-1)对其增殖具有促进作用(P<0.05),高浓度(102μmol·L-1)则显示抑制作用。各组HK-2细胞的形态未发现明显变化。相较于DMEM组,AngⅡ组,细胞上清液中TGF-β1、FGF-2含量和HPSE活性均显著增加(P<0.01,P<0.001),SCD1的含量显著下降(P<0.01);细胞中α-SMA、TGF-β1、FGF-2、HPSE蛋白的表达水平显著上升(P<0.001),E-cadherin和SCD1蛋白的表达水平显著下降(P<0.001)。给予不同剂量的SalB处理细胞24h,上清液中TGF-β1、FGF-2含量和HPSE活性明显降低(P<0.05,P<0.01,P<0.001),SCD1含量升高(P<0.05,P<0.01);细胞中α-SMA、TGF-β1、FGF-2、HPSE蛋白的表达水平下降以及E-cadherin和SCD1蛋白的表达水平上升(P<0.05,P<0.01),且呈剂量依赖性。3.荧光显微镜和Western blotting检测结果显示,Homo HPSE质粒转染HK-2细胞的适宜浓度为3μg·mL-1。相较于Blank组,转染Homo HPSE后,细胞中HPSE mRNA和蛋白表达水平均显著增加(P<0.001)。AngⅡ诱导HPSE基因过表达的HK-2细胞48h,再用SalB处理24h发现,相较于NC+AngⅡ组,NC组的HK-2细胞HPSEmRNA以及α-SMA、TGF-β1、FGF-2、HPSE蛋白表达水平均较低(P<0.05,P<0.01,P<0.001),E-cadherin和 SCD1 蛋白表达水平则较高(P<0.001);上清液中TGF-β1、FGF-2含量和HPSE活性均较低(P<0.05,P<0.01),SCD1的含量则较高(P<0.05)。HomoHPSE+AngⅡ组中 HPSEmRNA 以及α-SMA、TGF-β1、FGF-2、HPSE 蛋白表达水平均显著上升(P<0.05,P<0.01,P<0.001),E-cadherin 和SCD1蛋白表达水平显著下降(P<0.01);上清液中TGF-β1、FGF-2含量和HPSE活性明显增加(P<0.05),SCD1含量显著降低(P<0.05)。给予1μmol·L-1丹酚酸B处理24h后,其HPSE mRNA以及α-SMR、TGF-β1、FGF-2、HPSE蛋白表达水平均显著下降(P<0.05,P<0.01),E-cadherin和SCD1蛋白表达水平显著上升(P<0.01,P<0.001),但SCD1mRNA表达水平在各组中并无明显变化;上清液中TGF-β1、FGF-2含量和HPSE活性均降低(P<0.05,P<0.01),SCD1的含量增加(P<0.01)。结论:1.SalB有可能通过下调HPSE酶活性和蛋白表达水平,抑制SCD1的HS侧链的断裂,减少FGF-2和TGF-β1促纤维化因子的释放,来延缓单侧输尿管梗阻所致的肾间质纤维化的进程,且呈时间和剂量依赖性。2.SalB有可能通过下调HPSE酶活性和蛋白表达水平,抑制SCD1的HS侧链的断裂,减少FGF-2和TGF-β1促纤维化因子的释放,来抑制AngⅡ诱导的HK-2细胞EMT的发生以及ECM的沉积,且呈剂量依赖性。3.SalB可能通过直接下调HPSE表达水平以调控HPSE/SCD1轴,减少FGF-2和TGF-β1等细胞因子释放,改善AngⅡ介导的肾小管上皮细胞EMT以及ECM的沉积。更多还原

【Abstract】 Objective:To investigate the effect of salvianolic acid B(SalB)on the HPSE,SCD1 and HPSE/SDC1 axis in renal interstitial fibrosis in mice induced by unilateral ureteral obstruction(UUO)and in epithelial-mesenchymal transition(EMT)in human renal tubular epithelial cells(HK-2)induced by angiotensinⅡ(AngⅡ).To explore the effect of SalB on renal interstitial fibrosis and its possible mechanism in vivo and in vitro.Methods:1.C57 mice were randomly divided into 7 days and 14 days two groups,each group was divided into sham group,UUO group and three different doses of SalB groups(6.25mg·kg-1·d-1 of low,12.50mg·kg-1·d-1 of middle and 25.00mg kg-1·d-1 of high).Mice with left ureteral ligation under sterile conditions in UUO group and SalB groups.The sham group was the same operation method as the UUO group except the ligation.After first days of operation,the SalB groups was injected with the corresponding dose of SalB,the mice in sham group and UUO group were injected with the same volume of normal saline.The changes of body weight of mice were observed.Each group of mice were treated with 7d and 14d,respectively.The kidneys,blood and urine were taken on 7th day and 14th day after administration.Then,detect the levels of serum creatinine(Cr),blood urea nitrogen(BUN);transforming growth factor-β1(TGF-β1)fibroblast growth factor-2(FGF-2),syndecan-1(SCD1)and the activity of heparanase(HPSE).Hematoxylin eosin staining(HE staining)and collagen staining(Masson staining)to detect the changes of renal pathology.The expression of a-smooth muscle actin(a-SMA),HPSE and SDC1 were detected by immunohistochemistry(IHC).Westem Blotting(WB)is mainly detect a-SMA,E-cadherin,TGF-β1,FGF-2,HPSE and SDC1 expression of the protein in renal tissue.2.MTT assay different concentrations of AngⅡ(0,10-4,10-3,10-2,10-1,1,101,102μmol·L-1)on the HK-2 cells cultivate for 48h to determine the optimum concentration of Ang Ⅱ.MTT assay different concentrations of SalB(0,10-3,10-2,10-1,1,101,102,103,104μmol·L-1)on the HK-2 cells cultivate induced by AngⅡ(μmol·L-1)for 24h to determine the appropriate concentrations of SalB and observe the cell morphology of HK-2 cells by microscope.ELISA kits were used to detect the levels of TGF-(31,FGF-2,SCD1 and the activity of HPSE in the supernatant.WB and Immunofluorescence(IF)is mainly detect a-SMA,E-cadherin,TGF-β1,FGF-2,HPSE and SDC1 expression of the protein in HK-2 cells.3.To synthesize recombinant plasmid of human HPSE(Homo HPSE)gene andempty vector of control plasmid.After transfection of HK-2 cells with different concentrations of Homo HPSE(0,2,3,4,5μg·mL-1)for 24h,the expression level of HPSE protein in the cells was detected by fluorescence microscopy and WB to determine the appropriate transfection concentration of Homo HPSE.Cultured HK-2 cells were randomly divided into control group(Blank group),negative control group(group NC),Homo HPSE transfected group.Real-time PCR and WB were used to detect the expression of HPSE gene and protein in Homo transfected HK-2 cells and normal HK-2 cells in order to verify whether the HPSE gene was overexpressed in HK-2 cells.The HK-2 cells were randomly divided into NC group,NC+AngⅡ group,HomoHPSE+AngⅡgroup,HomoHPSE+AngⅡ+SalB group,using Real-time PCR and WB to detect the expression levels of HPSE,SCD1 gene and protein in HK-2 cells.ELISA kits were used to detect the levels of TGF-β1,FGF-2,SCD1 and the activity of HPSE in the supernatant.Results:1.After mice were treated with unilateral ureteral ligation for 7 and 14 days,compared with the sham group,the weight of UUO mice was decreased(P<0.05,P<0.001),and then slowly increased;the kidney weight and kidney index increased significantly(P<0.001);the levels of Cr,BUN,TGF-(31,FGF-2 and HPSE activity in serum were significantly increased(P<0.01,P<0.001),but the level of SDC1 was significantly decreased(P<0.01).The pathological results showed that UUO mice left kidney volume was significantly larger than the right,which is full of effusion,thinner renal parenchyma,pyelocaliectasis,dilation of renal tubule degeneration and necrosis,infiltration of inflammatory cells,renal collagen blue staining increased significantly,renal interstitial fibrosis area widened,with the obstruction time prolonged,the pathological changes were more significant.The expression levels of a-SMA,TGF-β1,FGF-2 and HPSE proteins increased significantly in the kidneys of mice(P<0.01,P<0.001),but the expression levels of E-cadherin and SCD1 protein significantly decreased(P<0.001),compared with 7d groups,the changes of proteins expression levels of 14d groups were more significant.After intraperitoneal injection of different doses of SalB in UUO mice,compared with UUO group,the mice weight has increased,but not significantly(P>0.05),kidney weight and kidney index decreased significantly(P<0.05,P<0.01,P<0.001);the serum Cr,BUN,TGF-beta 1,FGF-2 content and HPSE activity were significantly decreased(P<0.05,P<0.01,P<0.001),but the level of SDC1 was increased significantly(P<0.05,P<0.001),and these changes were positively correlated with the administration time and dose of SalB.The pathological results showed that SalB had a significant improvement in the renal injury of the UUO mice,which was dose and time dependent.After intraperitoneal injection of different doses of SalB in UUO mice,the expression levels of α-SMA,E-cadherin,TGF-β1 FGF-2 and HPSE in renal tissue were decreased(P<0.05,P<0.01,P<0.001),but the expression levels of E-cadherin and SCD1 were increased(P<0.05,P<0.01,P<0.001),with the prolongation of SalB administration time and the increase of dose,the effect of UUO on the expression of these proteins in kidney was significantly improved.2.The results of MTT showed that,compared with DMEM group,the treatment of HK-2 cells with 1μmol·L-1AngⅡ for 48h could significantly inhibit the proliferation of HK-2 cells(P<0.05);then HK-2 cells were treated with different concentrations of SalB for 24h,the low concentration of SalB(0.1μmol·L-1)could promote its proliferation(P<0.05),but the high concentration of SalB(102μmol·L-1)showed inhibitory effect.The morphology of HK-2 cells in each group did not change significantly.Compared with DMEM group,the levels of TGF-β1,FGF-2 and HPSE activity were significantly increased(P<0.01,P<0.001),but the level of SCD1 was significantly decreased in the supernatant of AngⅡ group(P<0.01).The expression levels of α-SMA,E-cadherin,TGF-β1,FGF-2 and HPSE protein in cells were significantly increased(P<0.001),but the expression levels of E-cadherin and SCD1 protein were significantly decreased(P<0.001).After cells were treated with different doses of SalB for 24h,the levels of TGF-β1,FGF-2 and HPSE activity were significantly decreased(P<0.05,P<0.01,P<0.001),but the level of SCD1 was significantly increased in the supernatant(P<0.05,P<0.01)in a dose-dependent manner of SalB;The expression levels of a-SMA,E-cadherin,TGF-β1,FGF-2 and HPSE protein in cells were decreased but the expression levels of E-cadherin and SCD1 protein were increased(P<0.05,P<0.01)in a dose-dependent manner of SalB.3.Fluorescence microscopy and WB results showed that the appropriate concentration of Homo HPSE plasmid transfected HK-2 cells was 3μg·ML-1L Compared with blank group,the expression of mRNA and protein of HPSE increased significantly after transfection with Homo HPSE in HK-2 cells(P<0.001).After the overexpression of HPSE gene in HK-2 cells were induced by AngⅡ for 48h,treated with SalB for 24h,compared with the NC+AngⅡ group,the expression levels of HPSE mRNA,α-SMA,E-cadherin,TGF-β1 FGF-2 and HPSE protein in HK-2 cells of NC group were lower(P<0.05,P<0.01,P<0.001),the expression levels of E-cadherin and SCD1 protein were higher(P<0.001);The levels of TGF-β1 FGF-2 and HPSE activity in supernatant were lower(P<0.05,P<0.01),the level of SCD1 was higher(P<0.05).The expression levels of HPSE mRNA,a-SMA,E-cadherin,TGF-β1 FGF-2 and HPSE protein in HK-2 cells of HomoHPSE+AngⅡ group were significantly increased(P<0.05,P<0.01,P<0.001),the expression levels of E-cadherin and SCD1 protein were significantly decreased(P<0.01);The levels of TGF-β1,FGF-2 and HPSE activity in supernatant were significantly increased(P<0.05),but the level of SCD1 was significantly decreased(P<0.05).After the treatment of HK-2 cells with concentration of 1μmol L-1 SalB for 24h,the expression levels of HPSE mRNA,a-SMA,E-cadherin,TGF-β1 FGF-2 and HPSE protein were significantly decreased(P<0.05,P<0.01),the expression levels of E-cadherin and SCD1 protein increased significantly(P<0.01,P<0.001),but the expression level of SCD1 mRNA in each group did not change significantly;The levels of TGF-β1,FGF-2 and HPSE activity in supernatant were decreased(P<0.05,P<0.01),but the level of SCD1 was increased(P<0.01).Conclusion:1.SalB may reduce the HS side chain cleavage of SCD1 and reduce the release of FGF-2 and TGF-β1 by decreasing the activity and the protein expression level of HPSE,in order to delay the progression of renal interstitial fibrosis caused by unilateral ureteral obstruction,in a time-and dose-dependent manner.2.SalB may reduce the HS side chain cleavage of SCD1 and reduce the release of FGF-2 and TGF-β1 by decreasing the activity and the protein expression level of HPSE,in order to inhibit the occurrence of EMT induced by AngⅡ and the deposition of ECM in HK-2 cells,in dose-dependent manner.3.SalB may regulate the HPSE/SCD1 axis by directly regulating the expression level of HPSE,decrease the release of cytokines such as FGF-2 and TGF-1,in order toimprove the EMT induced by AngⅡ and the deposition of ECM in HK-2 cells.更多还原