非酶糖基化终末产物抑制大鼠睾丸Leydig细胞睾酮的生成

目的:研究非酶糖基化终末产物受体(RAGE)在大鼠睾丸Leydig细胞上的表达及非酶糖基化终末产物(AGEs)对大鼠睾丸Leydig细胞睾酮合成的抑制作用。方法:原代培养大鼠睾丸Leydig细胞,RT-PCR和免疫荧光技术检测RACE在大鼠Leydig细胞上表达,不同浓度AGEs处理Leydig细胞(25、50、100、200μg/ml),ELISA法测定睾酮分泌量。结果:RT-PCR和免疫荧光结果表明RAGE在大鼠睾丸Leydig细胞上表达,不同浓度AGEs处理后,人绒毛膜促性腺激素(hCG)诱导的Leydig细胞睾酮合成量呈剂量浓度依赖性下降,与对照组相比,50、100、200μg/ml AGEs处理组差异显著(P0.01)。结论:大鼠Leydig细胞上存在RAGE受体,AGEs显著抑制原代培养大鼠Leydig细胞睾酮的分泌。     

青蒿琥酯对脂多糖诱导的大鼠肾小球系膜细胞凋亡及Bcl-2基因表达的影响

目的：观察青蒿琥酯（Art）对脂多糖（LPS）诱导的大鼠肾小球系膜细胞（glomerular mesangial cells,GMC）凋亡及Bcl-2基因表达的影响,并探讨其可能的作用机制。方法：用不同浓度的青蒿琥酯刺激脂多糖诱导增殖的大鼠肾小球系膜细胞,HE染色观察凋亡细胞形态,DAPI荧光染色观察细胞核凋亡情况,用流式细胞仪检测细胞凋亡率,免疫细胞化学结构和计算机图文分析系统检测Bcl-2的表达。结果：青蒿琥酯对系膜细胞凋亡有明显诱导作用,呈一定剂量依赖性;并能明显地抑制脂多糖诱导上调的Bcl-2基因的表达,且高浓度组的抑制作用强于低浓度组。结论：青蒿琥酯能够剂量依赖性的诱导大鼠系膜细胞凋亡,其机制可能与调控Bcl-2的表达有关。     

中药复方含药血清对体外培养的肾小球系膜细胞的作用

目的:观察糖基化终末产物(AGEs)对大鼠肾小球系膜细胞ROS、RAGE mRNA和结缔组织生长因子(CTGF)表达的影响,以及中药复方(由太子参、生黄芪、白术、川连、泽兰、丹参、全瓜蒌5 g等组成)的干预作用。方法:原代培养大鼠肾小球系膜细胞,细胞在DMEM/F12培养液中培养传代(37℃,5%CO2),取5~8代细胞用于实验。随机分为正常糖浓度组(含5.5 mmol/L)、高糖浓度组(30 mmol/L)、AGE-BSA组、中药复方+高糖组、中药复方+AGE-BSA组。分别于培养12、24、48 h时用0.25%胰蛋白酶消化液消化并收集细胞,采用CCK-8法检测细胞增殖,RT-PCR法检测细胞RAGE mRNA、CTGF mRNA的表达,流式细胞仪检测细胞内ROS表达。结果:高糖作用24 h和48 h的系膜细胞增殖显著高于正常组(P0.05),AGE-BSA组12 h、24 h和48 h的细胞增殖均有明显增加(P0.05),中药复方加高糖组较高糖组细胞增殖显著降低(P0.05);中药复方加AGE-BSA组较AGE-BSA组细胞增殖也有明显下降(P0.05)。高糖组和AGE-BSA组作用12 h、24 h和48 h的系膜细胞ROS表达均明显升高(P0.05);中药复方加高糖组作用12 h、24 h、48 h较高糖组细胞ROS表达均有下降(P0.05)。高糖组和AGE-BSA组作用12 h、24 h和48 h的系膜细胞CTGF mRNA表达均明显升高(P0.05),中药复方加高糖组作用48 h较高糖组细胞CTGF mRNA表达均有下降(P0.05)。中药复方加AGE-BSA组作用48 h较AGE-BSA组细胞CTGF mRNA也有明显下降(P0.05)。高糖组和AGE-BSA组作用12 h、24 h和48 h的系膜细胞RAGE mRNA表达均明显升高(P0.05);中药复方加高糖组作用12 h、24 h、48 h较高糖组细胞RAGE mRNA表达均有下降(P0.05);中药复方加AGE-BSA组作用48 h较AGE-BSA组细胞RAGE mRNA也有明显下降(P0.05)。结论:AGEs可能部分通过诱导细胞内ROS,促进肾小球系膜细胞表达CTGF,从而引起肾小球系膜基质增生,最终导致肾小球硬化,而中药复方可能通过调节AGEs-RAGE介导的氧化应激过程,从而阻止肾小球纤维化的发展进程。     

前列地尔调控Wnt/β-catenin通路对脂多糖诱导的肾小球上皮细胞氧化应激及凋亡的影响

目的:观察前列地尔对脂多糖(LPS)诱导的肾小球上皮细胞(GEC)氧化应激和凋亡的影响,并探讨其机制是否与调控Wnt/β-catenin通路有关.方法:体外培养GEC细胞,分为对照组(正常培养的细胞),LPS组(用含有LPS终浓度为80μg/ml的培养液处理细胞12 h)、LPS+前列地尔组(用终浓度为2μg/ml的前...     

晚期糖基化终末产物通过Wnt/β-catenin信号通路调控足细胞自噬和迁移

目的探讨晚期糖基化终末产物(AGEs)诱导Wnt/β-catenin信号通路活化对足细胞自噬和迁移的影响。方法将小鼠永生化肾足细胞系分为牛血清白蛋白组(100mg/L BSA)、糖基化终末产物组(100 mg/L BSA-AGEs)和糖基化终末产物+DKK-1组(100mg/L BSA-AGEs+100 DKK-1 mg/L),以未加干涉的正常足细胞为对照组。Western blot检测足细胞β-catenin蛋白、自噬膜蛋白微管相关蛋白轻链3-II(LC3-Ⅱ)以及自噬底物p62蛋白表达水平,免疫荧光观察足细胞自噬情况,ELISA检测细胞上清液中Nephrin蛋白含量,划痕试验检测足细胞迁移能力。用AGEs和AGEs+RAGE(糖基化终末产物受体)中和抗体处理足细胞系,Western blot检测足细胞β-catenin蛋白的表达。结果与对照组相比,AGEs组p-β-catenin、p62表达升高(P0.05),LC3-Ⅱ表达降低(P0.05)。AGEs+DKK-1组p-β-catenin、p62蛋白表达较AGEs组降低,LC3-Ⅱ表达升高(P0.05),但与对照组比较差异无显著性(P0.05)。BSA组各指标与对照组均无显著性差异(P0.05)。免疫荧光检测发现AGEs组足细胞中LC3荧光强度较对照组明显减弱,AGEs+DKK-1组较AGEs组LC3荧光强度增强,BSA组与对照组比较差异无显著性(P0.05)。AGEs组细胞培养上清中Nephrin含量较对照组明显降低(P0.05),AGEs+DKK-1组较AGEs组升高(P0.05),但仍低于对照组(P0.05),BSA组与对照组差异无显著性(P0.05)。与对照组相比,AGEs组足细胞迁移能力降低(P0.05),AGEs+DKK-1组、BSA组与对照组比较,差异无显著性(P0.05)。AGEs+RAGE中和抗体处理的足细胞β-catenin蛋白水平较AGEs组显著降低(P0.05)。结论 AGEs通过RAGE激活Wnt/β-catenin信号通路,抑制足细胞自噬和迁移能力,可能是糖尿病患者足细胞损伤机制之一,在糖尿病肾病的发生和进展中发挥重要调控作用。     

雷帕霉素对肾小球系膜细胞增殖及凋亡的影响

目的 观察不同浓度雷帕霉素对大鼠肾小球系膜细胞增殖及凋亡的影响,探讨其可能的作用机制。 方法 使用不同浓度雷帕霉素（1 μg/L、2 μg/L、4 μg/L、8 μg/L、16 μg/L）处理大鼠肾小球系膜细胞,并设正常对照组。MTT法测不同浓度雷帕霉素干预24 h、48 h、72 h后对细胞增殖的影响,并描记生长曲线。干预72 h后,采用RT-PCR和Western印迹法分别检测各浓度雷帕霉素组细胞周期负调蛋白p27和p53的mRNA和蛋白表达变化;用流式细胞计量术检测各浓度雷帕霉素组细胞周期及凋亡率。 结果 小剂量雷帕霉素（1 μg/L）对系膜细胞增殖即具有明显的抑制作用,停滞于G1期的细胞数明显增加,但对系膜细胞凋亡无明显影响。较大剂量雷帕霉素(8~16 μg/L)能够明显增加肾小球系膜细胞凋亡。雷帕霉素能够增加肾小球系膜细胞p27、p53 mRNA和蛋白表达,且呈剂量依赖性。 结论 雷帕霉素可能通过增加p27、p53表达抑制肾小球系膜细胞增殖和促进系膜细胞凋亡。     

全反式维甲酸对环孢素诱导的肾小球系膜细胞增殖与凋亡的影响及其机制

目的探讨全反式维甲酸(ATRA)对环孢素(Cs A)诱导的大鼠肾小球系膜细胞增殖及凋亡的影响及机制。方法采用不同剂量ATRA作用于不同剂量Cs A诱导下的肾小球系膜细胞。采用MTT比色法检测细胞增殖,Hoechst 33258荧光染色观察细胞凋亡形态,后用流式细胞仪检测细胞凋亡率,免疫荧光法检测线粒体促凋亡的Smac蛋白的表达。结果与对照组比较,Cs A剂量为0.5μg/m L及以上可抑制细胞増殖,Cs A剂量为1.0μg/m L及以上可诱导细胞凋亡,且Smac蛋白的表达随Cs A剂量的增加而增加,具有剂量和时间依赖的特征(均为P0.05)。而与Cs A组比较,Cs A+ATRA处理组对细胞增殖的抑制作用更为明显,而加入ATRA可呈剂量依赖性抑制Cs A诱导的肾小球系膜细胞凋亡及Smac蛋白的表达(均为P0.05)。结论 Cs A可抑制肾小球系膜细胞增殖,诱导其凋亡并刺激Smac蛋白的表达。ATRA可以抑制Cs A诱导的肾小球系膜细胞凋亡,且可能是通过Smac信号通路介导的。     

晚期糖基化终末产物对人血管内皮细胞增殖及凋亡的影响

目的研究晚期糖基化终末产物(AGEs)对人血管内皮细胞增殖及凋亡的影响,探讨糖尿病难愈创面新生血管化障碍或延迟的机理.方法不同作用时间及浓度AGE修饰的人血清白蛋白(AGE-HSA)与人血管内皮细胞(ECV304)在体外共同培养,用四唑盐(MTT)比色试验和细胞计数检测AGE-HSA对血管内皮细胞的增殖抑制作用,并用台盼蓝排斥试验检测细胞活力.采用FITC Annexin-V及碘化丙碇(PI)染色,用流式细胞仪检测凋亡细胞百分率,同时应用荧光共聚焦显微镜观察凋亡或死亡细胞FITC Annexin-V和PI的荧光染色,并用透射电镜和光镜观察凋亡细胞特异性的形态学改变.结果内皮细胞在12.5、25和50μg/ml AGE-HSA培养条件下,第2天细胞增殖数量与对照组比较无明显差异性,第4天和第6天则显著低于对照组(P＜0.01);而内皮细胞经100或200μg/ml AGE-HSA干预6h,MTT法测其OD值与对照组比较有明显差异(P＜0.01),同时内皮细胞出现特异性的凋亡形态学变化以及FITC Annexin-V阳性和/或PI阳性的细胞逐渐增多,且凋亡或死亡细胞数量随AGE-HSA作用时间及浓度的增加而增多.结论AGE-HSA能抑制内皮细胞增殖,并可以诱导其凋亡,而且与作用时间及浓度相关.提示AGEs可能是引起糖尿病难愈创面中新生血管化障碍或者延迟的机制之一.     

金雀异黄素对终末糖基化产物刺激大鼠系膜细胞终末糖基化产物受体表达的影响

目的 研究金雀异黄素(Gen)对终末糖基化产物(AGEs)刺激下其系膜细胞受体(RAGE)表达的影响。方法 以AGEs刺激高糖培养的SD大鼠系膜细胞,采用细胞免疫组化法检测系膜细胞RAGE表达,逆转录聚合酶链式反应(RT-PCR)检测细胞内.RAGEmRNA水平;比色法检测细胞内丙二醛(MDA)水平。结果 AGEs刺激高糖培养的系膜细胞后RAGE表达及RAGEmRNA水平明显增加(P<0.05);MDA水平显著增加(P<0.01)。Gen能有效抑制RAGE及其:mRNA的表达,减少MDA水平,且呈时间和浓度依赖效应,以200μmol／L作用48 h效果最显著(P<0.01)。结论 体外模拟糖尿病环境下,AGEs刺激使。肾系膜细胞内RAGE高表达。Gen可以下调AGEs刺激后系膜细胞RAGE的异常表达,其可能的机制是阻断了氧化应激介导的AGEs-RAGE之间的正反馈途径,表现为细胞内MDA含量减低。     

洛伐他汀对系膜细胞凋亡及Bcl-2和Bax基因表达的影响

目的 观察洛伐他汀对系膜细胞凋亡及Bcl-2和Bax基因表达的影响。并探讨其作用的可能机制。方法 用不同浓度的洛伐他汀处理大鼠系膜细胞，用流式细胞仪观察细胞凋亡指数，吖啶橙荧光活体染色观察细胞凋亡率，电镜观察凋亡细胞形态，免疫印迹法及细胞免疫结合图文分析观察洛伐他汀对系膜细胞Bcl-2和Bax基因表达的影响。结果 洛伐他汀对系膜细胞凋亡有明显诱导作用，呈一定剂量依赖性；并能明显抑制Bcl-2基因表达，促进Bax基因表达，致使两者比例明显下降。结论 洛伐他汀可剂量依赖性地诱导大鼠系膜细胞凋亡，其机制可能通过对凋亡抑制或促进基因的调控，改变Bcl-2/Bax比值而诱导MC的凋亡。     

Advanced glycation end products induce mitochondrial pathway apoptosis in glomerular mesangial cells

Objective To investigate whether advanced glycation end products (AGEs) can induce the expression of Ros, JC-1 and its apoptosis-related proteins in glomerular mesangial cells under high glucose environment, induce apoptosis and injury of glomerular mesangial cells. Methods Rat glomerular mesangial cell line HBZY-1 was cultured in vitro. The cells were cultured with different concentrations of AGEs for 0, 12, 24 and 48 hours respectively. MTT assay was used to observe the cell proliferation ability. After the optimal time and concentration of AGEs were selected, the caspase enzyme inhibitor Z-VAD-fmk and reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC) were cultured and the apoptosis rate was detected by cell death detection apoptosis ELISA plus and Annexin V-FITC/PI kit. JC-1 staining was used to detect the changes of mitochondrial membrane potential (MMP). Cell ROX deep red flow cytometry was used to detect the total ROS level. The expression of anti-apoptotic protein Bcl-2, pro-apoptotic protein BAX, caspase-9, caspase-3 and poly ADP-ribose polymerase (PARP)-activated fragments was detected by Western blotting. Results AGEs could decrease the activity of glomerular mesangial cells in a time and concentration-dependent manner, and induce cell death. The percentage of apoptotic cells in glomerular mesangial cells was significantly increased after treatment with 250 mg/L AGEs for 24 h (P＜0.01), and Z-VAD-fmk could significantly alleviate AGEs-induced glomerular mesangial cell apoptosis (P＜0.01). Compared with the control group, AGEs increased the level of intracellular reactive oxygen species and decreased MMP in a time-dependent manner, and the two time points that AGEs significantly caused the change were 1 h and 2 h (all P＜0.01). AGEs also reduced the expression of antiapoptotic protein Bcl-2 and increased the expression of pro-apoptotic protein Bax, cleaved caspase-9, cleaved caspase-3 and cleaved PARP (all P＜0.01). Compared with AGEs group, NAC could significantly stabilize MMP (P＜0.01), increase Bcl-2 expression (P＜0.01), and decrease the expression of BAX, cleaved caspase-9, cleaved caspase-3 and cleaved PARP (all P＜0.01). Conclusion AGEs induce mitochondrial pathway apoptosis in glomerular mesangial cells by increasing intracellular ROS level and destroying MMP.     

Gentamicin treatment induces simultaneous mesangial proliferation and apoptosis in rats

BACKGROUND: Gentamicin (G)-induced acute renal failure is characterized by an impairment of glomerular function without apparent changes in glomerular structure. However, G stimulates reactive oxygen species (ROS)-mediated mesangial cell proliferation in vitro. We studied whether G promotes mesangial cell apoptosis in vitro, and if apoptosis and proliferation in parallel may occur in glomerular cells in vivo after a renal damage induced by G treatment. METHODS: For in vivo studies, rats were treated with G (100 mg/kg body weight/day) for 6 days, and functional and histologic studies were performed. For in vitro studies, mesangial cell proliferation and apoptosis were evaluated after 24, 48, and 72 hours of 10(-5) mol/L G incubation. RESULTS: After G injections, the number of nuclei per glomerulus did not change, whereas proliferating and apoptotic cell numbers increased. G increases DNA synthesis and cell number in cultured mesangial cells, and increases markedly the apoptotic cell number. ROS scavengers superoxide dismutase and catalase reduce G-induced mesangial cell apoptosis, whereas the incubation with the ROS donor system xanthine plus xanthine oxidase increases apoptosis to levels similar to G. G-induced cellular proliferation and apoptosis either in vitro or in vivo is associated to an early increase in the pro-apoptotic protein Bax and a delayed increase in the survival protein Bcl-2. CONCLUSION: G simultaneously induces proliferation and apoptosis of mesangial cells in vitro and glomerular mesangial cells in vivo. ROS may mediate G-induced mesangial apoptosis in vitro. The equilibrium proliferation/apoptosis may maintain mesangial cell number within normal limits after a G-induced glomerular insult.     

补肾通络方对rIL-1β诱导的大鼠肾小球系膜细胞增殖及TGF-β_1、CTGF的影响

目的：研究补肾通络方含药血浆对重组大鼠白细胞介素-1β（r IL-1β）干预的大鼠肾小球系膜细胞（GMCs）增殖与TGF-β1和CTGF表达的影响,以探讨补肾通络方抗肾脏纤维化的分子作用机制。方法：体外培养GMCs,制备补肾通络方含药血浆,用白细胞介素对GMCs进行干预,MTT比色法检测GMCs增殖、ELISA检测TGF-β1蛋白表达,RT-PCR检测CTGF mRNA的表达。结果：与空白对照组比较,白细胞介素能诱导GMCs的增殖、促TGF-β1生成、上调CTGF表达;补肾通络方含药血浆能下调由白细胞介素介导的GMCs增殖、TGF-β1蛋白和CTGF mRNA的表达量,与白细胞介素组比差异有统计学意义（P〈0.01）,且呈时间依赖性。结论：补肾通络方抗肾脏纤维化作用与抑制TGF-β1和CTGF的表达,调节GMCs的功能密切相关。     

当归补血汤对高糖条件下系膜细胞增殖及NF—κB表达的影响

目的：观察当归补血汤对培养在高糖条件下肾小球系膜细胞（GMC）增殖及GMC中核转录因子－κB（NF—κB）蛋白的表达的影响,探讨其在糖尿病肾病（DN）防治中的意义。方法：（1）利用噻唑蓝（MTT）比色法动态观察各种处理因素的作用下各组GMC增殖情况;（2）用Western blot的方法检测各种处理因素的作用下各组GMC中NF-κB蛋白的表达情况。结果：高糖能明显刺激体外培养的GMC增殖,同时细胞中NF-κB蛋白表达也增加（P〈0．05）。而当归补血汤能明显抑制GMC增殖及细胞中NF出蛋白表达也相应减少（在相同的观察点,与高糖组相比P〈0．01,与其他对照组相比P〈0．05）,并呈一定量效关系,即随糖或药物浓度的增加,GMC增殖及细胞中NF-κB蛋白表达也相应增加或减少。结论：高糖可促进GMC增殖及GMC中NF—κB蛋白的表达的增加,当归补血汤能明显抑制高糖条件下GMC增殖及其细胞中NF-κB蛋白表达的增加,从而发挥预防和治疗肾小球硬化,进而延缓DN的进展。     

尼卡地平对大鼠Thy1系膜增生性肾小球肾炎的作用及其对肾内Bcl-2/BaX表达的影响

目的：研究尼卡地平诱导系膜细胞调亡在大鼠Thy1系膜增生性肾小球肾炎模型（Thy1模型）中的作用及其与Bcl-2/Bax的表达的关系。方法：（1）应用兔抗大鼠Thy1.1血清建立大鼠Thy1模型;（2）建立模型第7d,腹腔注射尼卡地平（6mg/kg),以等量生理盐水为对照;（3）应用浸条法测定尿蛋白;（4）应用原位末端标记法检测细胞凋亡;（5）应用免疫组化检测Bcl-2/Bax蛋白的表达。结果：注射尼卡地平组尿蛋白程度明显减轻,恢复较生理盐水组快,常规HE染色显示尼卡地平组肾小球内有核细胞数较生理盐水组少,同时肾小球内凋亡细胞数也增多,尼卡地平组肾小球内Bcl-2蛋白阳性细胞数减少,而Bax蛋白阳性细胞数无明显变化,且Bcl-2阳性细胞数与肾小球内细胞数呈负相关。结论：尼卡地平通过诱导系调亡减轻大鼠Thy1模型的肾小球损害,其机制可能与Bcl-2蛋白表达减少有关。     

15-Deoxy-Delta12,14-prostaglandin J2 regulates mesangial cell proliferation and death

BACKGROUND: Proliferation of intrinsic glomerular cells is a common response to renal injury. Acutely, proliferation may be beneficial, but sustained glomerular hypercellularity after injury is associated with progressive renal failure. To identify endogenous factors that may be responsible for regulating glomerular cell number, the effects of J-series cyclopentenone prostaglandins (PGs) on human glomerular mesangial cell proliferation and death were examined. METHODS: Human mesangial cells were grown in the presence or absence of PGJ2 or its metabolite 15-Deoxy-Delta12,14-PGJ2 (15dPGJ2). The number of viable cells was measured by the reduction of the tetrazolium MTS to a colored formazan product. Apoptosis was assessed by caspase-3 activation and DNA fragmentation. RESULTS: PGJ2 at concentrations up to 10 micromol/L caused mesangial proliferation. 15dPGJ2 also caused mesangial proliferation at low concentrations (< or =2.5 micromol/L), but induced mesangial cell death at higher concentrations (>5 micromol/L). Cell death occurred in part through apoptosis, measured as an increase in caspase-3 activity and DNA fragmentation in 15dPGJ2-treated cells. Cell death was associated with a decline in baseline phosphorylation of the survival factor Akt and increased Akt degradation, whereas 15dPGJ2-induced mesangial proliferation was blocked by inhibition of the PI 3-kinase/Akt pathway. 15dPGJ2 is a potent PPARgamma agonist. Like 15dPGJ2, treatment of mesangial cells with thiazolidinedione-type PPARgamma ligands (10 to 20 micromol/L) caused significant cell death, but at lower concentrations also caused a small degree of proliferation. CONCLUSIONS: J-series prostaglandins thus may be involved in the initiation of glomerular hypercellularity through Akt-dependent proliferation, and restoration of normal glomerular architecture through PPARgamma-mediated apoptosis. Manipulation of these prostaglandins may be relevant to the treatment of progressive glomerular disease.     

基于NLRP3/caspase-1通路研究丹参提取物对系膜增生性肾小球肾炎大鼠的保护作用

目的基于NLRP3/caspase-1通路探讨丹参提取物对系膜增生性肾小球肾炎(mesangial proliferative glomerulonephritis,MsPGN)大鼠的保护作用。方法取48只大鼠建立MsPGN模型,随机分为4组:(1)模型组:不给予药物治疗;(2)丹参提取物组:每日给予丹参提取物(10 mL/kg)灌胃;(3)VX-765组:每日给予VX-765(NLRP3炎性小体抑制剂,50 mg/kg)腹腔注射;(4)丹参提取物+VX-765组:每日给予丹参提取物(10 mL/kg)灌胃,并给予VX-765(50 mg/kg)腹腔注射。另取12只大鼠设为假手术组。各组均持续给药28 d。末次给药结束24 h后,收集各组大鼠24 h尿液,检测24 h尿蛋白含量;检测各组大鼠血清中BUN、Scr、IL-1β、IL-18水平;观察各组大鼠肾组织病理变化,并根据肾小球系膜细胞增殖程度、肾小管-间质病变程度进行病理评分;采用实时荧光定量PCR及免疫印迹法检测各组大鼠肾组织中NLRP3、caspase-1的mRNA和蛋白表达。结果与假手术组相比,模型组大鼠出现肾小球体积变大、内部细胞数量增多,系膜细胞弥漫性增生、细胞基质增多,肾间质有炎性细胞浸润等肾组织病理损伤症状,肾小球系膜细胞增殖程度评分、肾小管-间质病理评分、24 h尿蛋白含量、血清BUN、Scr、IL-1β、IL-18、肾组织NLRP3及caspase-1表达显著升高(P<0.05);与模型组比较,丹参提取物组、VX-765组、丹参提取物+VX-765组大鼠上述病理损伤症状减轻,肾小球系膜细胞增殖程度评分、肾小管-间质病理评分、24 h尿蛋白含量、血清BUN、Scr、IL-1β、IL-18、肾组织NLRP3及caspase-1表达均显著降低(P<0.05);与丹参提取物组及VX-765组分别比较,丹参提取物+VX-765组大鼠上述病理损伤症状进一步减轻,肾小球系膜细胞增殖程度评分、肾小管-间质病理评分、24 h尿蛋白含量、血清BUN、Scr、IL-1β、IL-18、肾组织NLRP3及caspase-1表达均显著降低(P<0.05)。结论丹参提取物可以减轻MsPGN大鼠炎症反应及系膜增生性病变,保护大鼠肾组织,修复大鼠肾功能,可能通过下调NLRP3/caspase-1通路实现。     

晚期糖基化终末产物与其受体相互作用对足细胞凋亡的影响

目的 探讨可溶性与复合型晚期糖基化终末产物(AGE)与晚期糖基化终末产物受体（RAGE）的相互作用对足细胞凋亡的影响。 方法 以可溶性（CML-BSA、AGE-BSA）和复合型（AGE修正胶原Ⅳ）AGE刺激小鼠足细胞，并用浓度分别为10、50、100 mg/L的AGE刺激细胞，应用TUNEL染色和荧光激活细胞分类（FACS）法来计数凋亡和坏死的足细胞。用RAGE iRNA转染足细胞后，以同样剂量的可溶性和复合型AGE刺激足细胞，观察凋亡情况的改变。 结果 可溶性和复合型AGE均可诱导小鼠足细胞凋亡，复合型AGE引起的足细胞凋亡是可溶性AGE的2~3倍（均P < 0.01）。AGE呈剂量依赖性引起足细胞凋亡。用RAGE iRNA转染足细胞，降低60%~70%RAGE基因活性后，可溶性AGE引起的凋亡率明显下降，复合型AGE诱导的凋亡有下降趋势，但不明显。只有在AGE 100 mg/L刺激后才发生细胞坏死。结论 可溶性AGE主要通过与RAGE相互作用引起足细胞凋亡，复合型AGE部分通过与RAGE相互作用诱导足细胞凋亡。减少AGE生成和RAGE表达可能是预防肾脏病进展的重要途径。     

PDGF signal transduction inhibition ameliorates experimental mesangial proliferative glomerulonephritis

BACKGROUND: Platelet-derived growth factor (PDGF) has been consistently implicated in the cell proliferation and extracellular matrix accumulation, which characterize progressive glomerular disease. In the present study, the effects of a potent and selective inhibitor of PDGF receptor tyrosine kinase, STI 571, were examined in vitro and in vivo. METHODS: Cultured mesangial cells were incubated with PDGF (50 ng/mL) and fibroblast growth factor-2 (FGF-2; 50 ng/mL) and treated with STI 571 (0.13 to 2.0 micromol/L). Experimental mesangial proliferative glomerulonephritis was induced in male Wistar rats with monoclonal OX-7, anti-rat Thy-1.1 antibody with rats randomized to receive either STI 571 (50 mg/kg intraperitoneally daily) or vehicle. Animals were examined six days later. RESULTS: In vitro, both PDGF and FGF-2 induced a threefold increase in mesangial cell 3H-thymidine incorporation. STI 571 reduced PDGF but not FGF-2-stimulated mesangial cell proliferation in a dose-dependent manner, with complete abolition at 0.4 micromol/L. In animals with Thy-1.1 glomerulonephritis, PDGF receptor tyrosine kinase blockade was associated with significant reductions in mesangial cell proliferation (P < 0.001), the number of activated (alpha-smooth muscle positive) mesangial cells, and glomerular type IV collagen deposition (P < 0.001). CONCLUSION: The amelioration of the pathological findings of experimental mesangial proliferative glomerulonephritis by blockade of PDGF receptor activity suggests the potential clinical utility of this approach as a therapeutic strategy in glomerular disease.