黄芪当归合剂对慢性肾病鼠肾脏细胞表型及MAPK信号转导通路的影响

目的　在慢性嘌呤霉素肾病 (PAN)大鼠模型中观察黄芪当归合剂 (A&A)对肾脏固有细胞表型及MAPK信号通路各亚类激酶 (ERK、JNK和p38)表达与活性的影响 ,初步探讨A&A治疗作用的细胞生物学机制。方法　肾组织标本来源于对照组、PAN组、A&A组和ACEI组大鼠。采用常规病理染色和免疫组化技术 ,观察各组肾组织不同部位的病理改变、细胞数量、细胞外基质积聚程度、不同部位α SMA表达变化及肾组织内MAPK表达及活化情况。结果　PAN大鼠肾小球内的α SMA表达量与肾小球系膜细胞增殖、细胞外基质的积聚程度密切相关 ;肾小管间质区的α SMA表达量与肾间质浸润细胞数相关 ,同时伴有肾小管上皮变性、萎缩和扩张。A&A治疗可使PAN大鼠上述部位的α SMA表达和病理变化明显减轻。在本实验条件下 ,PAN组与对照组肾组织内JNK、ERK、p38的蛋白表达量及表达部位无差别 ,肾组织内ERK、p38亦未被激活 ;PAN大鼠肾小球、肾小管及肾间质细胞的JNK均明显活化 ,A&A可明显抑制上述部位的JNK活化。结论　黄芪当归合剂可显著减轻嘌呤霉素肾病的肾脏损伤 ,它对肾脏固有细胞 (尤其是系膜细胞 )表型转化的抑制作用可能是其作用的主要环节之一 ,该作用可能与其下调JNK信号通路活化有关     

Cyclosporin reduces proteinuria in rats with aminonucleoside nephrosis.

The effect of cyclosporin (CS) was assessed in Sprague-Dawley rats with puromycin aminonucleoside (PA) nephrosis induced by a single intraperitoneal injection of PA. Three groups of rats were injected intraperitoneally with CS (10 mg/kg body weight) daily, beginning 1 day before PA administration, or 5 or 10 days after PA administration, for 10 days. CS significantly reduced proteinuria in rats with PA nephrosis in comparison with untreated nephrotic controls. After discontinuation of the CS treatment, proteinuria gradually increased, reaching values similar to those in control nephrotic rats. CS pretreatment did not prevent the induction of PA-induced nephrotic syndrome. Light microscopy and assessment of anionic sites in the glomerular basement membrane revealed no differences between normal rats, nephrotic controls, and CS-treated rats. These results show that CS can reduce proteinuria in PA nephrosis, but cannot ameliorate the glomerular changes.     

CAPTOPRIL MAGNIFIES THE INCREASE IN ANGIOTENSIN I-CONVERTING ENZYME ACTIVITY IN RATS WITH AMINONUCLEOSIDE NEPHROSIS

1. Serum, tissue and urine angiotensin I-converting enzyme (ACE) activity was estimated in the following groups of rats: saline-injected rats (controls); captopril-treated (CAP) control animals (CONTROL-CAP); puromycin aminonucleoside (PAN)-induced nephrotic syndrome (NS); and CAP-treated animals with NS (NS-CAP). 2. Serum ACE activity increased in the CONTROL-CAP, NS, and NS-CAP groups. The increase in the NS-CAP group was significantly higher compared with the NS or CONTROL-CAP groups. 3. In the CONTROL-CAP group, tissue ACE decreased in brain, heart and adrenal glands, and remained unchanged in the lung, testis, kidney, small intestine and liver. In the NS group, tissue ACE activity increased in the lung and testis, decreased in the brain and heart, and remained unchanged in the small intestine, adrenal glands, kidney and liver. Tissue ACE activity increased significantly in the NS-CAP group compared with the other groups. This increase in tissue ACE may contribute to an increase in the serum ACE activity in the NS-CAP group compared with the NS group. 4. Urine ACE activity increased in the NS and NS-CAP groups, although the rise in the NS-CAP group was significantly higher. The urine ACE correlated significantly with the circulating levels of this enzyme in the NS and NS-CAP groups. The loss of ACE in the urine in the presence of an increased serum ACE activity indicates that the biosynthesis of tissue ACE and its release into the bloodstream must be elevated.     

槐耳浸膏对嘌呤霉素氨基核苷所致体外小鼠肾小球足细胞损伤的保护作用及其机制

目的：探讨槐耳浸膏对嘌呤霉素氨基核苷（PAN）处理小鼠体外肾小球足细胞滤过率、活动力和细胞骨架重排的影响,阐明槐耳浸膏对足细胞损伤的保护作用及机制。方法：体外培养的足细胞随机分为对照组、模型组和实验组,其中模型组足细胞以50 mg·L^-1 PAN作用24 h,实验组足细胞经10 g·L^-1槐耳浸膏处理1 h后再换用含50 mg·L^-1 PAN的培养液作用24 h。采用两室弥散系统检测足细胞对异硫氰酸荧光素标记的白蛋白（FITC-BSA）的滤过率,细胞划痕实验检测足细胞划痕修复率,Transwell细胞迁移实验检测穿膜细胞数,激光共聚焦显微镜观察Invitrogen鬼笔环肽直接荧光标记足细胞纤维状肌动蛋白（F-actin）后细胞骨架的重排情况。结果：与对照组比较,模型组足细胞FITC-BSA滤过率明显升高（P<0.01）;与模型组比较,实验组足细胞FITC-BSA滤过率明显降低（P<0.01）。与对照组比较,模型组足细胞划痕修复率和穿膜细胞数均明显升高（P<0.05）;与模型组比较,实验组足细胞划痕修复率和穿膜细胞数均明显降低（P<0.05）。与对照组比较,模型组足细胞F-actin表达水平明显降低（P<0.01）,F-actin重排率明显升高（P<0.01）,足细胞骨架结构紊乱;与模型组比较,实验组足细胞F-actin表达水平明显升高（P<0.01）,F-actin重排率明显降低（P<0.01）,骨架重排情况明显缓解。结论：槐耳浸膏能够降低病理状态下的体外足细胞对牛血清白蛋白的滤过率,其机制可能与槐耳浸膏降低了足细胞活动力,进而改善体外足细胞骨架的重排有关。     

Novel proteomic approach (PUNCH-P) reveals cell cycle-specific fluctuations in mRNA translation

Monitoring protein synthesis is essential to our understanding of gene expression regulation, as protein abundance is thought to be predominantly controlled at the level of translation. Mass-spectrometric and RNA sequencing methods have been recently developed for investigating mRNA translation at a global level, but these still involve technical limitations and are not widely applicable. In this study, we describe a novel system-wide proteomic approach for direct monitoring of translation, termed puromycin-associated nascent chain proteomics (PUNCH-P), which is based on incorporation of biotinylated puromycin into newly synthesized proteins under cell-free conditions followed by streptavidin affinity purification and liquid chromatography-tandem mass spectrometry analysis. Using PUNCH-P, we measured cell cycle-specific fluctuations in synthesis for >5000 proteins in mammalian cells, identified proteins not previously implicated in cell cycle processes, and generated the first translational profile of a whole mouse brain. This simple and economical technique is broadly applicable to any cell type and tissue, enabling the identification and quantification of rapid proteome responses under various biological conditions.     

雷帕霉素对PAN肾病小鼠肾脏病变和VEGF及受体表达的影响

目的 探讨雷帕霉素对嘌呤霉素氨基核苷(PAN)肾病模型小鼠肾脏病变和肾小球VEGF及受体(VEGFR)表达的影响.方法 24只BALB/c小鼠随机分为PAN 组(单次尾静脉注射PAN造模,n=8)、雷帕霉素干预组(单次尾静脉注射PAN造模+雷帕霉素干预,n=8)和对照组(单次尾静脉注射PBS,n=8).各组动物留取24 h尿液,BCA蛋白定量试剂盒测定24 h尿蛋白排泄量.于造模后第10天处死动物取肾脏制备肾组织标本,透射电子显微镜观察各组肾小球足突结构改变;分别采用Real-time PCR、Western b1otting和免疫组织化学方法检测各组肾组织VEGF、VEGFRI、VEGFR2 mRNA以及VEGF、VEGFR2蛋白表达.结景24 h尿蛋白排泄量为PAN组>雷帕霉素干预组>对照组,组间差异均有统计学意义(P<0.05).电子显微镜观察发现PAN 组肾小球上皮细胞足突广泛融合.各组肾组织VEGF、VEGFRI、VEGFR2 mRNA及蛋白表达为PAN组>雷帕霉素干预组>对照组(P<0.05),且各组VEGF、VEGFRI、VEGFR2 mRNA及VEGF蛋白表达与24 h尿蛋白排泄量呈正相关(P<0.05).结论 雷帕霉素能减轻PAN肾病小鼠蛋白尿的程度,作用机制可能与下调肾小球VEGF及其受体基因表达有关.     

柴胡皂甙对肝细胞增殖及基质合成的实验研究

目的研究柴胡皂甙对原代培养肝细胞（ＨＣ）增殖及合成细胞外基质（ＥＣＭ）的作用，为进一步阐明柴胡皂甙防治肝损伤和抗肝纤维化的机理提供实验依据。方法电镜观察、免疫细胞化学及图像分析仪检测ＨＣ内Ⅰ、Ⅲ、Ⅳ型胶原和纤维粘连蛋白（ＦＮ）含量，流式细胞仪测定ＨＣ内的ＤＮＡ含量和３Ｈ－脯氨酸掺入量未检测ＨＣ内胶原蛋白总量。结果ＴＫＣｎｃｍ各组对细胞损伤得到改善，各实验组ＨＣ内３Ｈ－脯氨酸掺入量均明显升高，各治疗组ＨＣ内ＤＮＡ含量显著升高，而３Ｈ－脯氨酸掺入量均呈现降低趋势。ＨＣ内Ｉ型胶原含量明显减少，其余各组基质成分含量呈下降趋势。结论柴胡皂甙对肝细胞具有保护作用；表现为与相应实验组ＨＣ内ＤＮＡ含量内呈上升趋势；胶原蛋白总量及Ⅰ、Ⅲ、Ⅳ型胶原和ＦＮ含量及其合成受到抑制，从而抑制了ＨＣ对ＥＣＭ的合成     

Ultrastructural study on nephrin expression in experimental puromycin aminonucleoside nephrosis.

BACKGROUND: Nephrin is a recently identified protein that is a key component of the slit diaphragm. This protein may play a crucial role in maintaining the glomerular filtration barrier, and mutations in the gene for nephrin reportedly lead to congenital nephrosis. However, the expression of nephrin in acquired glomerular disease has not yet been fully clarified. To address this issue, we analysed the expression and localization of nephrin by morphological analysis based on immunoelectron microscopy in normal glomeruli and in glomeruli from proteinuric experimental models. METHODS: Twenty rats were divided into three experimental groups (n = 16 total) and a control group (n = 4). Rats in the experimental groups received a single intravenous injection of puromycin aminonucleoside (PAN), and were sacrificed at 1 (n = 4), 2 (n = 6) and 3 weeks (n = 6) post-injection. Nephrin expression was assessed by immunoelectron microscopy using a polyclonal antibody against nephrin and gold particles. It was quantified by counting the gold particles and the slit diaphragms and by measuring the average foot process width in microphotographs. RESULTS: The average foot process width in the 1 week group (5924.5 +/- 1523.9 nm) was far greater than that of controls (1112.9 +/- 79.8 nm), but decreased thereafter. The average number of total gold particles per unit length (10 000 nm) of the glomerular basement membrane (GBM) underlying the foot processes was reduced at 1 week (26.0 +/- 9.5), compared with controls (335.3 +/- 125.5), but increased thereafter. Also, the average number of junctional gold particles per unit length of the GBM was lower than controls (208.4 +/- 1.7) at 1 week (10.1 +/- 3.5), but increased thereafter. There were no significant differences between the numbers of junctional gold particles per slit diaphragm among the groups, but significant differences were observed in the distributions of gold particles among the groups. Gold particles were more frequently seen in cytoplasm at 1 week. CONCLUSIONS: The present ultrastructural studies showed that nephrin expression and its distribution were altered in PAN-treated rats, and this occurred in parallel with foot process effacement. Nephrin expression returned to normal with improved resolution of the effacement. Nephrin expression was found to be rather preserved in areas without foot process effacement, even in PAN-treated rats. The significance of the above findings in terms of proteinuria and foot process effacement needs further clarification.     

Differential effects of probucol on two distinct experimental rat nephrosis models

We compared an antiproteinuric effect of a lipid‐lowering agent probucol on two distinct types of experimental nephrosis in rats, i.e. mercuric–chloride (HgCl₂)‐induced autoimmune glomerulonephritis in Brown Norway (BN) rats and puromycin aminonucleoside (PA)‐nephrosis in Wistar rats. The rats were fed either standard rat chow or chow containing 5% probucol. BN rats were treated with HgCl₂ according to a standard protocol (HgCl₂ 1 mg/kg subcutaneously three times/week). Probucol treatment did not ameliorate proteinuria, renal histology or a strong linear staining for IgG and an intercellular adhesion molecule, ICAM‐1, in glomeruli. Wistar rats were made nephrotic with an intraperitoneal injection of PA (100 mg/kg). In this model, in contrast to the BN rat model, no glomerular deposition of IgG or ICAM‐1 was observed. Probucol treatment ameliorated proteinuria significantly. These findings suggest that the response to probucol differs in different experimental nephrosis models. As probucol only affects PA‐nephrosis, in which ICAM‐1 expression is negative, the ICAM‐1 attenuation is not likely to be involved in the antiproteinuric effect of probucol.