Double deficiency of cathepsin B and L in the mouse pancreas alters trypsin activity without affecting acute pancreatitis severity

BackgroundPremature intracellular trypsinogen activation has long been considered a key initiator of acute pancreatitis (AP). Cathepsin B (CTSB) activates trypsinogen, while cathepsin L (CTSL) inactivates trypsin(ogen), and both proteins play a role in the onset of AP.MethodsAP was induced by 7 hourly intraperitoneal injections of cerulein (50 μg/kg) in wild-type and pancreas-specific conditional Ctsb knockout (Ctsb^Δpan), Ctsl knockout (Ctsl^Δpan), and Ctsb;Ctsl double-knockout (Ctsb^Δpan;Ctsl^Δpan) mice. Pancreatic samples were collected and analyzed by histology, immunohistochemistry, real-time PCR, and immunoblots. Trypsin activity was measured in pancreatic homogenates. Peripheral blood was collected, and serum amylase activity was measured.ResultsDouble deletion of Ctsb and Cstl did not affect pancreatic development or mouse growth. After 7 times cerulein injections, double Ctsb and Ctsl deficiency in mouse pancreases increased trypsin activity to the same extent as that in Ctsl-deficient mice, while Ctsb deficiency decreased trypsin activity but did not affect the severity of AP. Ctsb^Δpan;Ctsl^Δpan mice had comparable serum amylase activity and histopathological changes and displayed similar levels of proinflammatory cytokines, apoptosis, and autophagy activity compared with wild-type, Ctsb^Δpan, and Ctsl^Δpan mice.ConclusionDouble deletion of Ctsb and Ctsl in the mouse pancreas altered intrapancreatic trypsin activity but did not affect disease severity and inflammatory response after cerulein-induced AP.     

蓝莓花青素对人胃腺癌BGC-823细胞增殖、侵袭的影响及机制研究

目的:探讨蓝莓花青素(BA)对人胃腺癌BGC-823细胞增殖、侵袭的影响及相关机制。方法:低、中、高剂量实验组与对照组人胃腺癌BGC-823细胞分别以25、50、100μg/mL BA与等量生理盐水处理,分别处理12 h、24 h、48 h。采用噻唑蓝(MTT)与流式细胞仪检测人胃腺癌BGC-823细胞增殖与凋亡;采用Transwell小室实验与免疫细胞化学法检测人胃腺癌BGC-823细胞侵袭与P53、半胱氨酸蛋白酶-3(Caspase-3)蛋白表达。结果:干预48 h时,对照组、低、中、高剂量实验组人胃腺癌BGC-823细胞P53与Caspase-3蛋白阳性细胞百分比分别为(11.12±1.01)%,(24.31±2.18)%,(47.56±2.91)%,(66.49±5.14)%与(15.04±1.23)%,(28.22±3.09)%,(69.36±5.42)%,(86.52±6.15)%。实验组胃腺癌BGC-823细胞增殖抑制率随BA浓度增大而逐渐升高;与对照组比较,低、中、高剂量实验组人胃腺癌BGC-823细胞凋亡率、P53与Caspase-3蛋白阳性细胞百分比均显著升高,侵袭率降低,呈浓度依赖性(均P0.05)。结论:BA可有效抑制胃腺癌BGC-823细胞增殖,诱导其凋亡,并降低侵袭能力,其机制可能与上调P53与Caspase-3蛋白表达有关。     

Oxidatively modified glyceraldehyde-3-phosphate dehydrogenase in neurodegenerative processes and the role of low molecular weight compounds in counteracting its aggregation and nuclear translocation

A number of independent studies have shown the contribution of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the pathogenesis of several neurodegenerative disorders. Indeed, GAPDH aggregates have been found in many post-mortem samples of brains of patients diagnosed with Alzheimer’s and Parkinson disease. Currently, it is accepted that GAPDH-mediated cell death pathways in the neurodegenerative processes are associated with apoptosis caused by GAPDH nuclear translocation and excessive aggregation under oxidative stress conditions. Also the role of GAPDH in neurodegenerative diseases is linked to it directly binding to specific amyloidogenic proteins and petides such as β-amyloid precursor protein, β-amyloid peptide and tau protein in Alzheimer’s disease, huntingtin in Huntington’s disease and α-synuclein in Parkinson disease. One of the latest studies indicated that GAPDH aggregates significantly accelerate amyloidogenesis of the β-amyloid peptide, which implies that aggregates of GAPDH may act as a specific aggregation “seed” in vitro.Previous detailed studies revealed that the active-site cysteine (Cys152) of GAPDH plays an essential role in the oxidative stress-induced aggregation of GAPDH associated with cell death. Furthermore, oxidative modification of this cysteine residue initiates the translocation of the enzyme to the nucleus, subsequently leading to apoptosis. The crystallographic structure of GAPDH shows that the Cys152 residue is located close to the surface of the molecule in a hydrophilic environment, which means that it can react with low molecular weight compounds such as hydroxynonenal or piceatannol. Therefore, it is highly possible that GAPDH may serve as a target for small molecule compounds with the potential to slow down or prevent the progression of neurodegenerative disorders.Recently appearing new evidence has highlighted the significance of low molecular weight compounds in counteracting the oxidation of GAPDH and consequently its aggregation and other unfavourable pathological processes. Hence, this review aims to present all recent findings concerning molecules that are able to interact with GAPDH and counteract its aggregation and translocation to the nucleus.     

血清胱抑素C在动态监测移植肾功能中的价值

目的探讨血清胱抑素C（ScysC）在监测肾脏移植患者肾小球滤过率中的应用价值。方法收集26例肾移植病例，分别于术前、术后1、3、5、7d，2周，4周采集血标本，分别测定ScysC、血清肌酐（Scr）、血清尿素（Urea）、内生肌酐清除率（CCr），比较ScysC、Scr与CCr的相关性。结果患者术后随着移植肾功能的恢复，ScysC值持续下降，与术前相比，差异有显著统计学意义（P〈0．01）。与术前相比，术后第1天，ScysC下降达51％，明显早于Scr（下降达40％），移植术前与术后不同时期，患者的ScysC与Scr呈正相关，r=0．945；与CCr呈负相关，r=-0．878。当50ml·min-1．73m。〈CCr≤80ml·min-1．73m-时，ScysC与CCr的相关性明显优于Scr，二者的相关系数分别为-0．856、-0．645（P〈0．05）。其ROC曲线下的面积分别为0．972±0．032、0．926±0．055（P〈0．05）。结论ScysC检测能比较准确评价移植肾的肾小球滤过率，优于Scr，具有临床应用价值。     

Tariquidar inhibits P-glycoprotein drug efflux but activates ATPase activity by blocking transition to an open conformation

P-glycoprotein (P-gp, ABCB1) is a drug pump that confers multidrug resistance. Inhibition of P-gp would improve chemotherapy. Tariquidar is a potent P-gp inhibitor but its mechanism is unknown. Here, we tested our prediction that tariquidar inhibits P-gp cycling between the open and closed states during the catalytic cycle. Transition of P-gp to an open state can be monitored in intact cells using reporter cysteines introduced into extracellular loops 1 (A80C) and 4 (R741C). Residues A80C/R741C come close enough (<7 Å) to spontaneously cross-link in the open conformation (<7 Å) but are widely separated (>30 Å) in the closed conformation. Cross-linking of A80C/R741C can be readily detected because it causes the mutant protein to migrate slower on SDS-PAGE gels. We tested whether drug substrates or inhibitors could inhibit cross-linking of the mutant. It was found that only tariquidar blocked A80C/R741C cross-linking. Tariquidar was also a more potent pharmacological chaperone than other P-gp substrates/modulators such as cyclosporine A. Only tariquidar promoted maturation of misprocessed mutant F804D to yield mature P-gp. Tariquidar interacted with the transmembrane domains because it could rescue a misprocessed truncation mutant lacking the nucleotide-binding domains. These results show that tariquidar is a potent pharmacological chaperone and inhibits P-gp drug efflux by blocking transition to the open state during the catalytic cycle.     

甲钴胺对脑缺血-再灌注大鼠皮层神经细胞凋亡的预防作用

目的 探讨甲钴胺对缺血-再灌注(I-R )大鼠大脑皮层神经细胞凋亡的影响.方法Wistar大鼠135只随机均分为五组 :A组为空白对照 ;B、C、D和 E组分别用0.9% 氯化钠20 ml · kg-1 ·d-1 、尼莫地平1mg·kg-1 ·d-1 、甲钴胺50μg·kg-1 ·d-1和100μg·kg-1 ·d-1灌胃1周后建立脑I-R模型.观察再灌注24 h后神经功能缺损评分.再灌注6、12和24 h ,用T U N EL法检测大脑皮层细胞凋亡 ,Western blot检测大脑皮层半胱氨酸天冬氨酸酶3(Caspase-3)蛋白表达.结果 再灌注24 h后 ,C、E组神经功能评分低于B、D组( P＜0 .05或P＜0 .01 ).C、D和E组神经细胞凋亡数均少于B组 ,E组少于D组(P＜0 .01).C、D和E组大脑皮层Caspase-3表达均低于B组 ,D组高于C组(P＜0 .01).结论 预用甲钴胺对大鼠脑I-R损伤有保护作用 ,可能与抑制大脑组织Caspase-3蛋白表达有关.     

老年帕金森病患者血清同型半胱氨酸及血尿酸水平与认知功能障碍的相关性

目的探讨血清同型半胱氨酸（Hcy）和血尿酸（UA）的水平与帕金森病（PD）患者认知功能障碍的相关性。方法检测58例帕金森病患者和50例健康老人的血清同型半胱氨酸及血尿酸水平,采用简易精神状态检查（MMSE）量表对老年 PD 患者进行认知功能评分。结果老年帕金森病组的 Hcy 浓度[（25．89±3．01）μmol／L]显著高于健康老年组[（13．29±2．13）μmol／L]（P ＜0．01）;血尿酸水平显著低于健康老年组[（267．34±68．43）μmol／L 比（341．26±52．75）μmol／L,P ＜0．01],并且 PD 伴认知障碍组的 Hcy 浓度显著高于 PD 无认知障碍组[（29．54±4．35）μmol／L 比（20．78±3．17）μmol／L ,P ＜0．05],PD 伴认知障碍组的血尿酸水平显著低于 PD 无认知障碍组[（245．10±55．61）μmol／L 比（302．21±52．31）μmol／L ,P ＜0．05]。结论老年帕金森病患者会出现血清同型半胱氨酸水平升高及血尿酸水平的降低,其变化与程度认知功能障碍的发生有一定关联度。     

甲状腺癌组织中Caspase-9、Livin表达的临床意义

目的探讨甲状腺癌组织中半胱氨酸天冬氨酸蛋白酶(caspase)-9和凋亡抑制蛋白因子(Livin)表达的临床意义。方法检测65例甲状腺癌组织和40例正常甲状腺组织中Caspase-9、Livin表达情况,分别比较Caspase-9、Livin在两种不同组织中表达的阳性率,分析Livin、Caspase-9在甲状腺癌组织中表达情况与临床病理因素的关系,应用Spearman等级相关检验分析Livin、Caspase-9在甲状腺癌组织表达的相关因素。结果 Livin、Caspase-9阳性染色主要位于细胞质内,表现为小片状分布的棕黄色或黄褐色颗粒;甲状腺癌组织中Caspase-9阳性表达率低于正常甲状腺组织(χ2=38.149,P=0.000),Livin阳性表达率高于正常甲状腺组织(χ2=43.161,P=0.000);Livin、Caspase-9在甲状腺癌组织中的表达与患者的年龄、性别及肿瘤的大小、病理类型无关(P0.05),而与肿瘤的临床分期、淋巴转移和包膜浸润有关(P0.05);Livin、Caspase-9在甲状腺癌组织中的表达呈负相关(r=-0.346,P0.05)。结论甲状腺癌组织中Caspase-9表达下调,Livin表达升高,二者的异常表达参与甲状腺癌的发生和发展过程,Livin、Caspase-9联合检测可作为判断甲状腺癌患者预后的重要参考指标。     

血浆同型半胱氨酸与冠状动脉病变程度的相关性研究

【目的】探讨血浆同型半胱氨酸（Hcy）与冠状动脉病变程度的相关性。【方法】检测2014年1～7月在湖南省人民医院行冠脉造影的126例患者血浆 Hcy水平,利用Gensini评分系统对患者的冠脉病变程度进行评估。根据造影结果设为冠心病（CHD）组（90例）,包括稳定型心绞痛（SAP）组（40例）及非ST段抬高型急性冠脉综合征（NSET‐ACS ）组（50例）,另设为正常对照组（36例）;根据 Hcy水平＞10μmol／L ,设为高同型半胱氨酸（HHcy）组（94例）及同型半胱氨酸正常（非HHcy）组（32例）;利用直线相关分析,探讨 Hcy水平与Gensini评分的相关性。【结果】 CHD组的 Hcy水平明显高于对照组（ P ＜0．01）,Hcy水平随Gensini评分升高而升高,两者呈正相关。H Hcy组的Gensini评分显著高于非 H Hcy组（ P ＜0．01 ）,三支及双支病变组Hcy水平显著高于单支病变组（ P ＜0．01）。【结论】血浆 Hcy水平与冠状动脉病变程度呈正相关。     

糖尿病肾病同型半胱氨酸水平与炎症因子的相关性分析

目的探讨糖尿病肾病（DN）患者同型半胱氨酸水平（Hcy）及其与炎症因子的关系。方法在本院就诊的2型糖尿病（T2DM ）的患者120例,按DN不同进展期分为：DM正常白蛋白尿组（DM A组）;早期糖尿病肾病组（DMB组）;临床糖尿病肾病组（DMC组）各40例。另以同期健康体检正常者40例为对照组。检测四组研究对象血浆 Hcy、高敏 C 反应蛋白（hs‐CRP）、肿瘤坏死因子‐α（TNF‐α）、白介素‐6（IL‐6）水平。结果与健康对照组比较,DN 患者的血浆 Hcy水平、炎症因子指标明显升高,差异有统计学意义（ P ＜0．01）;随着DN尿蛋白的升高,血浆 Hcy及hs‐CRP、TNF‐α、IL‐6水平显著递增,差异有统计学意义（ P ＜0．01）;DN患者血浆Hcy升高与hs‐CRP、TNF‐α、IL‐6的水平呈正相关（ r分别为0．420、0．461、0．429,P ＜0．01）。结论高Hcy血症及炎症因子参与了DN的发生和发展过程,Hcy可能通过促进机体炎症状态的产生促进DN的发生和发展。