姜黄素对TGF-β1诱导人肾小管上皮细胞转分化及分泌细胞外基质成分的影响

目的：通过研究姜黄素（curcumin,Cur）对转化生长因子-β1（TGF-β1）诱导人肾小管上皮细胞（HK-2）转分化及分泌细胞外基质（ECM）成分的影响,探讨姜黄素在防治肾小管-间质纤维化方面可能的作用机制。方法：应用不同浓度Cur处理经TGF-β1诱导活化的HK-2细胞,通过显微镜观察细胞形态改变,免疫组化技术检测α-平滑肌肌动蛋白（α-SMA）和E-钙黏蛋白（E-cadherin）的表达;应用酶联免疫吸附法（ELISA）检测细胞培养上清Ⅰ型胶原（ColⅠ）、Ⅲ型胶原（ColⅢ）和纤连蛋白（FN）的分泌。结果：（1）正常HK-2细胞表达E-cadherin,不表达α-SMA,经TGF-β1刺激后细胞表型发生转变,E-cadherin的表达明显减弱,α-SMA表达明显增强;（2）不同浓度Cur组与单纯TGF-β1刺激组相比α-SMA的表达有所减弱,而E-cadherin的表达增强;（3）不同浓度Cur抑制了ColⅠ、ColⅢ和FN的分泌,与单纯TGF-β1刺激组相比有统计学差异（P〈0.05）。结论：姜黄素能抑制TGF-β1诱导人肾小管上皮细胞转分化及细胞外基质成分ColⅠ、ColⅢ和FN的合成,在一定程度上具有防治肾小管-间质纤维化的作用。     

虫草素通过下调TGF-β抑制高糖诱导的大鼠肾小管上皮细胞转分化

目的：探讨虫草素对高糖诱导的大鼠肾小管上皮细胞转分化的影响。方法：体外培养大鼠近端肾小管上皮细胞株（NRK52E细胞株）,分为正常对照组（葡萄糖5.5 mmol/L,NG组）、高糖组（葡萄糖30 mmol/L,HG组）、高糖＋虫草素组（葡萄糖30 mmol/L＋虫草素10μg/ml,HG＋C组）。分别于刺激12 h,24 h,48 h后收集细胞。应用定量RT-PCR测定NRK52E TGF-β,E-cadherin,α-SMA mRNA的表达;Western印迹方法检测TGF-β、E-cadherin、α-SMA蛋白的表达。结果：高糖刺激后NRK52E细胞的TGF-β和α-SMA mRNA及蛋白表达明显高于正常糖组（P〈0.01）,而虫草素组TGF-β和α-SMA mRNA及蛋白表达显著低于高糖组（P〈0.05）;高糖诱导的NRK52E细胞E-cadherin mRNA及蛋白水平明显降低（P〈0.01）;而虫草素组NRK52E细胞E-cadherin mRNA及蛋白水平显著高于高糖组（P〈0.05）。结论：虫草素可以明显抑制高糖诱导的大鼠肾小管上皮细胞转分化,其机制可能是通过下调TGF-β实现。     

高糖及糖基化终末产物对肾小管上皮细胞转分化的影响及小檗碱的干预

目的:探讨小檗碱对糖基化终末产物(advanced glycation end product,AGE)和高糖诱导下肾小管上皮细胞转分化的影响及机制。方法:人肾小管上皮细胞株(HK-2细胞)于含10%胎牛血清的D-MEM/F12培养基中进行体外培养。细胞增殖达到70%左右,换无血清培养液继续培养12 h后,进行分组处理。分别用高糖(30 mmol)、AGE(100μg/ml)、小檗碱(10μmol)处理48 h后,倒置显微镜下观察细胞形态,原位细胞免疫组化检测AGE受体(RAGE)、α-SMA、Ⅰ型胶原蛋白(Col-Ⅰ)、平滑肌肌动蛋白(α-SMA)表达,Western blot检测RAGE、α-SMA、转化生长因子(TGF-β1)的蛋白翻译水平。结果:经高糖刺激细胞48 h后,细胞体积逐渐变大,形态变为长梭形,排列紊乱,少部分细胞脱落,高糖+AGE组细胞形态与高糖组相似;小檗碱干预后,细胞形态改变较少见,排列紊乱现象较轻微。免疫组化结果显示对照组及高糖组几乎未见RAGE阳性表达,但高糖+AGE组RAGE呈强阳性表达,Col-Ⅰ和α-SMA显著升高。小檗碱干预后可抑制细胞肥大,并使RAGE和α-SMA的水平有一定下降,同时Col-Ⅰ也有所降低;蛋白印迹法分析显示高糖和AGE可使RAGE蛋白表达显著上调,并刺激TGF-β1表达水平升高,小檗碱可抑制RAGE、TGF-β1的蛋白表达。结论:持续的高糖和AGE作用下可引起肾小管上皮细胞肥大,促进细胞外基质合成,并诱导肾小管上皮细胞转分化。小檗碱能改善高糖和AGE引起的细胞肥大和胶原蛋白合成,抑制肾小管上皮细胞转分化,其机制与下调RAGE,抑制TGF-β1表达有关。     

黄芪通过c-met调控TGF-β1诱导的肾小管上皮细胞转分化

AB 目的：探讨黄芪对TGF-β1诱导的肾小管上皮细胞转分化及细胞外基质分泌的作用及机制。方法：体外培养正常大鼠肾小管上皮细胞（NRK52E）,应用倒置相差显微镜观察NRK52E细胞形态学变化;免疫组织化学染色法及实时荧光定量PCR法检测α-平滑肌肌动蛋白（α-SMA）,肝细胞生长因子HGF受体（c-met）的表达;ELISA法定量检测细胞上清液中胶原Ⅰ（Col-Ⅰ）,胶原Ⅲ（Col-Ⅲ）和纤维黏连蛋白（FN）的水平。结果：TGF-β1可诱导肾小管上皮细胞肌成纤维细胞转分化（TEMT）,TGF-β1诱导组细胞肥大、拉长,呈长梭形,α-SMA表达明显增强,Col-Ⅰ、Col-Ⅲ和FN分泌增加（P〈0.05）。加入不同浓度黄芪后,细胞形态接近正常肾小管上皮细胞形态,α-SMA表达、Col-Ⅰ、Col-Ⅲ和FN分泌均较TGF-β1诱导组明显抑制（P〈0.05）,c-met表达较TGF-β1诱导组增加（P〈0.05）且呈剂量依赖性。结论：TGF-β1可以诱导肾小管上皮细胞肌成纤维细胞转分化,增加细胞外基质成分Col-Ⅰ、Col-Ⅲ和FN的分泌;黄芪能够抑制TGF-β1诱导的NRK52E细胞转分化以及细胞外基质的分泌;黄芪抑制细胞转分化的机制可能与其增强c-met的表达有关。     

高糖通过转化生长因子β1-Smad信号传递途径诱导肾小管上皮细胞转分化

目的 通过观察高糖对肾小管上皮细胞转分化(EMT)的作用，探讨其与转化生长因子β1 (TGF-β1)的关系及糖尿病肾病肾小管间质纤维化的发病机制。 方法 以人肾小管上皮细胞株HKC细胞和高表达Smad7蛋白的HKC转染细胞株为研究对象。蛋白印迹法检测高糖(葡萄糖浓度分别为25和50 mmol/L)对α平滑肌肌动蛋白(α-SMA)、E钙黏蛋白 (E-cadherin)和纤连蛋白(FN)表达的影响。酶联免疫吸附(ELISA)法检测TGF-β1的水平。Boyden小室检测HKC细胞的迁移能力。抗TGF-β1抗体中和实验分析高糖对肾小管EMT作用及与TGF-β1的关系。 结果 持续的高糖作用(96 h)能够导致HKC表达α-SMA蛋白，其中25和50 mmol/L的葡萄糖分别增加表达α-SMA 2.8倍和8.2倍；降低E-cadherin的表达；刺激合成FN。25和50 mmol/L的葡萄糖刺激HKC 12 h后，细胞培养上清液中TGF-β1浓度分别为(408.5±198.6)和(939.3±311.8) ng/L，呈剂量依赖性。抗TGF-β1抗体能够显著抑制高糖导致的HKC高表达α-SMA蛋白和FN及降低E-cadherin表达的作用。高表达Smad7蛋白的HKC转染细胞株在高糖的持续作用下，不能表达α-SMA和FN蛋白，E-cadherin也未见降低。细胞迁移实验表明，25和50 mmol/L高糖能够增加HKC迁移至Boyden小室膜下侧面的细胞数[(12.4±3.7)和（18.6±4.4)细胞/HP）]，与正常对照组[(3.0±0.8)细胞/HP]差异有统计学意义(P < 0.01)。抗TGF-β1多克隆抗体能够部分抑制高糖(50 mmol/L)造成的HKC细胞向Boyden小室膜下侧面的迁移[(11.9±5.2)细胞/HP]。高表达Smad7蛋白的HKC转染细胞株在高糖培养条件下迁移至Boyden小室膜下侧面的细胞数[（4.3±1.2）细胞/HP]与正常对照组差异无统计学意义。 结论 高糖能够诱导肾小管EMT，此作用与高糖刺激该细胞合成TGF-β1有关，阻止Smad信号途径能够拮抗TGF-β1介导的肾小管EMT的作用。     

红细胞生成素对高糖诱导下肾小管上皮细胞转分化的影响

目的 探讨红细胞生成素（EPO）对高糖诱导下的肾小管上皮细胞转分化的影响。 方法 体外培养人肾小管上皮细胞株（HK-2细胞）,分为正常对照组、渗透浓度对照组、高糖组、高糖＋EPO（5 U/ml）组和高糖＋EPO（10 U/ml）组。RT-PCR法检测各组细胞α平滑肌肌动蛋白（α-SMA）、转化生长因子β1（TGF-β1）、Smads信号蛋白2（Smad2）及整合素连接激酶（ILK）的mRNA表达。细胞免疫荧光法检测细胞TGF-β1及α-SMA的蛋白表达。 结果 RT-PCR结果显示,相对于对照组,高糖组细胞α-SMA、TGF-β1、Smad2、ILK的mRNA表达均显著上调（P ＜ 0.01）。细胞免疫化学也显示,高糖组TGF-β1和α-SMA的蛋白表达较对照组显著上调（P ＜ 0.01）,而高糖＋EPO（5 U/ml）组和高糖＋EPO（10 U/ml）组上述指标的表达均显著低于高糖组（均P ＜ 0.01）。 结论 EPO能抑制高糖诱导的肾小管上皮细胞转分化,可能与其抑制TGF-β1、Smad2及ILK表达有关。     

"慢性肾衰基本方"含药血清对人肾小管上皮细胞转分化的影响

目的:观察经验方"慢性肾衰基本方"对转化生长因子β1(TGF-β1)所诱导的肾小管上皮细胞(TECs)转分化及细胞外基质分泌的影响,以进一步证实并初步探讨其延缓慢性肾衰竭肾小管-间质纤维化的作用机制.方法:给予家兔中药灌胃3 d后制备"慢性肾衰基本方"含药兔血清,应用不同浓度含药血清处理经TGF-β1诱导活化的HK-2细胞,然后应用免疫细胞化学方法、流式细胞技术测定HK-2细胞α-平滑肌肌动蛋白(α-SMA)的表达率;应用酶联免疫吸附检测法(ELISA)测定培养上清纤维连结蛋白(FN)及Ⅳ型胶原(Col-Ⅳ)水平,并进而计算单个细胞FN及Col-Ⅳ分泌水平.结果:含药兔血清呈剂量依赖方式逆转TGF-β1诱导的HK-2细胞α-SMA的表达,并呈剂量依赖方式抑制单个细胞FN及Col-Ⅳ的分泌.结论:"慢性肾衰基本方"至少部分是通过抑制肾小管上皮细胞转分化及细胞外基质的过度分泌而对防治肾小管-间质纤维化发挥作用.     

三七总皂苷对TGF-β1诱导的HK-2细胞表型转分化的影响

目的:探讨三七总皂苷(PNS)对转化生长因子-β1(TGF-β1)诱导的人肾小管上皮细胞(HK-2)转分化的影响.方法:采用流式细胞仪(FCM)结合免疫荧光法(IF)检测PNS对TGF-β1诱导的HK-2细胞α-平滑肌肌动蛋白(α-SMA)阳性细胞百分率的影响;逆转录-聚合酶链反应(RT-PCR)检测PNS对TGF-β1诱导的HK-2细胞α-SMA信使核糖核酸(mRNA)表达.结果:流式细胞仪(FCM)结合免疫荧光法(IF)示:PNS 200～800 mg/L可使TGF-β1诱导的HK-2细胞增加的α-SMA阳性细胞百分率回降;RT-PCR示:PNS 200～800 mg/L剂量和时间依赖性地使TGF-β1诱导的HK-2细胞增加的α-SMA mRNA的基因表达回降.结论:PNS可通过抑制TGF-β1诱导的肾小管上皮细胞表型转分化,从而参与延缓肾间质纤维化的进程.     

Smad7对高糖介导大鼠肾小管上皮细胞转分化和胶原Ⅰ合成的影响

目的探讨Smad7对高糖介导肾小管上皮细胞转分化和胶原(Col)Ⅰ合成的影响。方法体外培养转染Smad7的大鼠近端肾小管上皮细胞株(NRK52E细胞),分为强力霉素(Dox)诱导组和未加强力霉素的对照组。前者予Dox诱导24 h后,给予高糖刺激;后者不加Dox诱导。采用免疫细胞化学方法检测磷酸化(p)-Smad2/3核转位情况;RT-PCR检测Smad7的表达;Western印迹方法检测不同时间点Smad7、α-SMA、E-钙黏蛋白(cadherin)和ColⅠ蛋白的表达水平。结果成功构建了Dox调控的可上调Smad7表达的NRK52E细胞。NRK52E细胞在基础状态下可表达低水平p-Smad2/3(16．1％),与未加Dox的对照组比较,Dox诱导组可显著抑制高糖刺激的NRK52E细胞TGF-β受体调控信号蛋白Smad2/3的活化和核转位(30．3％比58．5％, P<0．01)。上调表达Smad7可显著抑制高糖介导的NRK52E细胞α-SMA和ColⅠ蛋白的表达;逆转高糖介导的NRK52E细胞E-cadherin蛋白的下调表达。结论基因转染上调表达Smad7可通过TGF-β受体调控信号蛋白Smad2/3的活化和核转位而阻抑高糖介导的肾小管上皮细胞转分化及细胞外基质的合成。     

高糖通过转化生长因子β信号诱导肾小球内皮细胞向肌成纤维细胞转分化

目的 探讨高糖(HG)能否通过转化生长因子β(TGF-β)途径诱导大鼠肾小球内皮细胞向肌成纤维细胞转分化(EndMT).方法 体外培养大鼠肾小球内皮细胞(GEnC),分为正常对照组(NG,5.5 mmol/L)、高糖组(HG,15、30 mmol/L)、TGF-β抑制剂组(HG+LY36,30 mmol/L葡萄糖+10 μmol/L LY364947)以及高渗对照组(M,5.5 mmol/L葡萄糖+25.5 mmol/L甘露醇)和溶剂对照组(D,5.5 mmol/L葡萄糖+1 ml/LDMSO).采用Western印迹法检测各组细胞内皮细胞标志物claudin5和肌成纤维细胞标志物α-SMA表达变化;实时定量PCR法检测细胞TGF-β1和TGF-β2 mRNA表达改变;免疫荧光法观察细胞形态学变化以及血管内皮细胞标志物VE-cadherin和肌成纤维细胞标志物α-SMA的表达.结果 与NG组比较,HG组claudin5蛋白的表达量随葡萄糖浓度增加而降低(P＜0.05),α-SMA蛋白表达量随葡萄糖浓度增加而升高(P＜0.05),TGF-β1和TGF-β2 mRNA表达均升高(P＜0.05).与HG组比较,TGF-β抑制剂组claudin5蛋白表达量升高(P＜0.05),α-SMA蛋白表达降低(P＜0.05).高渗对照组和溶剂对照组改变差异无统计学意义.激光共聚焦免疫荧光结果显示,高糖处理可引起细胞形态由卵圆形向梭形改变,VE-cadherin表达减少,α-SMA表达增加;TGF-β抑制剂组细胞形态无明显改变.与HG组比较,TGF-β抑制剂组VE-cadherin表达增加,α-SMA表达降低(P＜0.05).结论 高糖诱导大鼠肾小球内皮细胞TGF-β表达增加及内皮细胞-肌成纤维细胞转分化.抑制TGF-β可抑制高糖引起的转分化,提示TGF-β参与了高糖引起的肾小球内皮细胞转分化过程.     

Suppression of the production of extracellular matrix and α-smooth muscle actin induced by transforming growth factor-β1 in fibroblasts of the flexor tendon sheath by hepatocyte growth factor

We examined the effectiveness of hepatocyte growth factor (HGF) in blocking production of transforming growth factor (TGF)-β1-induced collagen I, fibronectin, and α-smooth muscle actin (α-SMA) in the flexor tendon sheath of rabbits in vitro. Fibroblasts were obtained from the sheaths. Cell culture was supplemented with TGF-β1 5 ng/ml and increasing doses of HGF (10-40 ng/ml). The production of collagen I and fibronectin in supernatants culture were examined using an enzyme-linked immunosorbent assay (ELISA). α-SMA expression was assessed by western blot. TGF-β1 stimulated production of collagen I, fibronectin, and α-SMA greatly, while HGF significantly (p<0.05) reduced production of all components induced by TGF-β1 in a dose-dependent manner. This suggests that HGF effectively antagonises the action of TGF-β1 in cultured fibroblasts from flexor tendon sheaths. The results provide a cellular and molecular basis for HGF acting as a therapeutic agent for adhesions in flexor tendons.     

低氧对SD大鼠阴茎海绵体平滑肌纤维化的影响

目的:探讨低氧对SD大鼠阴茎海绵体平滑肌纤维化的影响。方法:体外培养阴茎海绵体平滑肌细胞,免疫组化鉴定细胞;常规氧浓度(21%O2浓度)分别培养12、24、48、72h作为对照,低氧(1%O2浓度)干预12、24、48、72h,RT-PCR分别测定各组TGF-β1、Ⅰ型胶原、Ⅲ型胶原的相对表达量。结果:体外培养的阴茎海绵体平滑肌细胞生长良好,抗平滑肌α-肌动蛋白单克隆抗体免疫组化染色阳性;RT-PCR结果提示TGF-β1、Ⅰ型胶原、Ⅲ型胶原的相对表达量在48h内与低氧时间成正相关,时间进一步延长不能增加其相对表达量。结论:在低氧环境下,SD大鼠阴茎海绵体平滑肌细胞的TGF-β1、Ⅰ型胶原、Ⅲ型胶原的相对表达量随时间的延长逐渐增加,48h达到最大值。低氧可导致SD大鼠阴茎海绵体平滑肌纤维化。     

血管内皮生长因子抑制肾小管上皮间充质转化及其与结缔组织生长因子和PI3K-Akt信号通路的关系

目的　探讨血管内皮生长因子（VEGF）对转化生长因子β1（TGF-β1）诱导的肾小管上皮间充质转化（EMT）的作用,及其与结缔组织生长因子（CTGF）、PI3K-Akt信号通路的关系。 方法 （1）将体外培养的HK2细胞分为正常对照组、TGF-β1（5 μg/L,下同）组、VEGF组（100 μg/L,下同）、TGF-β1＋VEGF组。HK2细胞体外培养48 h,用免疫组化双染方法检测肾小管上皮细胞α平滑肌肌动蛋白（α-SMA）和E钙黏蛋白的表达。（2）将体外培养的HK2细胞分为正常对照组、TGF-β1组、VEGF组、TGF-β1＋VEGF组、PI3K-Akt信号通路阻断剂LY294002组（25 μmol/L,下同）、TGF-β1＋LY294002组、VEGF＋LY294002组、TGF-β1＋VEGF＋LY294002组。HK2细胞体外培养48 h,用Western印迹和RT-PCR方法检测α-SMA和CTGF的表达;用ELISA方法检测培养上清中纤连蛋白（FN）和I型胶原（ColⅠ）的表达。 结果 免疫组化结果显示,TGF-β1组α-SMA表达比正常对照组增强,而E钙黏蛋白表达减弱; TGF-β1＋VEGF组α-SMA表达比TGF-β1组显著减弱,而E钙黏蛋白表达增强。 TGF-β1组α-SMA、CTGF蛋白和 mRNA及FN、ColⅠ表达比正常对照组显著增强（均P < 0.05）;TGF-β1＋VEGF组α-SMA、CTGF蛋白和mRNA及FN、ColⅠ表达比TGF-β1组显著减弱（均P < 0.05）;TGF-β1＋VEGF＋LY294002组α-SMA、CTGF蛋白和 mRNA及FN、ColⅠ表达比TGF-β1＋VEGF组显著增强（均P < 0.05）。 结论　VEGF能抑制TGF-β1诱导的体外培养的HK2细胞发生EMT,其机制可能与VEGF下调HK2细胞CTGF表达及减少细胞外FN、ColⅠ合成有关。VEGF的这种作用可能部分通过PI3K-Akt信号转导通路实现,其确切机制有待进一步研究。     

Effect of tet methylcytosine dioxygenase 2 on the regulation of transforming growth factor-β1 expression in mesangial cells induced by high glucose

Objective To investigate the role of tet methylcytosine dioxygenase 2 (TET2) in the regulation of transforming growth factor-β1 (TGF-β1) expression in human glomerular mesangial cells induced by high glucose. Methods Cultured human glomerular mesangial cells were divided into normal control group (5.5 mmol/L glucose) and high glucose group (30.0 mmol/L glucose) which was cultured for 12 h to 72 h. The gene expression of TET2 in mesangial cells were inhibited by small molecule chemical called SC1, and which were divided into high glucose group (30.0 mmol/L glucose+DMEM), DMSO group (30.0 mmol/L glucose+0.1%DMSO) and SC1 group (30.0 mmol/L glucose+3 μmol/L SC1). The mRNA and protein expression of TGF-β1, TET1 to 3 and α-smooth muscle actin (α-SMA) was detected by quantitative real-time PCR and Western blotting. Methylation of CpG islands in the regulation region of TGF-β1 was detected by bisulfite sequencing PCR (BSP). The activity of mesangial cell proliferation was assessed by colorimetry of thiazolyl blue (MTT). Results Compared with normal control group, the mRNA and protein expression of TET2 in mesangial cells induced by high glucose was increased significantly in a time-dependent manner (all P＜0.05), but the expression of TET1 and TET3 was not affected. Meanwhile methylation rate of 4 CG sites from 24 h to 72 h were decreased in the first exon of TGF-β1 (P＜0.01), but not in the promoter. Compared with high glucose group, when the expression of TET2 was inhibited by SC1, the methylation rate of TGF-β1 was recovered evidently (P＜0.05), the mRNA and protein expression of TGF-β1 and α-SMA was suppressed, and the proliferation of mesangial cells was decreased (all P＜0.05). Conclusions Demethylation of the CpG island mediated by TET2 may play an important role in the expression of TGF-β1 and mesangial cell phenotype transformation induced by high glucose.     

Fluorofenidone reduces renal fibrosis in diabetic kidney disease mice

Objective To investigate the effects of fluorofenidone (AKF-PD) on diabetic kidney disease in db/db mice and its possible mechanisms. Methods (1) Fifty-six mice aged 8 weeks (half male and half female), including 42 db/db mice and 14 wild-type mice were studied. Forty-two db/db mice randomly were divided into model group (mock-treated diabetic db/db mice), AKF-PD (250 mg?kg-1?d-1) treatment group and losartan (20 mg?kg-1?d-1) treatment group. Wild-type mice and model mice were treated with vehicle (0.5% sodium carboxymethylcellulose), while the treatment groups received either AKF-PD or losartan. After 18 weeks, the blood glucose and urinary albumin were measured, the pathological changes of kidney were observed by PAS staining. The protein expressions of type Ⅳ collagen and fibronectin (FN) in kidney tissue were detected by immunohistochemistry. (2) Mouse glomerular mesangial cells (MES-13 cells) were divided into six groups: normal glucose group (5.5 mmol/L glucose), hypertonic group (5.5 mmol/L glucose+19.5 mmol/L mannitol), high glucose group (25.0 mmol/L glucose), AKF-PD group (25.0 mmol/L glucose+400 mg/L AKF-PD) and losartan group (25.0 mmol/L glucose+2 μmol/L losartan). After 72 h treatment, the expressions of type Ⅰ collagen, type Ⅳ collagen and transforming growth factor-β1 (TGF-β1) mRNA were detected by real-time PCR, and the content of TGF-β1 protein in the culture supernatant was detected by ELISA. Results (1) Compared with the wild type mice, model mice had increased weight, blood glucose and glomerulosclerosis index (all P＜0.01), accompanied with heavy albuminuria, glomerular hypertrophy, mesangial area expansion and deposition of collagen type Ⅳ and FN (all P＜0.01). Compared with model mice, in AKF-PD and losartan groups 24 h urinary albumin and glomerulosclerosis index decreased (all P＜0.01), glomerular hypertrophy and mesangial area expansion alleviated, and the protein expressions of collagen type Ⅳ and FN were inhibited (all P＜0.01). (2) Compared with the normal glucose group, the mRNA expressions of type Ⅰ collagen and type Ⅳ collagen increased in high glucose group, meanwhile the mRNA and protein expressions of TGF-β1 increased (all P＜0.01). In AKF-PD and losartan groups the expressions of type Ⅰ collagen, type Ⅳ collagen and TGF-β1 were inhibited as compared with high glucose group (all P＜0.05). Conclusion Fluorofenidone may play an anti-fibrotic effect in db/db mice by reducing the expression of TGF-β1 and inhibiting collagen synthesis in glomerular mesangial cells.     

过氧化物酶体增殖物激活受体γ配体抑制大鼠肝纤维化的实验研究

目的 研究过氧化物酶体增殖物激活受体γ(peroxisome proliferator activated receptorgamma,PPARγ)配体对大鼠肝纤维化的作用.方法 将Wistar大鼠40只随机分为两组,对照组(20只)和罗格列酮组(20只).所有动物使用饮水中加人质量比0.3‰硫代乙酰胺的方法 制作肝纤维化模型.对照组喂饲普通颗粒饲料.罗格列酮组喂饲含200 ppm罗格列酮的颗粒饲料.喂饲6个月后,用RT-PCR方法 检测肝纤维化大鼠肝脏PPARγ、TGF-β 1 及Ⅰ型前胶原mRNA表达,用Westernblot法检测PPARγ、TGF-β 1 、Ⅰ型胶原及α平滑肌肌动蛋白(α-SMA)表达,用Van Gieson(VG)染色的方法 检测肝组织切片的胶原表达情况.结果 罗格列酮组与对照组相比,PPARγmRNA表达显著增强(t=6.93,P<0.01),TGF-β 1 mRNA(t=3.89,P<0.01)和Ⅰ型前胶原mRNA表达显著降低(t=5.67,P<0.01).PPARγ、TGF-β 1 及Ⅰ型胶原蛋白表达所得结果 与RT-PCR结果 相一致.罗格列酮组与对照组相比,α-SMA表达显著降低(t=3.12,P<0.01).罗格列酮组肝组织切片的胶原染色低于对照组(t=3.47,P<0.01).结论 PPARγ配体能够抑制大鼠纤维化肝脏的胶原产生,在体内具有一定的抗肝纤维化作用.     

骨髓间充质干细胞对大鼠慢性胰腺炎中星状细胞的影响

目的 观察骨髓间充质干细胞(BMSCs)对慢性胰腺炎(CP)中星状细胞(PSC)的活化、增殖和分化的影响.方法 将80只SD大鼠随机分为空白组(20只,注射无菌生理盐水)、模型组[20只,尾静脉注射二氯二丁基酯(DBTC)制作大鼠CP模型]、治疗组(20只,于CP模型制作成功后尾静脉注射体外培养的同种异体GFP-MSCs)、假治疗组(20只,于CP模型制作成功后尾静脉注射等量的无菌生理盐水).取大鼠胰腺检测PSC活化表达的Desmin、胶质纤维酸性蛋白(GFAP)、α-平滑肌肌动蛋白(α-SMA)水平,组织中Ⅰ、Ⅲ胶原含量及白细胞介素(IL)-10、肿瘤坏死因子(TNF)-α、转化生长因子(TGF) -β1表达水平.结果 治疗组PSC表达的Desmin、GFAP、α-SMA,组织中Ⅰ (0.135±0.030) ng/g、Ⅲ胶原含量(0.029±0.008) ng/g,TGF-β1 (0.020±0.006) μg/L浓度均低于假治疗组(P＜0.05),但IL-10 (603.799±89.374) g/ml,TNF-α(507.45±90.13) μg/L均高于假治疗组(P＜0.05),后者与模型组之间的差异无统计学意义(P＞0.05),空白组未见明显异常.结论 BMSCs可以通过对细胞因子的调控减少PSC的活化,抑制其增殖及分化,降低细胞外基质的沉积.     

Effects of pirfenidone on renal fibrosis in mice with diabetic nephropathy and its mechanisms

Objective To investigate effects of pirfenidone (PFD) on diabetic nephropathy model in db/db mice and to explore its possible mechanisms. Methods (1) Wild-type mice were as the normal control group, and db/db mice were divided into model group and PFD group, with 6 mice in each group. In the PFD group mice were administered continuously by 250 mg?kg-1?d-1 PFD for 18 weeks, and mice in the other two groups were administered by 0.5% sodium carboxymethyl cellulose. Blood glucose and 24 h urinary albumin were measured. The pathological changes of renal tissue were evaluated by PAS staining, PASM staining, Masson staining and Sirius red staining. The expression of collagen type Ⅳ in kidney tissues was detected by immunohistochemistry. (2) Mouse mesangial cells (SV40 MES-13 cells) were cultured as research objects. They were divided into control group, hyperosmolar group, high glucose (HG) group, and 50, 100, 200, 400, 800, 1600 mg/L PFD+HG group. BrdU cell proliferation test was used to evaluate cell proliferation rate. Cells were divided into control group, hyperosmolar group, HG group and PFD+HG group. The mRNA expressions of α-smooth muscle actin (α-SMA), collagen type Ⅰ, collagen type Ⅳ, transforming growth factor-β1 (TGF-β1), interleukin (IL)-1β, IL-6 and monocyte chemotactic protein-1 (MCP-1) were detected by real-time PCR. Results (1) Compared with normal control group, the model mice had higher weight, blood glucose and 24 h urinary albumin, accompanied with glomerular hypertrophy, mesangial area expansion, tubulointerstitial fibrosis and deposition of collagen type Ⅳ (all P＜0.05). Compared with those in model group, in PFD group 24 h urinary albumin decreased, glomerular hypertrophy, mesangial area expansion and tubulointerstitial fibrosis alleviated, and the protein expression of collagen type Ⅳ inhibited (all P＜0.05). (2) Compared with those in HG group, MES-13 cell proliferation rates of 100, 200, 400, 800, 1600 mg/L PFD+HG groups decreased (all P＜0.05), and the mRNA expressions of α-SMA, collagen type Ⅰ, collagen type Ⅳ, TGF-β1, IL-1β, IL-6 and MCP-1 reduced in 400 mg/L PFD+HG group (all P＜0.05). Conclusions PFD can inhibit high glucose-induced proliferation and activation of glomerular mesangial cells, decrease the expression of TGF-β1 and proinflammatory factors, as well as reduce the synthesis of collagen, which improve renal fibrosis of db/db mice.     

大黄酸抑制肾间质成纤维细胞激活的实验研究

目的 研究大黄酸(Rhein)抑制转化生长因子β1(TGF-β1)激活大鼠肾间质成纤维细胞(NRK-49F)作用及探讨大黄治疗肾脏病的作用机制。方法 以NRK-49F细胞为研究对象，采用细胞计数观察细胞生长，流式细胞仪分析细胞周期变化。α-平滑肌肌动蛋白(α-SMA)作为细胞活化指标，同时检测纤连蛋白(FN)表达的水平以评价细胞外基质合成的变化。结果 TGF-β1具有促进NRK-49F增殖的作用，大黄酸呈时间依赖性和剂量依赖性抑制该增殖作用。TGF-β1能够促进NRK-49F进入S期和G2/M期，而经大黄酸作用的细胞主要是G0/G1期细胞。大黄酸呈剂量依赖性显著抑制TGF-β1诱导的NRK-49F细胞α-SMA蛋白表达水平，同时，大黄酸还可减少TGF-β1诱导的NRK-49F细胞FN 蛋白的表达水平。结论 大黄酸能够阻止TGF-β1诱导的肾间质成纤维细胞由G0/G1期进入S期和G2/M期，抑制该细胞的增殖。同时，大黄酸还具有抑制TGF-β1激活肾间质成纤维细胞的作用，并拮抗TGF-β1导致的FN表达与合成。     

Change of autophagy in the transforming growth factor-β1-induced activation of renal fibroblasts in vitro

Objective To explore the changes of autophagy in the transforming growth factor (TGF)-β1-induced activation of renal fibroblasts in vitro. Methods (1) NRK-49F cells were cultured with 10 μg/L TGF-β1 for different times (0, 12, 24 h) in vitro. Morphological changes of the cells were observed under inverted microscope. The protein expressions of α-smooth muscle actin (α-SMA) and typeⅠcollagen (ColⅠ) in NRK-49F cells were measured by Western blotting. (2) NRK-49F cells were cultured with 0, 2, 5, 10, 15, 20 μg/L TGF-β1 for 1 hour and with 10 μg/L TGF-β1 for different times (0 min, 7 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h) in vitro. The protein expressions of microtubule-associated protein 1 light chain 3(LC3), p62, total- mammalian target of rapamycin (t-mTor), phospho-mammalian target of rapamycin (p-mTor) and Beclin 1 were detected by Western blotting. (3) NRK-49F cells were cultured with 10 μg/L TGF-β1 for different times (0, 1, 4 h) in vitro after cultured with serum-free medium for 2.5 hours. The protein expressions of LC3 and p62 in NRK-49F cells were measured by Western blotting. Results (1) The morphology of NRK-49F cells changed from stellate to spindle shape after cultured with TGF-β1. The expressions of cell activation markers α-SMA and ColⅠgradually increased as the extend of stimulation time (all P＜0.05). (2) TGF-β1 transiently increased the expressions of autophagy proteins p62 (peak value appeared after 4 h) and p-mTor (peak value appeared after 30 min), while decreased Beclin1 expression level (all P＜0.05). (3) TGF-β1 decreased the protein expression of LC3-Ⅱ in NRK-49F cells cultured with serum-free medium, whereas increased the protein expression of p62 at the same time (all P＜0.05). Conclusions The autophagy activity of renal fibroblasts is inhibited by the TGF-β1-induced cellular activation in vitro, which may contribute to the progression of renal interstitial fibrosis.