饰胶蛋白聚糖对大鼠肾系膜细胞生长的抑制作用及其信号转导途径的研究

目的 探讨饰胶蛋白聚糖(DCN)对大鼠系膜细胞(MsC)生长的抑制作用及其信号转导分子MAPKs和p21表达的影响&#65377;方法 经脂质体介导将DCN基因转染体外培养的大鼠MsC,筛选和鉴定后,收集阳性细胞克隆的培养上清(DCN上清),将其加入正常MsC的培养液中, 采用流式细胞仪检测细胞周期&#65377;用Western 印迹法分别检测MAPKs,包括细胞外调节激酶(ERK)1/2&#65380;应激活化蛋白激酶(SAPK)/氨基末端激酶(JNK)和p38和p21蛋白表达;用免疫荧光法观察p21在细胞中的表达&#65377;结果 DCN上清明显抑制正常MsC的增殖, G2-M期细胞数明显减少,仅为对照组的35%(P < 0.05); 磷酸化ERK1/2及SAPK/JNK表达增强, 分别为对照组的2.2倍&#65380; 1.4倍及1.7倍&#65380; 1.8倍;磷酸化p38无明显变化&#65377;DCN抗体呈浓度依赖性抑制磷酸化ERK1/2&#65380; SAPK/JNK的表达上调&#65377;DCN上清还可使细胞p21的表达明显增强,而DCN抗体也同样呈浓度依赖性地抑制其上调表达的作用, ERK1/2 及SAPK/JNK通路抑制剂U0126和circumin均可抑制其上调表达的作用,分别为对照组的64%和61%;而p38通路抑制剂SB203580则对其无影响&#65377;结论 DCN对肾MsC的生长有抑制作用,其机制可能经ERK1/2&#65380;SAPK/JNK和p21蛋白介导&#65377;     

白蛋白诱导的鼠肾小管细胞凋亡与牛磺酸的抗凋亡作用

目的：观察白蛋白是否诱导鼠肾小管上皮细胞凋亡和牛磺酸对此过程的影响，探讨其信号传导机制。方法：将培养的大鼠肾小管细胞（NRK-52E）与不同浓度的去脂无内毒素牛血清白蛋白（BSA）共同孵育6、12、18、24h。然后用电镜、共聚焦激光显微系统和流式细胞仪检测细胞凋亡；将不同浓度的牛磺酸与NRK-52E细胞孵育1h后加入30mg／ml的白蛋白（BSA）再孵育12h，然后以流式细胞仪检测细胞凋亡，以Westenblot法测定p38、JNK和ERK磷酸化水平。结果：白蛋白以时间和剂量依赖方式导致肾小管细胞凋亡；牛磺酸使白蛋白诱导的NRK-52E细胞凋亡率减少，呈剂量依赖性；牛磺酸以剂量依赖方式降低p38和JNK磷酸化水平，增加ERK磷酸化水平。结论：白蛋白以时间和剂量依赖方式诱导肾小管细胞凋亡；牛磺酸通过抑制白p38和JNK磷酸化，促进ERK磷酸化拮抗白蛋白诱导的肾小管细胞凋亡。     

代谢综合征及其代谢因子与慢性肾损害相关性的临床研究

目的 探讨代谢综合征及其各组成因子与包括轻度肾损害在内的慢性肾损害的相关性&#65377; 方法 收集我院2003年1月至2003年12月心内科&#65380; 肾内科和内分泌科符合入选标准的住院患者966例进行回顾性分析&#65377;按有&#65380; 无慢性肾脏病(CKD)或轻度肾损害分组, 比较代谢综合征各因素与慢性肾脏损害的关系&#65377; 统计学处理包括单变量t检验&#65380;卡方检验和Logistic多因素回归分析&#65377;结果 (1)CKD组的年龄&#65380; 身体质量指数(BMI)&#65380; 总胆固醇(TC)&#65380; 甘油三酯(TG)&#65380; 高血糖&#65380; 高血压和尿酸水平, 冠心病&#65380; 脑卒中的患病率均高于无CKD组, 而高密度脂蛋白(HDL)水平明显低于无CKD组患者; (2)随着代谢综合征因子数量的增多, CKD发病率上升; (3)代谢综合征中各因子并存较各因子单独存在的CKD的危险性增加, 与高血糖并存的频率最高; (4)BMI增加也是CKD的重要危险因素; (5)高血糖患者发生轻度肾损害的风险最大(优势比OR=7.698)&#65377;结论 代谢综合征及其各组成因子是包括轻度肾损害在内的CKD的重要危险因素&#65377;随着代谢综合征因子的增多, CKD的危险也随之增加&#65377; 除了高血糖和高血压, BMI增加也是其中重要的影响因子&#65377;     

糖基化终产物对肾间质成纤维细胞DNA损伤的影响

目的 探讨糖基化终产物(AGE)对肾间质成纤维细胞DNA损伤的影响及抗氧化剂的干预作用&#65377; 方法 不同浓度AGE修饰的牛血清白蛋白(AGE-BSA)作用于NRK-49F细胞24 h,普通培养基和牛血清白蛋白 (BSA)作为对照&#65377;N-乙酰半胱氨酸(NAC)预处理细胞以观察抗氧化剂的干预作用&#65377;AlamarBlue还原法测定细胞增殖活力,单细胞凝胶电泳(彗星实验)测定细胞DNA损伤&#65377; 结果 与对照组比较,AGE作用后的细胞增殖活力下降;DNA迁移距离(彗星尾长)增加,差异有统计学意义,且2者呈剂量依赖关系&#65377;各浓度BSA作用后对细胞增殖活力无明显影响;彗星尾长无明显变化&#65377;与相同浓度BSA相比, 400&#65380;800 mg/L AGE分别使AlamarBlue还原率下降14%和15%(P < 0.05);200&#65380;400&#65380;800 mg/L AGE组彗星尾长分别为BSA组的1.45&#65380;2.12&#65380;2.71倍(P < 0.05)&#65377;与未用NAC预处理组相比,NAC预处理可使AlamarBlue还原率上升[(45.15±0.93)% 比(38.40±0.81)%,P < 0.05];彗星尾长变短[(10.02±4.54) μm比(13.48±5.32) μm,P < 0.05]&#65377; 结论 AGE可导致肾间质成纤维细胞DNA损伤,使用抗氧化剂可有效减轻AGE导致的DNA损伤&#65377;细胞内氧化应激增强可能是AGE引起肾间质成纤维细胞DNA损伤的作用机制之一。     

脱氢抗坏血酸对高糖诱导肾小管上皮细胞产生氧自由基的影响

目的 探讨脱氢抗坏血酸(DHA)对高糖诱导肾小管上皮细胞产生氧自由基的影响&#65377;方法 (1)传代培养肾小管上皮细胞;(2)以Fe3+还原法检测细胞内抗坏血酸(AA)浓度,观察肾小管上皮细胞摄取AA和DHA的情况及葡萄糖和葡萄糖转运蛋白(GLUT)抑制剂细胞松弛素B(cytochalasin B)对其影响;(3)激光扫描共聚焦显微镜检测细胞内氧自由基,观察高糖诱导肾小管上皮细胞氧自由基产生的情况及不同浓度DHA对其影响&#65377;结果 (1)AA不能由细胞外进入肾小管上皮细胞,而DHA可以进入,并且随着细胞外葡萄糖浓度的增加,其进入速度减慢&#65377;细胞松弛素B则完全抑制了DHA进入到肾小管上皮细胞, 而固定葡萄糖浓度(25 mmol/L)时,随着DHA浓度的增加,DHA进入细胞内的速度逐渐增快&#65377;(2)高糖快速诱导了肾小管上皮细胞氧自由基产生增多,DHA抑制了高糖的这种作用,并且该抑制作用在浓度小于&#65380;等于4 mmol/L的范围内呈浓度依赖性&#65377;结论 (1)肾小管上皮细胞是依赖DHA利用VitC的细胞型,DHA进入该细胞依赖GLUT介导,高糖可抑制其进入细胞;(2)DHA可有效抑制高糖诱导的肾小管上皮细胞氧自由基产生增多,并在一定范围内呈浓度依赖性&#65377;     

依贝沙坦抑制造影剂诱导的肾小管上皮细胞凋亡

目的 探讨依贝沙坦在造影剂诱导肾小管上皮细胞凋亡中的作用及机制。方法 培养的大鼠肾小管细胞(NRK-52E)分别与不同碘浓度(25、50、100、150 mgI/ml)的安射力(非离子型造影剂)共孵育1 h;安射力(100 mgI/ml)先后刺激NRK-52E细胞0.5、1、2、4 h。与安射力(100 mgI/ml)相同渗透浓度的甘露醇(420 mmol/L)与NRK-52E细胞共孵育1 h作为阳性对照。不同浓度依贝沙坦(0.01、0.1、1 mmol/L)先与NRK-52E细胞共孵育1 h后,安射力(100 mgI/ml)刺激1 h。Hoechst染色和流式AnnexinV-FITC/PI双染法检测细胞凋亡。激光共聚焦显微镜检测细胞内活性氧(ROS)水平。RT-PCR检测Bax、Bcl-2 mRNA的表达。 结果 安射力呈浓度和时间依赖性诱导NRK-52E细胞凋亡。与安射力相同渗透浓度的甘露醇不能明显诱导细胞凋亡。安射力与NRK-52E细胞共孵育后,细胞内ROS产生增多,Bcl-2 mRNA的表达下调,Bax mRNA的表达上调。依贝沙坦呈浓度依赖性抑制安射力诱导的NRK-52E细胞凋亡、细胞内ROS的产生、Bcl-2 mRNA表达下调及Bax mRNA表达上调。细胞内ROS水平与细胞凋亡呈正相关。 结论 安射力呈浓度和时间依赖性诱导NRK-52E细胞凋亡,此作用与细胞内氧化应激及bcl-2 mRNA表达下调、Bax mRNA表达上调相关。依贝沙坦能抑制上述安射力的作用,呈浓度依赖性抑制NRK-52E细胞凋亡。     

肝细胞生长因子负性调控细胞转分化在肾间质纤维化中的作用

目的 研究肝细胞生长因子(HGF)对单侧输尿管梗阻(UUO)大鼠肾间质纤维化的保护作用及其可能机制&#65377;方法 大鼠随机分为UUO组&#65380;HGF治疗组和假手术组&#65377;用实时荧光定量RT-PCR&#65380;Western杂交和免疫组化检测术后大鼠肾组织结缔组织生长因子(CTGF)和骨形成蛋白7(BMP7)表达量&#65377;免疫组化检测大鼠肾组织TGF-β1&#65380;FN及α-SMA表达&#65377;结果 与假手术组相比,UUO组及HGF治疗组CTGF mRNA&#65380;TGF-β1&#65380;α-SMA&#65380;FN&#65380;CTGF蛋白表达均增高,且UUO组明显高于治疗组;UUO组及HGF治疗组BMP7 mRNA和蛋白表达均减少,且UUO组显著低于治疗组&#65377;结论 HGF能减轻肾间质纤维化,负性调控肾小管上皮细胞-肌成纤维细胞转分化,调节CTGF及BMP7表达可能是其作用途径&#65377;     

造影剂激活p38信号通路诱导肾小管细胞凋亡

目的探讨丝裂素激活蛋白激酶(MAPK)信号通路在造影剂诱导的肾小管细胞凋亡中的作用。方法培养大鼠肾小管细胞(NRK-52E),分别加入不同种类和浓度的造影剂刺激,部分实验同时加入SB203580(p38MAPK抑制剂)。台盼蓝排除试验检测细胞膜完整性。流式细胞仪DNA分析、FITC-AnnexinⅤbinding/PI染色以及电镜扫描检测细胞凋亡。Western印迹分析和免疫沉淀法检测MAPK的磷酸化水平和活性。结果高渗性造影剂泛影葡胺可诱导NRK-52E细胞凋亡,并且,在一定的渗透浓度范围内,随着渗透浓度的升高,细胞凋亡率越高;在一定的时间范围内,随着时间的延长,细胞凋亡率也越高。低渗性造影剂欧乃派克在实验浓度范围内则不能诱导NRK-52E细胞凋亡。泛影葡胺刺激NRK-52E细胞15min后,p38MAPK磷酸化水平及活性便明显增加,并持续至60min,120min时磷酸化水平及活性明显降低,几乎回到零水平。总的p38MAPK水平在整个实验期间内几乎无变化。SB203580可明显抑制泛影葡胺诱导的NRK-52E细胞凋亡。p44/p42MAPK的磷酸化水平在整个实验阶段则无明显变化。结论造影剂呈渗透浓度和时间依赖性诱导NRK-52E细胞凋亡,造影剂诱导细胞凋亡与其高渗有关。p38MAPK信号通路的激活介导了造影剂诱导的NRK-52E细胞凋亡。     

通过基因敲低探讨多个足细胞分子作用及分子间反应

目的 研究足细胞裂孔隔膜(SD)复合体分子nephrin&#65380;podocin和CD2AP,以及足细胞骨架蛋白α-辅肌动蛋白(actinin)-4的作用及分子间反应&#65377;方法 针对nephrin&#65380; podocin&#65380;CD2AP和α-actinin-4 mRNA序列分别设计并构建2个特异RNA干扰质粒-psiRNA-hH1GFPzeo,分别导入小鼠足细胞系MPC5以 “敲低”其表达&#65377;免疫荧光染色观察其分布方式&#65377;半定量RT-PCR和免疫蛋白印迹检测其mRNA和蛋白表达&#65377; 结果 (1) podocin敲低组(siPod966和siPod54):未检测到podocin及nephrin mRNA,其蛋白分别下降了92%&#65380;79%及82%&#65380;67%&#65377;而CD2AP mRNA和蛋白分别增加了62%&#65380;42%及71%&#65380;46%&#65377;α-actinin-4无变化&#65377;(2)nephrin敲低组(siNep492):未检测到nephrin mRNA和蛋白&#65377;而CD2AP mRNA和蛋白分别增加了35%&#65380;48%&#65377;Podocin和α-actinin-4无变化&#65377;(3)CD2AP敲低组(siCda744和siCda21):未检测到CD2AP mRNA,其蛋白分别下降了92%和83%&#65377;Nephrin mRNA和蛋白分别下降了60%&#65380;48%及76%&#65380;72%;而podocin mRNA和蛋白分别增加了38%&#65380;22%及56%&#65380;44%&#65377;Α-actinin-4无变化&#65377;(4)α-actinin-4敲低组(siAct1790和siAct319):α-actinin-4和nephrin 的mRNA分别下降了69%&#65380;58%及64%&#65380;49%;蛋白分别下降了81%和55%以及71%&#65380;64%&#65377;而podocin以及CD2AP mRNA分别增加了50%&#65380;34%及45%&#65380;28%;蛋白分别增加了64%&#65380;46%及65%&#65380;42%&#65377;(5)敲低nephrin&#65380;podocin和CD2AP后,这些表达量降低的分子的分布发生了明显改变,即以核周为主;而相应分子敲低后引起的podocin和CD2AP表达增加,其分布亦主要以核周染色增强为主&#65377;α-actinin-4即使表达降低,分布亦无变化,仍呈细丝状分布于胞质及足细胞伸出的突起中&#65377;结论 (1)在SD复合体分子中,nephrin可能具有相对独立的作用&#65377;(2) α-actinin-4对nephrin&#65380;podocin和CD2AP有直接或间接的作用&#65377;(3)足细胞分子间的作用和联系不总是“一致的”,可能是“单向的”&#65380;也可能是“双向的”&#65377;(4)nephrin&#65380;podocin&#65380;CD2AP和α-actinin-4在足细胞的分布有赖于其表达量的正常及正常的分子间反应&#65377;     

两种病理类型肾病患者尿IgG对人肾小管上皮细胞表达巨噬细胞移动抑制因子的影响

目的 分离纯化表现为肾病综合征(NS)的微小病变型(MCD)及膜性肾病(MN)患者尿IgG，比较它们对人近端小管上皮细胞(HK-2)表达巨噬细胞移动抑制因子(MIF)的影响。方法 采用硫酸铵沉淀&#65380;蛋白G亲和层析纯化尿中IgG，并经SDS-PAGE、Western印迹分析鉴定&#65377;用不同浓度(0&#65380;0.5&#65380;1.0&#65380;2.5&#65380;5.0&#65380;10.0 mg/ml)的上述两种患者的尿IgG分别刺激HK-2细胞6 h，应用RT-PCR检测细胞表达MIF mRNA的变化;应用Western印迹检测细胞中MIF的蛋白水平&#65377; 结果 纯化的尿IgG经SDS-PAGE分析显示其分解为4个片段,以兔抗人IgG抗体进行免疫印迹鉴定，证实这些蛋白条带均为IgG成分&#65377; 两种不同病理类型NS患者的尿IgG均可上调HK-2细胞MIF 的基因及蛋白表达，并呈剂量依赖性&#65377;MN患者的尿IgG 0.1 mg/ml即可明显上调HK-2细胞MIF mRNA和蛋白表达(P < 0.01);而MCD患者的尿IgG需达到2.5 mg/ml才具有显著上调效应&#65377; 结论 呈NS的MCD和MN患者尿IgG可上调HK-2细胞表达MIF&#65377;MN患者尿IgG的作用强于MCD患者,提示这两种不同病理类型患者尿IgG可能存在结构或功能上的差异&#65377;     

丝裂原活化蛋白激酶类和蛋白激酶C在转化生长因子β1诱导肾小管细胞结缔组织生长因子表达中的作用

目的了解转化生长因子β1（TGF-β1）诱导肾小管细胞结缔组织生长因子（CTGF）表达的机制，特别是蛋白激酶C（PKC）和丝裂原活化蛋白激酶（MAPK）在CTGF基因表达中的作用及其对Smad磷酸化的影响。方法分别应用PKC抑制剂G06850以及MAPK的3个组成成分ERK、JNK和p38MAPK的抑制剂PD98059、U0126、SP600125和SB203580阻断相应通路，观察其对TGF．131诱导的CTGF表达以及Smad2／Smad3磷酸化的影响。结果TGF-β1（5μg／L）以时间依赖方式诱导HK-2细胞中Smad2／Smad3的磷酸化，从基础值0．87±0．09上升至2h时高峰2．350±0.11。PKC抑制剂G06850（5μmol／L）和ERK抑制剂PD98059（10μmol／L）、U0126（10μmol／L）可部分抑制TGF-β1诱导的CTGF表达，而p38MAPK抑制剂SB203580（20μmol／L）和JNK抑制剂SP600125（10μmol／L）对TGF-β1诱导的CTGF的表达无影响。PKC抑制剂G06850（5μmol／L）可减少TGF-β1诱导的Smad2／Smad3磷酸化，而ERK抑制剂PD98059（10μmol／L）和U0126（10μmol／L）对Smad2／Smad3的磷酸化没有影响。结论在肾小管上皮细胞中，TGF-β1诱导CTGF的表达需要PKC和Ras／MEK／ERK的参与。PKC以Smad依赖的方式参与肾小管上皮细胞中TGF-β1诱导的CTGF的表达，而Ras／MEK／ERK对CTGF表达的调节不依赖于Smads。     

Role of IQGAP1 in mediating angiotensinⅡ-induced apoptosis of podocytes and its underlying mechanism

Objective To investigate the role of IQ domain GTPase-activating protein 1 (IQGAP1) in angiotensinⅡ(AngⅡ) -induced podocyte apoptosis and the underlying mechanism. Methods Differentiated mouse podocytes were exposed to AngⅡ at different concentrations for 6 h or at 10-8 mol/L for variable incubation time. Podocyte apoptosis was assessed by flow cytometry. Expression of IQGAP1 was analyzed by immunofluorescence and Western blotting. IQGAP1 siRNA and MAPK pathway inhibitors(10 μmol/L SB202190, 25 μmol/L SP600125, 10 μmol/L U0126) were further introduced to investigate the role of IQGAP1 and MAPK signalings in the process. And co-immunoprecipitation was used to evaluate the interaction between ERK1/2 and IQGAP1. Results (1) AngⅡ promoted podocyte apoptosis in a dose- and time-dependent manner. (2) IQGAP1 was located in celluar membrane and cytoplasm of cultured podocytes. Exposure to AngⅡ stimulated IQGAP1 expression in a dose- and time-dependent manner, and elevated phosphorylation of p38, JNK, and ERK1/2 simultaneously. (3) Pretreatment with SB202190, SP600125, or U0126 dramatically prevented AngⅡ-promoted podocyte apoptosis respectively (P＜0.05). However, the protein level of IQGAP1 was not altered. (4) Knockdown of IQGAP1 with siRNA obviously prevented AngⅡ-induced apoptosis of podocytes(P＜0.05) and reduced AngⅡ-induced phosphorylation of ERK1/2(P＜0.05), but not that of p38, JNK. This was accompanied by a reduced interaction between ERK1/2 and IQGAP1(P＜0.05). Conclusion IQGAP1 contributes to AngⅡ-induced podocyte apoptosis by interacting with the ERK1/2 signaling protein.     

Evidence for JNK-dependent up-regulation of proteoglycan synthesis and for activation of JNK1 following cyclical mechanical stimulation in a human chondrocyte culture model

OBJECTIVE: To examine the expression of mitogen-activated protein kinases (MAPKs) in human chondrocytes, to investigate whether selective activation of MAPKs is involved in up-regulation of proteoglycan (PG) synthesis following cyclical mechanical stimulation (MS), and to examine whether MS is associated with integrin-dependent or independent activation of MAPKs. METHODS: The C-28/I2 and C-20/A4 human chondrocyte cell lines were mechanically stimulated in monolayer cell culture. PG synthesis was assessed by [(35)S]-sulphate incorporation in the presence and absence of the p38 inhibitor SB203580, and the extracellular-regulated kinase (ERK1/2) inhibitor PD98059. Kinase expression and activation were assessed by Western blotting using phosphorylation status-dependent and independent antibodies, and by kinase assays. The Jun N-terminal kinase (JNK) inhibitor SP600125 and the anti-beta(1) integrin (CD29) function-blocking antibody were used to assess JNK activation and integrin dependence, respectively. RESULTS: Increased PG synthesis following 3 h of cyclic MS was abolished by pretreatment with 10 microM SB203580, but was not affected by 50 microM PD98059. The kinases p38, ERK1/ERK2 and JNKs were expressed in both stimulated and unstimulated cells. Phosphorylated p38 was detected at various time points following 0.5, 1, 2 and 3 h MS in C-28/I2, but not detected in C-20/A4 cell lines. Phosphorylation of ERK1 and ERK2 was not significantly affected by MS. Phosphorylation of the 54 and 46 kDa JNKs increased following 0.5, 1, 2 and 3 h of MS, and following CO(2) deprivation. MS-induced JNK phosphorylation was inhibited by SB203580 at concentrations > or =5 microM and activation of JNK1 following MS was blocked by SP600125 and partially inhibited by anti-CD29. CONCLUSIONS: The data suggest JNK, rather than p38 or ERK dependent increases in PG synthesis, and selective, partially integrin-dependent, activation of JNK kinases in human chondrocyte cell lines following cyclical MS. JNK activation is also very sensitive to changes in CO(2)/pH in this chondrocyte culture model.     

In vitro evidence for role of ERK, p38, and JNK in exocrine pancreatic cytokine production

Elucidation of mechanisms of acinar cell cytokine production is essential for a better understanding of acute pancreatitis pathogenesis. We hypothesize that the stress kinases ERK, p38, and JNK play an important role in acinar cell cytokine production. Rat pancreatic fragments were incubated with 100 nM concentration of the cholecystokinin analog caerulein or 100 nM caerulein and specific ERK inhibitor (100 μM PD98059), specific p38 inhibitor (10 μM SB203580), or specific JNK inhibitor (20 μM SP600125). After 3 hours of caerulein treatment, pancreatic fragments were homogenized and assayed for total and phosphorylated ERK, p38, and JNK, and for tumor necrosis factor-α or interleukin-1β concentrations (ELISA). Pancreatic fragments stimulated with caerulein showed activation of ERK, p38, and JNK and increased cytokine concentrations (ANOVA, P<0.05). Specific stress kinase inhibitors significantly attenuated caerulein-induced activation of the corresponding stress kinase and cytokine production; however, the effect of the JNK inhibitor was comparatively less convincing. Increased activation of ERK, p38, and JNK in pancreatic fragments was not associated with significant increases in total ERK, total p38, or total JNK concentrations. The stress kinases ERK and p38 play an important role in caerulein-stimulated exocrine pancreatic overproduction of cytokines. The role of JNK needs further evaluation in this experimental model. This work was presented at the Forty-Seventh Annual Meeting of The Society for Surgery of the Alimentary Tract, Los Angeles, CA, May 22, 2006. Dr. Samuel was supported for this research by an American College of Surgeons Faculty Research Fellowship (2003–2005) and a National Institutes of Health NIDDK Career Development Award (grant K08-DK062805).     

Modulation of vascular smooth muscle cell alignment by cyclic strain is dependent on reactive oxygen species and P38 mitogen-activated protein kinase

PURPOSE: The aim of this study was to investigate the molecular targets of reactive oxygen species (ROS) and to determine whether cyclic strain induces smooth muscle cell (SMC) alignment via the ROS system. We assessed stretch-induced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase activation and the redox sensitivity of cyclic strain-stimulated activation of the mitogen-activated protein kinase (MAPK) family. METHODS: SMCs were seeded on flexible collagen I-coated plates and exposed to cyclic strain. NAD(P)H oxidase activation was measured with lucigenin-enhanced chemiluminescent detection of superoxide. Activation of MAPK was detected by determining phosphorylation of extracellular signal-regulated protein kinase (ERK1/2), c-jun N-terminal kinase (JNK1/2), and p38 MAPK with immunoblotting. In other experiments, SMCs were exposed to diphenylene iodonium (DPI), an NAD(P)H inhibitor, 30 minutes before stretch. MAPK activation and cell orientation were then assessed. RESULTS: Cyclic strain elicits a rapid increase in intracellular NADH/NADPH oxidase in SMCs. There was also a rapid and robust phosphorylation of ERK1/2, JNK1/2, and p38 MAPK. Cyclic strain-induced intracellular NAD(P)H generation was almost completely blocked with DPI. DPI also inhibited the strain-induced phosphorylation of ERK1/2, JNK1/2, and p38 MAPK. Both the p38 MAPK specific inhibitor, SB 202190, and DPI blocked cyclic strain-induced cell alignment, but PD98059, an ERK1/2-specific inhibitor, and SP600125, an anthrazolone inhibitor of JNK, did not. CONCLUSION: Our results provide evidence that p38 MAPK is a critical component of the oxidant stress ROS-sensitive signaling pathway and plays a crucial role in vascular alignment induced by cyclic stain.     

JNK-c-Jun信号通路下调连接蛋白43的表达引起肾小管上皮细胞转分化

目的 观察c-Jun氨基末端激酶（JNK）-c-Jun通路对连接蛋白43（Cx43）表达的影响及在转化生长因子（TGF）β1诱导的肾小管上皮细胞-肌成纤维细胞转分化（TEMT）中的作用。 方法　大鼠肾小管上皮细胞（NRK-52E）随机分成3组：对照组、TGF-β1（10 μg/L）组和TGF-β1（10 μg/L）+JNK选择性抑制剂SP600125（50 μmol/L）组。用免疫细胞化学、Western印迹检测JNK、c-Jun、连接蛋白43（Cx43）、上皮细胞标志物E-钙黏蛋白（E-cadherin）和肌成纤维细胞标志物α-SMA的表达。用RT-PCR检测Cx43的mRNA水平。用激光共聚焦显微镜荧光漂白恢复（FRAP）技术检测NRK-52E细胞间通讯功能。 结果 TGF-β1引起肾小管上皮细胞α-SMA、JNK、c-Jun表达上调（均P < 0.05）,Cx43、E-cadherin表达下调（均P < 0.05）,Cx43的mRNA水平下降（P < 0.05）,细胞间通迅功能下降 （P < 0.05）。JNK抑制剂处理后,上述改变明显减轻。 结论 TGF-β1引起肾小管上皮细胞内JNK表达上调,增加c-Jun活性,从而抑制Cx43的表达和降低细胞间通迅功能,导致TEMT。     

转化生长因子β1对肾小管上皮细胞中结缔组织生长因子基因启动子活性的影响

目的探讨转化生长因子[β1（TCF-β1）对人近端肾小管上皮细胞系HK-2中结缔组织生长因子（CTGF）基因启动子活性的调控作用，以及丝裂原激活蛋白激酶（MAPK）途径对该生长因子作用的影响。方法构建含有人类CTGF基因启动子的报告基因pCTGF-luc，将其瞬时转染HK-2细胞。通过检测荧光素酶的活性观察TGF-β1和MAPK途径抑制剂对CTGF基因启动子活性的影响。结果TGF-β1以剂量和时间依赖方式上调HK-2中CTGF基因启动子的活性。最佳刺激浓度是5ng／ml，最佳刺激时间为12h，荧光素酶相对活性分别为对照组的1．82倍和2．10倍（P〈0．05）。应用PD98059、SB203580和SP600125分别特异性抑制MAPK途径的胞外信号调节蛋白激酶（ERK）、蛋白激酶p38（p38MAPK）和c-Jun-氨基末端激酶（JNK）通路，对TGF-β1上调CTGF启动子活性的作用有不同影响。PD98059显著增加HK-2中pCTGF-luc的基础活性．并在一定浓度范围内（0．5～10μmol／L）促进TGF-β1的上调作用。SB203580对pCTGF-luc基础活性无影响，但以剂量依赖方式显著抑制TGF-β1的激活效应。而SP600125对基础状态和TGF-β1刺激下CTGF基因启动子活性无影响。结论TGF-β1以剂量和时间依赖方式上调HK-2中CTGF基因启动子活性，在转录水平调节CTGF表达。MAPK途径的ERK和p38MAPK通路可影响TGF-β1的这一调控作用。     

Mitogen-activated protein kinases signal inhibition of apoptosis in lipopolysaccharide-stimulated neutrophils.

BACKGROUND: Neutrophil (PMN) apoptosis is critical to the resolution of infection and the limitation of inflammation. Bacterial endotoxin (lipopolysaccharide [LPS]) inhibits PMN apoptosis and activates the p38 mitogen-activated protein kinase (MAPK) signal cascade. The role of p38 and other MAPKs (ERK and SAPK/JNK) in regulating PMN apoptosis after LPS stimulation is unknown. We hypothesize that MAPK activation by LPS signals inhibition of PMN apoptosis. METHODS: PMNs were isolated from the blood of healthy human volunteers and incubated with PD98059 (ERK inhibitor), SB203580 (p38 inhibitor), or 0.1% dimethyl sulfoxide (vehicle) for 1 hour before treatment with LPS (0, 10, or 1000 ng/mL). Neutrophil MAPK activation was determined by Western blot analysis for phosphorylated p38, ERK, and SAPK/JNK. Apoptosis was quantified by flow cytometry with use of propidium iodide and annexin V. RESULTS: LPS inhibited PMN apoptosis and activated p38 and ERK in a dose- and time-dependent fashion. SAPK/JNK was not activated by LPS. Treatment of cells with ERK inhibitor before LPS stimulation abrogated LPS signaled inhibition of PMN apoptosis. Conversely, p38 inhibition with SB203580 augmented inhibition of apoptosis by LPS. CONCLUSIONS: These data demonstrate opposing roles of MAPKs in mediating PMN apoptosis after LPS stimulation. We conclude that LPS signal transduction by ERK inhibits PMN apoptosis while activation of p38 promotes apoptosis.     

c-Jun N-terminal kinase and nuclear factor ��B mediate nitric oxide-induced expression of matrix metalloproteinase-13

The purpose of this study was to investigate the mechanism of expression of matrix metalloproteinase-13 (MMP-13) induced by nitric oxide (NO). Human chondrocytes (HCs) were stimulated with a NO donor (MAHMA-NONOate), then mitogen-activated protein kinases’ (MAPKs) and nuclear factor κB’ (NF-κB) activations and MMP-13′ expression were assayed by Western blot analysis. Additionally, the intracellular signalling of NO was investigated using the inhibitors of MAPKs and NF-κB. NO-induced MMP-13 expression was not suppressed by extracellular signal-regulated kinase (ERK) inhibitor (PD98059) or inhibitors of p38 kinase (SB203580), but was inhibited by a c-jun terminal kinase (JNK) inhibitor (SP600125) and inhibitors of NF-κB (SN-50). Additionally, SP600125 treatment reduced NF-κB activation, but SN-50 treatment did not significantly affect JNK activation. These results suggest that NO induces MMP-13 expression by JNK and NF-κB activation in HCs.