高脂喂养大鼠肾小管上皮细胞SREBP-1、TGF-β1、α-SMA的表达和细胞外基质沉积

目的:探讨高脂喂养对大鼠肾脏小管上皮细胞SREBP-1、TGF-β1、α-SMA表达和细胞外基质(ECM)的影响。方法:高脂饲料喂养大鼠12周后,油红O检测肾脏脂质沉积,Masson染色检测肾小管间质细胞外基质沉积,免疫组化、Western blot和原位杂交检测SREBP-1、TGF-β1、α-SMA和FN的表达。结果:高脂喂养后大鼠体重明显增加,血糖、甘油三酯和胰岛素均升高,油红O检测显示大鼠肾小管上皮细胞内出现明显脂滴。SREBP-1蛋白和mRNA在肾小管上皮细胞内表达,高脂组高于正常对照组,分别是正常组的1.88倍和1.85倍;TGF-β1和α-SMA也定位于肾小管上皮细胞胞浆并出现上调。Masson染色显示高脂喂养大鼠肾间质ECM沉积增多,纤维粘连蛋白FN检测也显示模型组表达强于对照组。结论:高脂饮食喂养可能通过上调肾脏小管上皮细胞SREBP-1表达使细胞内脂滴沉积,并进一步诱导TGF-β1、α-SMA合成而导致细胞外基质堆积。     

依帕司他对单侧输尿管梗阻大鼠肾间质纤维化的干预作用及其机制

目的:观察依帕司他(EPS)对单侧输尿管梗阻(UUO)大鼠间质纤维化的保护作用及其机制。方法:实验设假手术组(Sham)组、UUO、UUO+EPS(50 mg/kg)及UUO+EPS(100 mg/kg)剂量组,每组n=8。左侧输尿管结扎制备UUO大鼠模型。造模后连续灌胃给药3周,sham和UUO组给予等体积的羟甲基纤维素钠。HE和Masson染色观察肾组织病理变化及胶原沉积情况。免疫组化法观察肾组织醛糖还原酶(AR)表达情况,分别采用real-time PCR和(或) Western blot检测肾脏I型胶原(collagen I)、III型胶原(collagen III)、α-平滑肌肌动蛋白(α-SMA)、成纤维细胞特异蛋白-1(FSP-1)、纤连蛋白(FN)、E-钙粘蛋白(E-cadherin)、转化生成因子-β1(TGF-β1)和AR mRNA及蛋白表达。结果:与Sham组相比,UUO组大鼠小管上皮细胞萎缩、空泡样变性,肾间质成纤维细胞及肌成纤维细胞大量增殖并伴大量炎症细胞浸润,胶原沉积明显增加,collagen I、collagen III、TGF-β1和AR mRNA及蛋白表达水平明显升高(P<0.01),同时EMT标志性蛋白α-SMA、FSP-1、FN mRNA及蛋白表达水平明显升高(P<0.01),而E-cadherin mRNA及蛋白表达水平明显降低。与UUO组相比,经EPS治疗3周后,肾间质纤维化程度明显减轻,胶原沉积明显减少,collagen I、collagen III、TGF-β1和AR mRNA及蛋白表达水平明显降低(P<0.01或P<0.05),另外α-SMA、FSP-1、FN mRNA及蛋白表达水平明显降低(P<0.01或P<0.05),而E-cadherin mRNA及蛋白表达水平明显升高(P<0.01或P<0.05),而且100 mg/kg剂量组上述指标的改变均好于低剂量组(P<0.05,P<0.01)。结论:依帕司他对肾间质纤维化具有一定的改善作用,其机制可能与其抑制TGF-β1介导的AR表达、进而抑制大鼠肾小管上皮细胞EMT有关。     

树突状细胞在肾小管间质纤维化中作用及缬沙坦的干预调节

探讨树突状细胞(DC)在肾纤维化大鼠肾小管间质中分布,以及缬沙坦对DC浸润聚集的干预作用。建立肾大部切除大鼠模型,随机分为正常组(n=18),假手术组(n=18),模型组(n=18),缬沙坦治疗组(n=18)。分别于建模1、4、12周取肾组织,采用HE和Masson染色评定各组肾小管间质纤维化(TIF)程度;采用免疫双染及荧光图像分析法,观察DC-SIGN DC在各组大鼠肾组织中分布变化;采用免疫组化方法,观察P-选择素以及TGF-β1、α-平滑肌肌动蛋白(α-SMA)、III型胶元(ColIII)、纤维连接蛋白(FN)在上述肾组织中表达;以及RT-PCR检测P-选择素、TGF-β1、α-SMA、ColIII、FN的mRNA水平。结果显示,(1)模型组DC-SIGN DC主要分布于肾小管、肾间质和肾血管,以肾间质最为明显;其分布数量于12周较1和4周呈明显增多,且与慢性肾功能减退呈正相关。(2)12周时手术组大鼠肾小管间质区P-选择素、TGF-β1、α-SMA、ColIII、FN mRNA转录水平和蛋白质表达均明显增加,并与TIF程度以及DC-SIGN DC分布数量呈正相关。(3)经缬沙坦治疗后,DC-SIGN DC分布减少,以及P-选择素、TGF-β1、α-SMA、ColIII、FN mRNA转录水平和蛋白质表达下降,TIF程度减轻及肾功能改善。研究结果表明,DC启动参与了肾小管间质纤维化形成,并与肾功能损害程度密切相关。缬沙坦对此具有明显的抑制和肾脏保护作用。     

肌肽对高糖环境下心肌成纤维细胞胶原表达的影响

目的:探讨肌肽对高糖环境下心肌成纤维细胞中胶原生成的影响及作用机制。方法:原代培养心肌成纤维细胞,将细胞分为正常糖组(NG,5.5mmol/L glucose)、高糖组(HG,25mmol/L glucose)、高糖+10mmol/L肌肽组、高糖+20mmol/L肌肽组、高糖+40mmol/L肌肽组、高糖+SB组(HG+10μmol/L SB203580)、高糖+PD组(HG+10μmol/L PD98059)。ELISA检测胶原Ⅰ、Ⅲ的含量,Western blot检测TGF-β1、p-p38 MAPK和p-ERK(1/2)等蛋白的表达。结果:与正常糖组相比,高糖组中胶原Ⅰ和胶原Ⅲ含量增加(P<0.01);TGF-β1、p-p38和p-ERK等表达增加(P<0.01);与高糖组相比,高糖+肌肽组中胶原Ⅰ、Ⅲ、TGF-β1、p-p38、和p-ERK等均降低(P<0.05);高糖+SB组和高糖+PD组中胶原Ⅰ、Ⅲ表达减少(P<0.05)。结论:肌肽对心肌成纤维细胞中胶原生成具有抑制作用,且通过MAPK通路实现。     

高脂高糖高胆固醇饮食对小型猪肾脏细胞外基质表达的影响

为研究高脂高糖高胆固醇(HFSCD)诱导的2型糖尿病小型猪肾脏细胞外基质(ECM)的表达情况,将10头广西巴马小型猪分别喂正常和HFSCD饲料,收集血、尿液,测定其生化指标,进行口服葡萄糖耐量试验。5个月后取部分肾组织,行H.E和Masson染色,免疫组织化学方法检测ECM相关蛋白的表达。结果表明,喂养HFSCD后,小型猪血糖、血脂和胰岛素明显升高,出现胰岛素抵抗和β细胞功能紊乱;尿糖和微量白蛋白升高;肾脏纤维连接蛋白(FN)和Ⅳ型胶原的表达明显增加,而Ⅰ型胶原无表达。这说明HFSCD喂养广西巴马小型猪5个月能成功建立早期糖尿病肾病模型,促进FN和Ⅳ型胶原的表达。     

Sirt1活化剂对肾间质纤维化的保护作用及机制研究

研究Sirt1活化剂对单侧输尿管结扎导致的小鼠肾间质纤维化及TGF-β1/CTGF信号通路的影响。CD-1小鼠随机分为假手术组(Sham)、单侧输尿管结扎组(unilateral ureteral obstruction,UUO)和UUO+SRT1720(Sirt1活化剂)组(SRT1720)。Masson染色后光镜下观察肾小管间质病变面积;分别用Western blot和Real-time PCR检测Sirt1及纤维化相关因子Col1、α-SMA、TIMP-1、PAI-1蛋白质和m RNA表达水平;TUNEL检测细胞凋亡程度,同时检测凋亡相关因子Bcl-2/Bax的变化;检测SOD、MDA、GPx、GSH水平以示氧化应激变化。与Sham组相比,UUO组显示肾间质纤维化病变面积显著升高(P0.01);Sirt1水平及活性显著下降;TGF-β1/CTGF水平、纤维化相关因子表达及凋亡水平显著上升;氧化应激水平显著升高(P0.01)。SRT1720干预能够显著降低UUO引起的肾间质纤维化病变面积(P0.05),同时改善TGF-β1/CTGF水平、纤维化相关因子及凋亡水平(P0.01),显著降低肾脏氧化应激水平(P0.01)。以上说明,Sirt1活化剂SRT1720能显著改善肾间质纤维化水平,该作用可能是通过TGF-β1/CTGF信号通路的抑制和氧化应激实现的。     

高糖背景下白蛋白抑制肾小管上皮细胞的自噬表达

目的:探讨高糖背景下白蛋白造成肾小管间质损伤的作用及其机制。方法:体外培养大鼠近端肾小管上皮细胞系NRK-52E细胞,观察高糖培养环境下细胞自噬表达的改变;同时观察低浓度牛血清白蛋白(BSA)单独刺激,对肾小管上皮细胞自噬蛋白表达的影响以及细胞凋亡蛋白的表达改变;接着在高糖培养环境下加入低浓度的白蛋白刺激,观察肾小管上皮细胞的损伤效应及自噬表达情况。结果:高糖培养条件下肾小管上皮细胞自噬蛋白Beclin-1表达增加(P0.05),低浓度白蛋白也诱导肾小管上皮细胞自噬蛋白Beclin-1、Atg12表达增加(P0.05),以及细胞凋亡蛋白cleaved caspase3的轻度增加,乳酸脱氢酶活性增加(P0.05);但高糖培养下,少量白蛋白却抑制了肾小管上皮细胞自噬蛋白Beclin-1、Atg12的表达,并且显著增加了肾小管上皮细胞的凋亡蛋白cleaved caspase3的表达(P0.05)。结论:自噬是细胞自身的一种保护机制。在高糖背景下,白蛋白通过影响自噬的自身调节机制,促进了肾小管间质的损害作用。     

苦参素对单侧输尿管梗阻大鼠肾间质纤维化的保护作用

目的探讨苦参素对单侧输尿管梗阻(UUO)大鼠肾间质纤维化的影响及可能机制。方法45只雄性SD大鼠随机分为3组:A假手术组,B单侧输尿管结扎(UUO)组,C治疗组。治疗组在UUO的基础上每天以苦参素100mg/Kg/d腹腔注射。B组和C组于术后第7,14,21,28分别处死5只大鼠,A组于第28天处死大鼠。用PAS及Masson染色法观察肾脏病理改变。用免疫组化法检测转化生长因子β1(transforming growth factor-beta1 TGF-β1),α-平滑肌肌动蛋白(alpha-smooth muscle actin-αSMA),Ⅰ型胶原(collagenⅠColⅠ)的表达。结果与UUO组相比,治疗组梗阻侧肾脏TGF-β1,-αSMA,ColⅠ的表达明显减少,肾小管损害和肾间质纤维化的程度也明显减轻。结论苦参素可通过下调TGF-β1,减少肾小管上皮细胞转分化而减轻肾间质纤维化。     

人脐带间充质干细胞缓解马兜铃酸诱导小鼠肾纤维化的作用及机制

该实验探究了人脐带间充质干细胞(human umbilical cord mesenchymal stem cell, hucMSC)缓解马兜铃酸(aristolochic acid, AA)诱导小鼠肾纤维化的作用及可能机制。我们将huc-MSC经尾静脉注射干预AA诱导的肾纤维化小鼠模型。HE、PAS和Masson染色观察肾脏形态变化, Western blot和免疫组化检测上皮间质转化相关标志物E-cadherin、N-cadherin和TGF-β/Smad信号通路蛋白TGF-β1和p-Smad2/3表达水平。组织形态学染色结果显示, AA可诱导小鼠出现肾小管扩张、结构破坏,肾间质区胶原纤维沉积,呈纤维化改变; Western blot和免疫组化结果显示,其E-cadherin表达降低,N-cadherin、TGF-β1及p-Smad2/3表达增高。huc-MSC干预后,肾脏形态明显改善,胶原纤维沉积减少,E-cadherin表达增高, N-cadherin、TGF-β1及p-Smad2/3表达受到降低。研究结果表明, huc-MSC能够通过抑制TGF-β/Smad信号通路减轻肾脏上皮间质转化,从而缓解马兜铃酸诱导的小鼠肾纤维化。     

Smad2/3上调促进大鼠梗阻性肾病的纤维化和凋亡

目的观察TGF-β1/Smads信号传导途径对大鼠肾间质纤维化表达的影响.方法 40只Wistar大鼠分为假手术组及模型组.单侧输尿管结扎术式(UUO)建立肾梗阻模型.分别于术后3、6、14和28d处死动物.应用免疫组织化学和流式细胞学技术来检测TGF-β1、Smad2/3、p-Smad2/3和观察细胞外基质以及细胞凋亡的改变.MASSON染色观察肾间质病变程度.结果与假手术组相比,模型组于术后3d梗阻肾TGF-β1、Smad2/3、p-Smad2/3开始升高,细胞凋亡百分率同时升高,I型胶原及α-平滑肌肌动蛋白(α-SMA)水平亦升高.第6d各项指标水平显著升高.第14、28d升高更加明显(P＜0.01).B3、B6、B14和B28组之间也随天数增加而显著升高(P＜0.01).肾间质病变面积增加显著(P＜0.01).结论在肾间质纤维化中TGF-β1、Smad2/3、p-Smad2/3及细胞凋亡明显上升,同时I型胶原及α-SMA表达升高及肾间质纤维化程度增加.TGF-β1/Smads信号传导途径在肾间质纤维化形成中可能起着重要促细胞外基质沉积和细胞凋亡的作用.     

A Diet High in Saturated Fat and Sucrose Alters Glucoregulation and Induces Aortic Fatty Streaks in New Zealand White Rabbits

A new and convenient animal model for studying peripheral vascular and coronary artery disease in diabetes was established in this study. Male New Zealand White rabbits weighing approximately 2 kg were divided into 2 groups: a normal control group fed standard laboratory chow and a diabetogenic diet–fed group received a high-fat/high-sucrose diet. The high-fat/high-sucrose diet (contained 10% lard and 37% sucrose) feeding was maintained for 6 months. Plasma total cholesterol, high-density lipoprotein (HDL) cholesterol, triglyceride, superoxide dismutase, nitric oxide, nitric oxide synthase, insulin, and glucose were quantitated at monthly or bimonthly intervals. The aortic fatty streak lesions were quantified following lipid staining with Sudan IV. The aortic samples were observed by electron microscopy. High plasma triglyceride and glucose concentrations were induced. At the end of 6 months, the aortic fatty streak lesions were present in the animals'' vascular specimens. As far as we know, this is the first report that demonstrates that New Zealand White rabbits can develop obvious aortic fatty streaks by feeding a high-fat/high-sucrose diet. Our results suggest that NewZealand White rabbits fed a high-fat/high-sucrose diet would provide a convenient model for studying peripheral vascular and coronary artery disease in diabetes.     

The antifibrotic effects of TGF-β1 siRNA on hepatic fibrosis in rats

Background/aims: Hepatic fibrosis results from the excessive secretion of matrix proteins by hepatic stellate cells (HSCs), which proliferate during fibrotic liver injury. Transforming growth factor (TGF)-β1 is the dominant stimulus for extracellular matrix (ECM) production by stellate cells. Our study was designed to investigate the antifibrotic effects of using short interference RNA (siRNA) to target TGF-β1 in hepatic fibrosis and its mechanism in rats exposed to a high-fat diet and carbon tetrachloride (CCL4). Methods: A total of 40 healthy, male SD (Sprague–Dawley) rats were randomly divided into five even groups containing of eight rats each: normal group, model group, TGF-β1 siRNA 0.125 mg/kg treatment group, TGF-β1 siRNA 0.25 mg/kg treatment group and TGF-β1 siRNA negative control group (0.25 mg/kg). CCL4 and a high-fat diet were used for 8 weeks to induce hepatic fibrosis. All the rats were then sacrificed to collect liver tissue samples. A portion of the liver samples were soaked in formalin for Hematoxylin–Eosin staining, classifying the degree of liver fibrosis, and detecting the expression of type I and III collagen and TGF-β1; the remaining liver samples were stored in liquid nitrogen to be used for detecting TGF-β1 by Western blotting and for measuring the mRNA expression of type I and III collagen and TGF-β1 by quantitative real-time polymerase chain reaction. Results: Comparing the TGF-β1 siRNA 0.25 mg/kg treatment group to the model group, the TGF-β1 siRNA negative control group and the TGF-β1 siRNA 0.125 mg/kg treatment group showed significantly reduced levels of pathological changes, protein expression and the mRNA expression of TGF-β1, type I collagen and type III collagen (P < 0.01). Conclusions: Using siRNA to target TGF-β1 can inhibit the expression of TGF-β1 and attenuate rat hepatic fibrosis induced by a high-fat diet and CCL4. A possible mechanism is through the down-regulation of TGF-β1 expression, which could inhibit HSC activation, as well as the proliferation and collagen production of collagen reducing, so that collagen deposition in the liver is reduced.     

A Lipoprotein Lipase–Promoting Agent,NO-1886, Improves Glucose and Lipid Metabolism in High Fat,High Sucrose–Fed New Zealand White Rabbits

The synthetic compound NO-1886 is a lipoprotein lipase activator that lowers plasma triglycerides and elevates high-density lipoprotein cholesterol (HDL-C). Recently, the authors found that NO-1886 also had an action of reducing plasma glucose in high-fat/high-sucrose diet–induced diabetic rabbits. In the current study, we investigated the effects of NO-1886 on insulin resistance and β-cell function in rabbits. Our results showed that high-fat/high-sucrose feeding increased plasma triglyceride, free fatty acid (FFA), and glucose levels and decreased HDL-C level. This diet also induced insulin resistance and impairment of acute insulin response to glucose loading. Supplementing 1% NO-1886 into the high-fat/high-sucrose diet resulted in decreased plasma triglyceride, FFA, and glucose levels and increased HDL-C level. The authors also found a clear increased glucose clearance and a protected acute insulin response to intravenous glucose loading by NO-1886 supplementation. These data suggest that NO-1886 suppresses the elevation of blood glucose in rabbits induced by feeding a high-fat/high-sucrose diet, probably through controlling lipid metabolism and improving insulin resistance.     

长春新碱对糖尿病兔肾小管间质损伤的影响

目的观察长春新碱对糖尿病兔肾小管-间质损伤的影响。方法 24只日本大白兔随机分成正常对照组（control组）、糖尿病组（DM组）、糖尿病长春新碱治疗组（DV组）;耳缘静脉注射四氧嘧啶造成糖尿病兔模型,DV组每周进行一次长春新碱耳缘静脉注射;胰岛素控制DM组、DV组血糖16.0～25.0mmol/L。8周时测各组兔血糖、肾重、体重、尿β2-MG、RBP、NAG,肾组织病理观察肾小管-间质变化。结果给药后7hDV组、DM组血糖明显升高形成稳定糖尿病模型;8周时,DV组体重明显高于DM组（P〈0.05）、肾重/体重值明显低于DM组（P〈0.05）;DM组、DV组尿β2-MG阳性反应率较低;DM组尿RBP、NAG明显高于control组（P〈0.05）,长春新碱可抑制糖尿病兔尿RBP、NAG增加（P〈0.05）,并减轻糖尿病兔肾小管间质损伤指数（P〈0.05）。结论长春新碱可减轻糖尿病兔肾小管间质损伤。     

Cation-independent mannose 6-phosphate receptor inhibitor (PXS25) inhibits fibrosis in human proximal tubular cells by inhibiting conversion of latent to active TGF-beta1

Hyperglycemia and hypoxia have independent and convergent roles in the development of renal disease. Transforming growth factor-β(1) (TGF-β(1)) is a key cytokine promoting the production of extracellular matrix proteins. The cationic-independent mannose 6-phosphate receptor (CI-M6PR) is a membrane protein that binds M6P-containing proteins. A key role is to activate latent TGF-β(1). PXS25, a novel CI-MPR inhibitor, has antifibrotic properties in skin fibroblasts, but its role in renal fibrosis is unclear. The aim was to study the role of PXS25 in matrix protein production under high glucose ± hypoxic conditions in human proximal tubule (HK-2) cells. HK-2 cells were exposed to high glucose (30 mM) ± 100 μM PXS25 in both normoxic (20% O(2)) and hypoxic (1% O(2)) conditions for 72 h. Cellular fibronectin, collagen IV, and matrix metalloproteinase-2 (MMP-2) and MMP-9 were assessed. Total and active TGF-β(1) were measured by ELISA. High glucose and hypoxia independently induced TGF-β(1) production. Active TGF-β(1), but not total TGF-β(1) was reduced with concurrent PXS25 in the presence of high glucose, but not in hyperglycemia+hypoxia conditions. Hyperglycemia induced fibronectin and collagen IV production (P < 0.05), as did hypoxia, but only hyperglycemia-induced increases in matrix proteins were suppressed by concurrent PXS25 exposure. High glucose induced MMP-2 and -9 in normoxic and hypoxic conditions, which was not modified in the presence of PXS25. High glucose and hypoxia can independently induce endogenous active TGF-β(1) production in human proximal tubular cells. PXS25 inhibits conversion of high glucose-induced release of active TGF-β(1), only in the absence of hypoxia.     

Dietary fiber enhances TGF-β signaling and growth inhibition in the gut

Dietary fiber intake links to decreased risk of colorectal cancers. The underlying mechanisms remain unclear. Recently, we found that butyrate, a short-chain fatty acid produced in gut by bacterial fermentation of dietary fiber, enhances TGF-β signaling in rat intestinal epithelial cells (RIE-1). Furthermore, TGF-β represses inhibitors of differentiation (Ids), leading to apoptosis. We hypothesized that dietary fiber enhances TGF-β's growth inhibitory effects on gut epithelium via inhibition of Id2. In this study, Balb/c and DBA/2N mice were fed with a regular rodent chow or supplemented with a dietary fiber (20% pectin) and Smad3 level in gut epithelium was measured. In vitro, RIE-1 cells were treated with butyrate and TGF-β(1), and cell functions were evaluated. Furthermore, the role of Ids in butyrate- and TGF-β-induced growth inhibition was investigated. We found that pectin feeding increased Smad3 protein levels in the jejunum (1.47 ± 0.26-fold, P = 0.045, in Balb/c mice; 1.49 ± 0.19-fold, P = 0.016, in DBA/2N mice), and phospho-Smad3 levels (1.92 ± 0.27-fold, P = 0.009, in Balb/c mice; 1.83 ± 0.28-fold, P = 0.022, in DBA/2N mice). Butyrate or TGF-β alone inhibited cell growth and induced cell cycle arrest. The combined treatment of butyrate and TGF-β synergistically induced cell cycle arrest and apoptosis in RIE-1 cells and repressed Id2 and Id3 levels. Furthermore, knockdown of Id2 gene expression by use of small interfering RNA caused cell cycle arrest and apoptosis. We conclude that dietary fiber pectin enhanced Smad3 expression and activation in the gut. Butyrate and TGF-β induced cell cycle arrest and apoptosis, which may be mediated by repression of Id2. Our results implicate a novel mechanism of dietary fiber in reducing the risk of colorectal cancer development.     

Protective effect of TRPV1 against renal fibrosis via inhibition of TGF-β/Smad signaling in DOCA-salt hypertension

To investigate the effects of the transient receptor potential vanilloid type 1 (TRPV1) channel on renal extracellular matrix (ECM) protein expression including collagen deposition and the transforming growth factor β (TGF-β)/Smad signaling pathway during salt-dependent hypertension, wild-type (WT) and TRPV1-null (TRPV1?/?) mutant mice were uninephrectomized and given deoxycorticosterone acetate (DOCA)-salt for 4 wks. TRPV1 gene ablation exaggerated DOCA-salt-induced impairment of renal function as evidenced by increased albumin excretion (μg/24 h) compared with WT mice (83.7 ± 7.1 versus 28.3 ± 4.8, P < 0.05), but had no apparent effect on mean arterial pressure (mmHg) as determined by radiotelemetry (141 ± 4 versus 138 ± 3, P > 0.05). Morphological analysis showed that DOCA-salt-induced glomerulosclerosis, tubular injury and macrophage infiltration (cells/mm2) were increased in TRPV1?/? compared with WT mice (0.74 ± 0.08 versus 0.34 ± 0.04; 3.14 ± 0.26 versus 2.00 ± 0.31; 68 ± 5 versus 40 ± 4, P < 0.05). Immunostaining studies showed that DOCA-salt treatment decreased nephrin but increased collagen type I and IV as well as phosphorylated Smad2/3 staining in kidneys of TRPV1?/? compared with WT mice. Hydroxyproline assay and Western blot showed that DOCA-salt treatment increased collagen content (μg/mg dry tissue) and fibronectin protein expression (%β-actin arbitrary units) in the kidney of TRPV1?/? compared with WT mice (26.7 ± 2.7 versus 17.4 ± 1.8; 0.93 ± 0.07 versus 0.65 ± 0.08, P < 0.05). Acceleration of renal ECM protein deposition in DOCA-salt-treated TRPV1?/? mice was accompanied by increased TGF-β1, as well as phosphorylation of Smad2/3 protein expression (%β-actin arbitrary units) compared with DOCA-salt-treated WT mice (0.61 ± 0.07 versus 0.32 ± 0.05; 0.57 ± 0.07 versus 0.25 ± 0.05; 0.71 ± 0.08 versus 0.40 ± 0.06, P < 0.05). These results show that exaggerated renal functional and structural injuries are accompanied by increased production of ECM protein and activation of the TGF-β/Smad2/3 signaling pathway. These data suggest that activation of TRPV1 attenuates the progression of renal fibrosis possibly via suppression of the TGF-β and its downstream regulatory signaling pathway.     

Epithelial�Cmesenchymal transition of rat peritoneal mesothelial cells via Rhoa/Rock pathway

The objective of this study was to investigate the role of the RhoA/Rock signaling pathway in the epithelial-mesenchymal transition (EMT) of rat peritoneal mesothelial cells (RPMCs). Primary SD rat peritoneal mesothelial cells were cultured in vitro. RPMCs were randomly assigned to four groups: group A (control), group B (TGF-β1, 10?μg/L), group C (10?μg/L TGF-β1?+?10?μmol/L Y-27632, an inhibitor of Rock that was pre-applied for 2?h before TGF-β1 stimulation), and group D (Y-27632 alone, 10?μmol/L). Our results were as follows: (1) TGF-β1 stimulation elicited a robust increase in RhoA activity in a time-dependent manner; the increase was 2.57?±?0.52 times larger than the activity observed for the control group (P?相似文献   

育阴软肝颗粒剂对肝纤维化大鼠TGF-β1表达的影响

目的：观测育阴软肝颗粒剂对大鼠肝纤维化模型的防治作用及对转化生长因子-β1（TGF-β1）表达的影响。方法：将Wistar大鼠分为6组（n=10）,注射四氯化碳、饲以高脂饲料并饮用20%乙醇6周复制肝纤维化大鼠模型,经6.2~24.8 g/kg育阴软肝颗粒剂干预（qd）6周后,测定肝纤维化大鼠血清丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）活性、透明质酸（HA）、Ⅲ型前胶原（PCⅢ）、Ⅳ型胶原（C-Ⅳ）及板层素（LN）含量,观测肝组织病理学及肝组织TGF-β1表达的变化,对育阴软肝颗粒剂防治肝纤维作用及机制进行研究。结果：实验第7周,模型组大鼠肝组织出现明显的纤维化病变（P<0.01）;与模型组比较,6.2~24.8g/kg的育阴软肝颗粒剂能明显降低肝指数以及血清ALT、AST活性与HA、PCⅢ、C-Ⅳ、LN含量,缓解肝组织纤维化病理变化,抑制纤维化肝组织TGF-β1的表达（P<0.05,0.01）。结论：育阴软肝颗粒剂对多因素复制肝纤维化大鼠造模具有明显的治疗作用,而抑制TGF-β1的表达可能是其作用机制之一。     

消痰化瘀利窍中药组方对改善慢性间歇性低氧大鼠心肌纤维化的作用及其机制*

目的: 探讨转化生长因子-β(TGF-β)信号通路在消痰化瘀利窍中药组方(XC)对改善慢性间歇性低氧(CIH)大鼠心肌纤维化中的作用。方法: 40只SD 大鼠,随机分为常氧组(Normoxia)、常氧+中药干预组(TCMC)、慢性间歇性低氧模型组(CIH)、CIH +中药干预组(TCMC+CIH),每组10只。通过向舱内充入氮气,使舱内氧体积分数在90 s内从21%下降到9%,随后90 s再充氧气使舱内氧体积分数逐渐上升到21%为一循环建立CIH模型。CIH 与 TCMC+CIH 组大鼠置于CIH装置, Normoxia 和TCMC组大鼠置于正常氧舱。此外TCMC+CIH 与 TCMC 组大鼠于每日XC生药(24 g/kg)煎制灌胃,而 CIH 组与 Normoxia 组大鼠给予等体积生理盐水。造模结束后,天狼星红染色观察大鼠心肌间质内胶原沉积情况;Western blot 法检测大鼠心肌间质中 CollagenⅠ、Collagen Ⅲ、Fibronectin、TGF-β、p-Smad2、p-Smad3的蛋白表达水平。采用Q-PCR法检测基质金属蛋白酶2(MMP-2)和基质金属蛋白酶抑制因子 2 (TIMP-2) 的 mRNA表达水平。结果: 与正常组比较,CIH大鼠心肌组织出现明显胶原的沉积,CollagenⅠ、Collagen Ⅲ和Fibronectin蛋白表达明显增多(P均＜0.01),TGF-β、p-Smad2、p-Smad3蛋白表达水平也明显增高(P均＜0.01);CIH大鼠心肌组织TIMP-2 mRNA上调导致MMP-2 mRNA明显减少(P均＜0.01)。给予XC干预后,CIH大鼠心肌组织胶原沉积明显减少,CollagenⅠ、Collagen Ⅲ和Fibronectin蛋白表达明显降低(P＜0.05,P＜ 0.01,P＜0.05);CIH大鼠心肌组织中TGF-β、p-Smad2、p-Smad3蛋白表达水平明显降低(P＜0.01,P＜0.05,P＜ 0.01)。心肌组织中TIMP-2明显基因减少致MMP-2增多(P均＜0.05)。 结论: 消痰化瘀利窍中药组方可抑制CIH大鼠心肌纤维化的形成,进而改善CIH大鼠心肌功能。其机制与该中药组方下调TGF-β/ Smad2/3信号通路及下调TIMP-2mRNA有关。