高糖通过PI3K-AKT-mTOR信号通路增加足细胞自噬

目的 研究高糖引起足细胞自噬变化及其相关的信号机制.方法 培养的足细胞被分为6组,正常浓度葡萄糖(NG)组、高浓度葡萄糖(HG)组、NG+雷帕霉素(Rap)组、HG+Rap组、NG+LY294002组和HG+LY294002组.观察自噬增强剂Rap和PI3K抑制剂LY294002对高糖条件下培养的足细胞自噬和凋亡的影响.电镜和吖啶橙染色观察细胞内自噬体的形成;Western印迹检测自噬标志蛋白微管相关蛋白1轻链3(LC3)和自噬血管基因Beclin-1的表达;通过阻断自噬的信号通路观察磷脂酰肌醇3激酶-蛋白激酶B-哺乳动物雷帕霉素靶蛋白(PI3K-AKT-mTOR)相关蛋白AKT和mTOR的磷酸化水平的改变.结果 高糖可导致足细胞凋亡增加,促进足细胞内自噬体和自噬相关蛋白表达增加(均P＜ 0.05).与高糖组相比,HG+ Rap组LC3-Ⅱ和Beclin-1的表达增加(均P＜0.05);LY294002部分抑制高糖导致的LC3-Ⅱ和Beclin-1表达增加(均P＜0.05).与高糖组相比,HG+ LY294002组足细胞内AKT磷酸化的水平增加(P＜0.05),mTOR的磷酸化水平降低(P＜0.01);HG+ LY294002组足细胞的AKT和mTOR磷酸化水平较高糖组均降低(均P＜0.05).结论 高糖可促进足细胞的自噬和凋亡,推测高糖诱导的足细胞自噬作用部分通过PI3K-AKT-mTOR信号通路调节实现的.     

尿足细胞与狼疮肾炎关系的研究

目的研究尿足细胞与狼疮肾炎（ LN）的关系，探讨尿足细胞评估 LN 肾损伤的临床价值。方法选取20例行肾活检的LN患者，收集相关临床资料，光镜观察肾组织病理改变；电镜观察足细胞超微结构变化；免疫组化检测肾足细胞蛋白 Nephrin 的表达；免疫荧光技术检测 Podocalyxin 表达来计数尿足细胞数量。结果20例LN病理显示Ⅲ型2例、Ⅳ型12例、Ⅳ型＋Ⅴ型4例、Ⅴ型2例，足细胞标志蛋白Nephrin 表达较正常组明显减低，且Ⅳ型、Ⅴ型降低更明显。尿足细胞阳性检出率约80％，尿足细胞数量与肾
　　足细胞相关蛋白 Nephrin 的表达呈负相关（r＝－0．627， P ＜0．01），与狼疮活动性呈正相关（r＝0．571， P ＜0．05），与24h蛋白定量无明显相关性（r＝0．215， P ＞0．05）。结论尿足细胞数量可监测狼疮活动及肾小球损伤程度，为LN病理分型提供参考。     

足细胞相关蛋白在糖尿病肾病中的表达及氯沙坦的干预

目的:研究Nephrin、Podocin在糖尿病肾病大鼠肾组织中表达的变化,并探讨氯沙坦的干预作用。方法:60只健康雄性SD大鼠,采用小剂量STZ(25mg/kg)联合完全弗氏佐剂腹腔注射建立DN大鼠模型,反复测量三次血糖值均≥16.7mmol/L视为造模成功。采用随机数字表的方法将造模成功的大鼠分配至对照组、模型组、氯沙坦组。12周末处死大鼠,收集大鼠尿液测定24h尿白蛋白排泄率;光镜观察肾脏病理;western blot检测肾组织中Nephrin、Podocin蛋白的表达。结果:(1)与对照组比较,模型组大鼠24h尿白蛋白排泄率(UARE)均明显升高(P<0.01);与模型组比较,氯沙坦组大鼠24hUARE明显下降(P<0.01);(2)肾脏病理显示对照组肾组织结构基本正常,模型组肾组织损伤明显,经氯沙坦干预后,肾脏病理损伤减轻;(3)western blot检测结果:模型组与正常对照组比较,肾组织中Nephrin、Podocin蛋白表达明显降低(P<0.01),氯沙坦组与模型组相比,肾组织Nephrin、Podocin蛋白均有明显的提高(P<0.01)。结论:(1)糖尿病肾病大鼠足细胞Nephrin、Podocin蛋白表达的减少;(2)氯沙坦对糖尿病肾病大鼠肾脏有明显的保护作用,可以减少蛋白尿。(3)氯沙坦可以通过上调DN大鼠肾组织Nephrin、Podocin蛋白的表达而减轻蛋白尿。     

益肾胶囊含药血清对高糖环境下肾小球足细胞自噬的影响

目的:观察益肾胶囊含药血清对高糖环境下小鼠肾小球足细胞自噬的影响。方法:制备不同浓度益肾胶囊含药血清,对体外培养的小鼠肾小球足细胞进行分组干预,细胞分为:正常组(NG)、高糖组(HG)、低浓度益肾胶囊含药血清组(LY)、中浓度益肾胶囊含药血清组(MY)、高浓度益肾胶囊含药血清组(HY)、贝那普利含药血清组(BP)。Western Blot检测自噬相关LC3蛋白及泛素结合蛋白p62(p62/SQSTMl)蛋白的表达变化。透射电镜下观察各组足细胞内自噬体的变化。结果:与NG组相比,HG组足细胞自噬相关蛋白LC3Ⅱ表达减少,泛素结合蛋白p62(p62/SQSTMl)表达增多(P0.05);与HG组相比,HY组足细胞自噬相关蛋白LC3Ⅱ表达增多,泛素结合蛋白p62(p62/SQSTMl)表达减少(P0.05)。透射电镜提示,与HG组相比,HY组足细胞自噬体增多(P0.05)。结论:高糖可导致足细胞自噬障碍,而益肾胶囊可通过改善其自噬障碍,进而发挥足细胞保护的作用。     

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。     

3-甲基腺嘌呤促进吡柔比星诱导膀胱癌BIU-87细胞凋亡及机制

目的 探讨3-甲基腺嘌呤(3-MA)自噬抑制剂联合吡柔比星(THP)对膀胱癌BIU-87细胞杀伤作用及机制.方法 膀胱癌BIU-87细胞分为对照组、3-MA(10 mmol/L)组、THP(5 mg/L)组、3-MA(10 mmol/L)+ THP(5 mg/L)组.噻唑蓝(MTT)法检测细胞增殖;流式细胞术检测细胞凋亡;Western blot检测各组细胞中自噬蛋白微管相关蛋白1轻链3(LC3)-Ⅱ、Beclin-1以及凋亡蛋白半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3和Caspase-9的表达.结果 药物作用24h后,各组细胞增殖率分别为(86.64±3.09)％、(63.70±5.62)％、(58.43±3.89)％、(37.55±4.17)％,其中3-MA+THP组细胞增殖下降最明显(P ＜0.05);3-MA与THP联合应用后,自噬蛋白LC3-Ⅱ、Beclin-1的表达明显减弱,而凋亡蛋白Caspase-3和Caspase-9的表达在各组中最强(P＜0.05).结论 3-MA抑制膀胱癌BIU-87细胞对化疗药物保护性自噬反应,增加膀胱癌细胞对THP细胞毒杀伤作用的敏感性.     

糖尿病肾病大鼠足细胞自噬的变化

目的:研究糖尿病肾病大鼠足细胞自噬标志物LC3、P62蛋白表达的改变,探讨糖尿病肾病可能的发病机制。方法:将清洁级雄性SD大鼠20只随机分为正常对照组(n=10只)、造模组(n=10只)。采用高糖高脂饲料结合一次性腹腔注射链脲佐菌素制备糖尿病动物模型。造模成功后于第12周结束时留取大鼠尿标本,检测大鼠尿微量白蛋白、尿肌酐,摘取肾脏称量重量,分别计算尿蛋白肌酐比和肾重体重比;HE染色、PAS染色观察肾脏组织病理变化;Western-blot检测肾小球LC3、P62蛋白表达。结果:与正常对照组相比,尿蛋白肌酐比、肾重体重比在造模组均显著增加(P<0.05);P62蛋白表达亦较正常组表达显著增加(P<0.05);造模组大鼠肾脏病理在光镜下可见明显的系膜增生、基质增多。结论:糖尿病肾病大鼠肾脏足细胞自噬较正常组减低,可能是糖尿病肾病发病的机制之一。     

调控自噬流对巨噬细胞粘附与迁移功能的影响

目的巨噬细胞浸润是包括糖尿病肾病(diabetic nephropathy, DN)在内的多种慢性肾脏疾病的重要组织病理特征。本研究通过调控巨噬细胞(RAW264.7)自噬流各阶段,探究其对巨噬细胞黏附迁移功能的影响。 方法体内实验,建立糖尿病肾病大鼠模型,于12周末分别处死正常大鼠、DN组大鼠,病理染色观察肾脏病理改变,检测肾组织巨噬细胞标志物及自噬相关标志物表达。体外实验,检测正常与高糖(30 mM)条件下,巨噬细胞自噬体数量变化,LC3、Beclin－1(自噬相关标志物)、P62(自噬体清除指标)的表达,以及巨噬细胞粘附迁移数量。分别加用自噬溶酶体降解抑制剂氯喹(CQ)、自噬体生成激活剂雷帕霉素(RAPA),观察其对巨噬细胞自噬标记物表达及其粘附迁移功能的影响,电镜观察巨噬细胞自噬体数量与自噬溶酶体形态的变化。 结果体内实验,DN大鼠肾脏损伤明显,肾小球体积增大,基底膜增厚,系膜基质增多,肾组织CD68(巨噬细胞标志物)、P62表达增加(t＝3.35、t＝16.27, P<0.05),LC3表达减少(t＝51.12, P<0.05);体外实验,在高糖组,电镜观察发现自噬体数量较正常组减少,Western印迹与免疫荧光显示自噬相关蛋白LC3、Beclin－1表达降低,P62表达升高(t＝27.02,t＝45.56、t＝32.71,P<0.05),巨噬细胞粘附和迁移数量增多(t＝6.87、t＝8.76,P<0.05)。用CQ处理后,电镜观察发现巨噬细胞自噬体溶酶体降解受到抑制,Western印迹与免疫荧光显示自噬相关蛋白LC3、Beclin－1表达降低,P62表达升高(t＝14.64、t＝12.45、t＝8.57,P<0.05);CQ进一步促进高糖诱导的巨噬细胞黏附迁移数量增多(t＝4.37、t＝7.27,P<0.05);RAPA增加巨噬细胞自噬体数量,Western与免疫荧光显示RAPA提高了被高糖抑制的巨噬细胞自噬水平,[LC3、Beclin－1表达升高,P62表达降低(t＝9.37、t＝11.53,t＝8.73;P<0.05)],减少高糖诱导的巨噬细胞黏附、迁移数量增多(t＝4.16、t＝5.74, P<0.05)。 结论高糖抑制自噬水平,促进巨噬细胞黏附迁移;抑制自噬溶酶体降解可降低自噬水平、促进巨噬细胞粘附迁移;激活自噬体生成能提高自噬水平,减轻巨噬细胞粘附迁移。     

风湿性心脏病患者左心耳组织中自噬相关蛋白与房颤的相关研究

目的通过风湿性心脏病(风心病)患者左心耳组织中自噬相关基因的检测,探讨其与风心病房颤的可能关系。方法将59例风心病患者分为窦律组(28例)和房颤组(31例),应用q-PCR检测自噬相关蛋白Beclin-1和微管相关蛋白轻链3(LC3)m RNA水平,同时用Western blot技术检测相应蛋白的表达水平,采用Pearson相关分析上述指标与风心病患者左心房内径的相关性。结果房颤组m RNA表达Beclin-1为(3.94±1.07)、LC3Ⅱ为(4.06±0.94),分别高于窦律组的(1.83±0.54)和(2.37±0.67),差异有统计学意义(P0.05)。房颤组相应蛋白表达Beclin-1为(1.49±0.31)、LC3Ⅱ为(1.84±0.37),分别高于窦律组的(0.53±0.11)和(0.93±0.10),差异有统计学意义(P0.05)。结论风心病房颤患者左心耳自噬相关蛋白Beclin-1和LC3-Ⅱ的表达增加,可能与风心病房颤的发生发展相关。     

雷帕霉素通过调节mTOR-ULK1信号通路减轻高糖诱导的足细胞损伤

目的探讨雷帕霉素对高糖环境下足细胞自噬和损伤作用的机制。 方法体外培养永生化小鼠肾小球足细胞(mouse podocyte cell 5,MPC5)并进行分组：甘露醇等渗组(mannitol isotonic group,MG组)、高糖组( high glucose group,HG组)、雷帕霉素组(rapamycin group,RG组)以及自噬相关蛋白5-siRNA组(SiG组)。PCR和Western印迹检测足细胞标志Synaptopodin、自噬相关的ULK1以及mTOR通路相关蛋白p70S6K的表达。 结果与MG组相比,HG组的Synaptopodin表达降低,自噬活性降低,p-ULK1以及p70S6K表达明显升高。与HG组相比,RG组的Synaptopodin表达升高,自噬活性较高,p-ULK1以及p70S6K表达较低。SiG组表现出与HG组相似的变化趋势。 结论雷帕霉素可能通过mTOR-ULK1信号通路调节足细胞内自噬反应、减轻高糖环境引起的足细胞损伤。     

Changes of autophagosomes of podocytes in idiopathic membranous nephropathy and its clinical significance

Objective To observe the quantity change of autophagosomes in podocytes and expressions of autophagy-related gene Beclin-1 and microtubule-associated protein 1 light chain 3 (LC3) in different pathological stages of idiopathic membranous nephropathy (IMN), and to explore how autophagy is related to podocyte injury, the occurrence of proteinuria and the disease progression in IMN. Methods Clinical data of 26 patients who were diagnosed as IMN (14 IMN stage 1 and 12 IMN stage 2) admitted to Zhejiang Provincial people's Hospital from January 2013 to December 2014 were retrospectively analyzed. Normal renal tissue from 15 cases of kidney neoplasms with nephrectomy was collected as control. The changes of kidney tissue pathology were detected after PAS and PASM staining by light microscope. The autophagosomes of podocyte were detected by transmission electron microscopy. Expressions of Beclin-1 and LC3 protein were detected by immunohistochemistry. Expressions of LC3 and synaptopodin were detected by immunofluorescence. The correlation of autophagosomes and clinical pathologic factors in IMN patiens was analyzed. Results There were fewer autophagosomes of podocytes and lower expression of Beclin-1 and LC3 protein in IMN group than those in control group (P=0.034, P=0.011, P=0.013, respectively). Moreover, these effects were more obvious with the development of IMN. Compared with those in control group, autophagosomes, Beclin-1 and LC3 protien were reduced in IMN stage 2 group (P=0.009, P=0.030, P=0.015); the number of autophagosomes and the expressions of LC3 and Beclin-1 were decreased in IMN stage 1 group as well, however statistically insignificant (P=0.352, P=0.087, P=0.128); Comparisons between IMN stage 2 patients and IMN stage 1 patients shown significant difference in the number of autophagosomes (P=0.030), but no significant difference in expressions of Beclin-1 and LC3 (P=0.355, P=0.181). Autophagosomes number was not correlated with serum creatinine, serum urea nitrogen, 24-hour urinary protein and eGFR (all P＞0.05). The expressions of synaptopodin and LC3 protein were lower in IMN group than those in control group. Conclusion Autophagy may contribute to podocyte injury and the production of protein urine in IMN, and may be closely related to the progression of disease.     

Autophagy alleviate podocytes injury induced by contrast media via oxidative stress

Objective To evaluate the effects of autophagy on oxidative stress induced by contrast media in podocytes. Methods The differentiated mouse podocytes were exposed to contrast media (Iopromide, 50 mg/L)、rapamycin (Rap, autophagy enhancer, 1 ng/L), 3-methyladenine (3-MA, autophagy inhibitor, 2 mmol/L) for 2 hours. The expression of autophagy protein LC3-Ⅱand Beclin-1 as well as oxidative stress-related proteins Catalase, MnSOD were detected by Western blot. The formations of autophagy were observed by MDC staining, and the levels of reactive oxygen species (ROS) by CM-H2DCFDA staining. Cell activity was evaluated by CCK8 assay. Results Both the levels of oxidative stress and autophagy in podocytes increased when stimulated by contrast media, the expression of LC3-Ⅱand Beclin-1 were enhanced, Catalase and MnSOD were inhibited (all P＜0.05). Rapamycin increased the expression of Catalase, MnSOD and cell activity of podocytes, reduced the generation of ROS (all P＜0.05), but in Rap group, cell activity showed no significant difference (P＞0.05). 3-MA decreased the expression of Catalase、 MnSOD and inhibited the cell activity of podocyte, increased the generation of ROS (all P＜0.05). Conclusion Autophagy protects podocyte from contrast media by the means of reducing oxidative stress.     

Role of autophagy in high glucose-induced podocyte lipid droplet accumulation

Objective To investigate the role of autophagy in high glucose-induced podocyte lipid droplet metabolism. Methods (1) Cultured, conditionally immortalized human podocytes (HPC) were divided into normal control group, high glucose group and mannitol group. Oil red O staining and oil red O staining extraction assay was used to observe the degree of lipid accumulation; Protein level of SREBP-1 was analyzed by Western blotting. (2) HPC were cultured and divided into normal control group, high glucose group, high glucose+3-methyladenine (3-MA) group, and mannitol group. Acridine orange staining was used to observe the formation of autophagosomes. Western blotting was used to detect the protein levels of beclin-1 and LC3-II/LC3-I. Oil red O staining and oil red O staining extraction assay was used to observe the degree of lipid accumulation; Western blotting was used to analyze the expression of SREBP-1. Results (1) Compared with the normal control group, the lipid accumulation in the high glucose group was increased and the lipid metabolism related molecule SREBP-1 was up-regulated (P＜0.05); There was no significant difference between the normal control group and the mannitol group in lipid accumulation (P＞0.05). (2) Compared with the normal group, the number of autophagosomes was increased and autophagy-related proteins beclin-1 and LC3-II/LC3-I were up-regulated in high glucose group (all P＜0.05). After intervened with 3-methyladenine, a significant decrease in autophagosomes was observed; Protein levels of autophagy-related proteins beclin-1 and LC3-II/LC3-I were decreased (all P＜0.05); The lipid droplets in the high glucose+3-MA group was decreased and lipid metabolism related molecule SREBP-1 was down-regulated (all P＜0.05). Conclusion Autophagy may be involved in the process of high-glucose-induced podocyte lipid accumulation by affecting SREBP-1 expression, and inhibition of autophagy can alleviate the high-glucose-induced podocyte lipid accumulation.     

落新妇苷对大鼠肝脏缺血再灌注损伤及自噬的影响

目的探讨落新妇苷对大鼠肝脏缺血再灌注损伤（HIRI）的保护作用及自噬的影响。 方法将54只SD大鼠随机分为假手术组（Sham组）、缺血再灌注组（HIRI组）以及落新妇苷组（40 mg·kg^-1·d^-1,连续7 d）,每组18只。建立大鼠HIRI模型,于再灌注4、8、16 h后取下腔静脉血及肝左外叶组织。全自动生化分析仪检测血清丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）水平;光学显微镜下观察肝细胞显微结构变化;Western blotting分析肝组织中自噬相关蛋白LC3、Beclin-1的表达;电镜下观察肝脏组织内自噬小体数量情况。 结果同检测时间点比较,Sham组和落新妇苷组的血清ALT和AST水平均低于HIRI组（P<0.05）,且肝细胞肿胀、炎性细胞浸润、汇管区结构损伤明显较轻。落新妇苷组LC3-Ⅱ/ LC3-Ⅰ灰度值比值及Beclin-1蛋白灰度值在术后4、8、16 h明显低于HIRI组（P<0.05）。落新妇苷组肝组织的自噬小体数量较HIRI组有所减少（3.68±0.42 vs 7.12±0.60,t=36.382,P<0.01）。 结论落新妇苷预处理可减轻大鼠HIRI,其作用机制可能与其抑制自噬有关。     

Dynamic changes of autophagy during hypertrophic scar formation and the role of autophagy intervention

BackgroundThe role of autophagy in the formation of hypertrophic scars (HS) remains unclear. This study aimed to explore the role and potential mechanism of autophagy during the development of HS.MethodsRNA and protein expression levels of Beclin-1, p62, and LC3II in normal skin tissues and HS specimens from different patients were examined. Autophagy inducers and inhibitors were used to cure established HS in rabbit ears, and the expression of Beclin-1, p62, and LC3II at the RNA and protein level was determined. Lastly, the effects of autophagy inducers and inhibitors on HS development were analyzed.ResultsCompared to normal skin tissues, the expression of LC3Ⅱ and Beclin-1 was higher (P<0.05), while that of p62 was lower (P<0.05) in HS tissues. In addition, the LC3II/LC3I ratio was increased during HS formation, and the altered expression of the three proteins stabilized after one year. Administration of autophagy inducers enhanced the formation of HS as well as the expression levels of LC3II and Beclin-1 but decreased p62 expression. Meanwhile, administration of autophagy inhibitors increased the expression of LC3II, Beclin-1, and p62, along with reduced HS formation.ConclusionAutophagic activity increased during HS initiation and subsequent stabilization. In addition, autophagy inhibitors were able to inhibit HS formation by suppressing autophagy, whereas autophagy inducers promoted scar hyperplasia by enhancing autophagy.     

Role and mechanism of Angiotensin Ⅱ-induced ChemR23 in podocyte injury

Objective To observe the expression of ChemR23 induced by Angiotensin Ⅱ (AngⅡ) in podocyte and its role in renal injury. Methods Conditionally immortalized mice podocytes were cultured in vitro. Immunofluorescence was used to observe the sub-cellular location of ChemR23. The expressions of ChemR23, Nephrin and Podocin stimulated by different concentrations of AngⅡ were detected by qRT-PCR and Western blotting. Lentivirus targeting ChemR23 was used. The expressions of Nephrin and Podocin and the phosphorylation state of NF-κB P65 were detected by Western Blot. The inhibitor of NF-κB P65 was added to the cultural medium for 2 h before AngⅡ stimulation. The effect of NF-κB P65 inhibitor on AngⅡ-induced expression of Nephrin and Podocin was detected by Western Blot. Results It is showed that ChemR23 was located in cytosol and membrane. Compared with the normal control, the expression of ChemR23 was significantly increased by AngⅡ in mRNA and protein level, while the expressions of Nephrin and Podocin were decreased (P＜0.05). When using Lentivirus vector to interfere the expression of ChemR23, AngⅡ-repressed expressions of Nephrin and Podocin were restored (P＜0.05). Western Blot showed the level of phosphorylated NF-κB P65 was significantly increased by AngⅡ stimulation (P＜0.05), which could be inhibited by interfering the expression of ChemR23. When adding the NF-κB P65 inhibitor, the low expression of Nephrin and Podocin induced by AngⅡ stimulation was restored (P＜0.05). Conclusions AngⅡ can induce ChemR23 expression, which activates NF-κB P65 signaling pathway, and then inhibits the expressions of Nephrin and Podocin. Targeting ChemR23 is a potential way to alleviate podocyte injury caused by AngⅡ.     

Tacrolimus对糖尿病大鼠肾脏足细胞损伤的保护作用及机制

目的探讨Tacrolimus（FK506）是否通过足细胞的保护作用减轻糖尿病大鼠尿白蛋白排泄。方法将40只大鼠按随机数表法分为对照组（C组）、糖尿病（diabetesmellitus,DM）模型组（DM组）、DM＋FKS060．5mg·kg^-1·d^-1给药组（FK5060．5组）及DM＋FK5061．0mg·kg^-1·d^-1给药组（FK5061．0组）,每组10只。采用链脲佐菌素（streptozotocin,STZ）腹腔注射建立糖尿病模型,FK506灌胃给药。4周后大鼠24h尿白蛋白测定采用酶联免疫方法,电镜下观察肾小球足细胞病理组织学改变,应用免疫荧光与Westernblot检测肾组织Nephrin和Podocin表达。结果DM组大鼠24h尿白蛋白排泄率（albuminexcretionrate,AER）明显高于对照组（P〈0．01）,FK5060．5与1．0mg／kg给药组大鼠AER水平明显低于模型组（P〈0．05,P〈0．01）。透射电镜观察DM组肾小球基底膜增厚、结构模糊不清,系膜基质增多,足细胞损伤,与DM组比较,FK5060．5、1．0组肾组织超微结构改变有不同程度改善。免疫荧光显示Nephrin和Podocin在C组大鼠肾小球呈线状均匀分布;DM组大鼠肾小球表达明显减少,且呈颗粒状不均匀分布;FK5060．5组和FK5061．0组Nephrin和Podocin表达不同程度增加,呈线状及颗粒状分布。Westernblot显示DM组Nephrin和Podocin较C组表达明显下降;FK5060．5组和FK5061．0组Nephrin和Podocin量较DM组明显增加（P〈0．01）。结论FK506能减少糖尿病大鼠尿白蛋白排泄,改善肾小球足细胞病变,其机制可能与上调Nephrin和Podocin表达有关。     

缺氧复氧对H9C2心肌细胞凋亡、自噬和焦亡的影响

目的:探讨缺氧复氧(hypoxia reoxygenation,HR)对H9C2心肌细胞凋亡、自噬和焦亡3种细胞死亡方式的影响。方法:将培养的H9C2心肌细胞按随机数字表法分为正常对照组(Ctrl组)和HR组,其中HR组H9C2心肌细胞在缺氧培养箱中进行氧糖剥夺8h,然后复氧12 h。通过检测细胞培养液中的乳酸脱氢酶(lactate dehydrogenase,LDH)含量及四甲基偶氮唑盐[3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide,MTT]比色法检测细胞活力来评估细胞损伤情况(每组6孔),Western blot检测(每组9孔)细胞凋亡相关蛋白[天门冬氨酸特异性半胱氨酸蛋白酶(cysteinyl aspartate-specific proteinase,caspase)-3、B细胞淋巴瘤/白血病-2(B-cell lymphoma/leukemia 2,Bcl-2)、Bcl相关蛋白(bcl-associate x protein,Bax)]、自噬相关蛋白[(轻链蛋白3(light chain 3,LC3)Ⅱ/Ⅰ、p62、Beclin-1、磷酸化哺乳动物雷帕霉素靶蛋白(phosphorylated mammalian target of ra-pamycin,p-mTOR)]、焦亡相关蛋白[Nod样受体蛋白-3(nod-like receptor pyrin domain3,NLRP3)、凋亡相关斑点样蛋白(apopto-sis associated speck-like protein,ASC)、caspase-1p20、IL-1β、IL-18]。通过免疫荧光染色进一步评估细胞凋亡、自噬及焦亡情况(每组6孔)。结果:与Ctrl组比较,HR组H9C2心肌细胞HR后细胞活力降低(P<0.05),LDH释放增加(P<0.05),活化的cas-pase-3及Bax的表达上调(P<0.05),Bcl-2表达下降(P<0.05),TUNEL染色显示HR后细胞凋亡显著增加(P<0.05),提示HR后细胞凋亡及损伤增加;而p-mTOR、p62均表达增加(P<0.05),但LC3Ⅱ/Ⅰ和Beclin-1蛋白降低(P<0.05),LC3Ⅱ/Ⅰ免疫荧光染色显示HR后细胞内的自噬小体增加(P<0.05),表明HR后H9C2心肌细胞自噬受到明显抑制;同时炎性小体NLRP3、ASC、cas-pase-1p20蛋白均显著上调(P<0.05),活化的炎症细胞因子IL-1β、IL-18表达增加(P<0.05),提示HR后NLRP3炎性小体活化,细胞焦亡增加。结论:H9C2心肌细胞在HR损伤过程中自噬被抑制,细胞焦亡及凋亡增加。