乳杆菌源性外囊泡对LPS诱导小胶质细胞活化的影响及蛋白质组学分析

目的评价乳杆菌源性细胞外囊泡(Lac-EVs)对脂多糖(LPS)诱导小胶质细胞活化的影响及蛋白质组学分析。方法取生长状态较好的小鼠BV2小胶质细胞, 采用随机数字表法分为3组(n=12):对照组(C组)、LPS组(L组)和LPS+Lac-EVs组(L+E组)。C组正常培养;L组LPS(终浓度1 μg/ml)孵育24 h;L+E组于LPS处理后24 h加入Lac-EVs(终浓度2.5 μg/ml)孵育24 h。随后采用免疫荧光染色法检测CD86和CD206的表达。取L组和L+E组细胞沉淀, 采用蛋白质组学方法筛选两组差异表达蛋白。对鉴定得到的差异表达蛋白进行生物信息学分析并采用RT-qPCR和Western blot法对载脂蛋白1(Apoa1)和G蛋白偶联受体激酶相互作用蛋白2(Git2)两个差异表达蛋白进行验证。结果与C组相比, L组CD86表达上调, CD206表达下调(P<0.05);与L组相比, L+E组CD86表达下调, CD206表达上调(P<0.05)。采用蛋白质组学法筛选出125个差异表达蛋白(FC=2.0, P<0.05), 其中66个蛋白表达上调, ...     

蛋白激酶C在大鼠机械通气相关性肺损伤中的作用

目的 探讨蛋白激酶C(PKC)在大鼠机械通气相关性肺损伤中的作用.方法 健康雄性Wistar大鼠30只,体重250 ～ 300 g,采用随机数字表法,将大鼠分为5组(n=6):对照组(C组)、小潮气量组(S组)、小潮气量+ PKC抑制剂组(S+P组)、大潮气量组(L组)、大潮气量+PKC抑制剂组(L+P组).大潮气量组VT42 ml/kg,小潮气量组VT7 ml/kg,呼吸频率40次/min,I∶E 1∶2,呼吸末正压为0,FiO221％,机械通气4h.S+P组、L+P组于麻醉前1h肌肉注射PKC抑制剂bisindolvlmaleimide Ⅰ 0.12 mg/kg.C组于气管切开后即刻,其它4组机械通气4 h时处死大鼠,取肺组织,计算湿/干重比(W/D比),观察病理学结果；采用Western blot法测定肺组织occludin蛋白表达.结果 与C组比较,其余4组肺组织W/D比升高,occludin蛋白表达下调(P＜0.05)；与 S组比较,L组肺组织W/D比升高,occludin蛋白表达下调,S+P组肺组织W/D比降低,occludin蛋白表达上凋(P＜0.01)；与L组比较,L+P组肺组织W/D比降低,occludin蛋白表达上调(P＜0.01).S+P组和L+P组肺组织病理学损伤较S组和L组减轻.结论PKC参与了大鼠机械通气相关性肺损伤.     

丙泊酚对脂多糖诱导人单核细胞表达Peroxiredoxin-2蛋白和超氧化物歧化酶蛋白的影响

目的 研究丙泊酚对脂多糖(lipopolysaccharide,LPS)诱导人单核细胞(THP-1)表达Peroxiredoxin-2及超氧化物歧化酶(SOD1)的影响.方法 将体外培养的THP-1细胞按照完全随机方法分为4组:正常对照组(C组);LPS刺激组(L组):加入LPS 1 mg/L;丙泊酚处理组(P组):给予丙泊酚15 mg/L和丙泊酚预处理合并LPS刺激组(P+L组):给予丙泊酚15 mg/L,1 h后再给予LPS 1 mg/L.通过Western blot法分别检测各组内Peroxiredoxin-2及SOD1含量的变化,用四唑盐快速比色法(MTT)观察各组THP-1细胞的存活率.结果 在LPS刺激12 h后收集细胞,Westernblot结果显示与正常对照组(C组)、丙泊酚处理组(P组)、丙泊酚预处理合并LP3刺激组(P+L组)3组相比,LPS刺激组(L组)中Peroxiredoxin-2和SOD1的表达量明显降低,而L组与(P+L)组比较,(P+L)组中Peroxiredoxin-2和SOD1的表达量则明显高于L组;MTT结果显示L组与(L+P)组相比较,L组细胞存活率明显低于(L+P)组细胞存活率(P<0.05)结论丙泊酚可以逆转LPS对THP1细胞表达抗氧化蛋白Peroxiredoxin-2及SOD1的抑制作用,其抗氧化作用可能与增加Peroxiredoxin-2及SOD1的表达有关.     

丙泊酚对脂多糖诱导人单核细胞表达Peroxiredoxin-2蛋白和超氧化物歧化酶蛋白的影响

目的 研究丙泊酚对脂多糖(lipopolysaccharide,LPS)诱导人单核细胞(THP-1)表达Peroxiredoxin-2及超氧化物歧化酶(SOD1)的影响.方法 将体外培养的THP-1细胞按照完全随机方法分为4组:正常对照组(C组);LPS刺激组(L组):加入LPS 1 mg/L;丙泊酚处理组(P组):给予丙泊酚15 mg/L和丙泊酚预处理合并LPS刺激组(P+L组):给予丙泊酚15 mg/L,1 h后再给予LPS 1 mg/L.通过Western blot法分别检测各组内Peroxiredoxin-2及SOD1含量的变化,用四唑盐快速比色法(MTT)观察各组THP-1细胞的存活率.结果 在LPS刺激12 h后收集细胞,Westernblot结果显示与正常对照组(C组)、丙泊酚处理组(P组)、丙泊酚预处理合并LP3刺激组(P+L组)3组相比,LPS刺激组(L组)中Peroxiredoxin-2和SOD1的表达量明显降低,而L组与(P+L)组比较,(P+L)组中Peroxiredoxin-2和SOD1的表达量则明显高于L组;MTT结果显示L组与(L+P)组相比较,L组细胞存活率明显低于(L+P)组细胞存活率(P<0.05)结论丙泊酚可以逆转LPS对THP1细胞表达抗氧化蛋白Peroxiredoxin-2及SOD1的抑制作用,其抗氧化作用可能与增加Peroxiredoxin-2及SOD1的表达有关.     

丙泊酚对脂多糖诱导人单核细胞表达Peroxiredoxin-2蛋白和超氧化物歧化酶蛋白的影响

目的 研究丙泊酚对脂多糖(lipopolysaccharide,LPS)诱导人单核细胞(THP-1)表达Peroxiredoxin-2及超氧化物歧化酶(SOD1)的影响.方法 将体外培养的THP-1细胞按照完全随机方法分为4组:正常对照组(C组);LPS刺激组(L组):加入LPS 1 mg/L;丙泊酚处理组(P组):给予丙泊酚15 mg/L和丙泊酚预处理合并LPS刺激组(P+L组):给予丙泊酚15 mg/L,1 h后再给予LPS 1 mg/L.通过Western blot法分别检测各组内Peroxiredoxin-2及SOD1含量的变化,用四唑盐快速比色法(MTT)观察各组THP-1细胞的存活率.结果 在LPS刺激12 h后收集细胞,Westernblot结果显示与正常对照组(C组)、丙泊酚处理组(P组)、丙泊酚预处理合并LP3刺激组(P+L组)3组相比,LPS刺激组(L组)中Peroxiredoxin-2和SOD1的表达量明显降低,而L组与(P+L)组比较,(P+L)组中Peroxiredoxin-2和SOD1的表达量则明显高于L组;MTT结果显示L组与(L+P)组相比较,L组细胞存活率明显低于(L+P)组细胞存活率(P<0.05)结论丙泊酚可以逆转LPS对THP1细胞表达抗氧化蛋白Peroxiredoxin-2及SOD1的抑制作用,其抗氧化作用可能与增加Peroxiredoxin-2及SOD1的表达有关.     

异丙酚对内毒素诱导人脐静脉内皮细胞过氧亚硝基阴离子生成的影响

目的 探讨异丙酚对内毒素诱导人脐静脉内皮细胞过氧亚硝基阴离子(ONOO-)生成的影响.方法 培养至活细胞计数大于95%的人脐静脉内皮细胞,随机分为7组(n=5),对照组(C组)不给予任何处理;LOS0.1组、LPS1组和LPS10组分别加入内毒素(LPS)至终浓度为0.1、1和10 μg/ml,于37℃5%CO2培养箱中孵育6 h;P4+LPS10组和P40+LPS10组预先加入异丙酚至终浓度为4、40μg/ml,I40+LPS10组预先加入脂质溶剂Introlipid至终浓度为40 μg/ml,于37℃ 5%CO2培养箱中孵育30 min,再分别加入LPS至终浓度为10μg/ml,于培养箱中继续孵育6 h.孵育6 h时,测定细胞活力和乳酸脱氢酶(LDH)释放率;采用免疫组化法和Western blot法测定硝基酪氨酸蛋白(NT)表达.结果 与C组比较,其余各组细胞活力降低,内皮细胞NT表达上调,LPS1组、LPS10组、I40+LPS10组、P4+LPS10组和P40+LPS10组LDH释放率升高(P<0.01);与LPS0.1组比较,LPS1组细胞活力、LDH释放率和内皮细胞NT表达差异无统计学意义(P>0.05),LPS10组细胞活力降低,LDH释放率升高,内皮细胞NT表达上调(P<0.01);与LPS10组比较,I40+LPS10组细胞活力、LDH释放率和内皮细胞NT表达差异无统计学意义(P>0.05),P4+LPS10组和P40+LPS10组细胞活力升高,LDH释放率降低,内皮细胞NT表达下调(P<0.01).结论 异丙酚可通过抑制ONOO'-的生成,减轻内毒素诱导人脐静脉内皮细胞损伤.     

L-精氨酸对内毒素诱导急性肺损伤大鼠肺表面活性物质的影响

目的 评价L-精氨酸对内毒素(LPS)诱导急性肺损伤大鼠肺表面活性物质的影响.方法 健康雄性SD大鼠48只,随机分为4组:正常对照组(C组,n=16)、LPS组(n=16)、LPS 3 h+L-精氨酸治疗组(L1组,n=8)和LPS 6 h+L-精氨酸治疗组(L2组,n=8).LPS组、L1组和L2组静脉注射LPS 5mg/kg,C组给予等容量生理盐水.L1组和L2组分别于给予LPS后3 h或6 h腹腔注射L-精氮酸500 mg/kg.L1组和L2组于给予L-精氨酸后3 h(C组和LPS组分别于给予生理盐水或LPS后6、9 h)取8只大鼠,取肺组织,测定表面活性物质结合蛋白A(SP-A)mRNA的表达水平、肺泡灌洗液(BALF)中总磷脂(TPL)和总蛋白(TP)浓度,光镜下观察肺组织病理学结果.结果 与C组比较,LPS组SP-A mRNA表达下调,BALF中TPL浓度降低,TP浓度升高(P＜0.01).与LPS组比较,L1组SP-A mRNA表达上调,BALF中TPL浓度升高,TP浓度降低(P＜0.05或0.01);L2组上述指标差异无统计学意义(P＞0.05).L1组肺损伤程度轻于LPS组和L2组.结论 L-精氨酸可促进肺表面活性物质合成,从而对大鼠内毒素诱导急性肺损伤具有治疗作用.     

异丙酚对LPS诱导BV-2小胶质细胞IL-1β和TNF-α释放的影响及TLR4在其中的作用

目的 评价异丙酚对LPS诱导BV-2小胶质细胞IL-1β和TNF-α释放的影响及Toll样受体4(TLR4)在其中的作用.方法 将体外培养的BV-2小胶质细胞接种于96孔培养板中,采用随机数字表法,将其随机分为4(n=12):对照组、LPS组、异丙酚组和LPS+异丙酚组.LPS组加入LPS1μg/ml孵育24h;异丙酚组加入异丙酚30 μmol/L孵育24 h;LPS+异丙酚组同时加入LPS 1 μg/ml和异丙酚30 μmol/L孵育24h.于孵育6h时,采用ELISA法检测细胞上清液TNF-α浓度,以此反映TNF-α的释放量,采用RT-PCR法测定TLR4 mRNA表达;于孵育24h时,采用ELISA法检测细胞上清液IL-1β浓度,以此反映IL-1β的释放量,采用Western Blot法检测TLR4蛋白表达.结果 与C组比较,LPS组和LPS+异丙酚组IL-1β和TNF-α的释放量升高,TLR4 mRNA及其蛋白表达上调(P＜0.05);与LPS组比较,LPS+异丙酚组IL-1β和TNF-α的释放量降低,TLR4 mRNA及其蛋白表达下调(P＜0.05).结论 异丙酚可抑制LPS诱导BV-2小胶质细胞IL-1β和TNF-α的释放,其机制与抑制TLR4的表达有关.     

NF-κB信号通路在异丙酚抑制脂多糖诱导RAW264.7细胞诱导型一氧化氮合酶基因表达上调中的作用

目的 探讨NF-κB信号通路在异丙酚抑制脂多糖(LPS)诱导RAW264.7细胞诱导型一氧化氮合酶( iNOS)基因表达上调中的作用.方法 体外培养RAW264.7细胞,以5×105/ml密度接种于6 cm培养皿(3 ml/皿)或6孔板(2 ml/孔),采用随机数字表法,将其随机分为3组(n=18):正常对照组(C组)、LPS组(L组)和LPS+异丙酚(LP组).C组不做任何处理;L组和LP组均加入1μg/mlLPS,LP组于加入LPS前2h加入50 μmol/L异丙酚.于LPS孵育30 min时,每组取6皿和6孔,收集细胞,分别采用免疫印迹法测定磷酸化IκB激酶(p-IKK)和NF-κB活性;于LPS孵育6h时,每组取6皿,收集细胞,测定iNOS mRNA表达.结果 与C组比较,L组p-IKK和iNOS mRNA表达上调,NF-κB活性升高(P＜0.05);与L组比较,LP组p-IKK和iNOS mRNA表达下调,NF-κB活性降低(P＜0.05).结论 NF-κB信号通路参与了异丙酚抑制脂多糖诱导的RAW264.7细胞iNOS基因表达上调.     

丙泊酚对脂多糖刺激人单核细胞丝裂原活化蛋白激酶信号通路的影响

目的 研究丙泊酚对脂多糖(lipopolysaccharide,LPS)刺激人单核细胞(human mononuclear macrophage cell,THP-1)丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路的影响.方法 将体外培养的THP-1细胞按完全随机方法分为4组:对照组(C组):给予脂肪乳20 mg/L;LPS刺激组(L组):给予LPS10mg/L;丙泊酚处理组(P组):给予丙泊酚20 mg/L;丙泊酚处理合并LPS刺激组(P+L组):给予丙泊酚20mg/L及LPS10 mg/L.在刺激后0.5、1、2、6h4个时间点通过免疫蛋白印迹分析(Western blot)法检测磷酸化p38MAPK (p-p38MAPK),磷酸化细胞外信号调节激酶(P-extracellular-signal regulated protein kinase,p-ERK)1/2及磷酸化c-Jun氨基末端激酶(p-c-Jun amino-terminal kinase,p-JNK)1/2含量的变化.结果 给予LPS刺激THP-1细胞0.5 h时,L组p-p38MAPK、p-ERK1/2及p-JNK1/2的相对灰度值分别为14.67±0.82、1.34±0.05、4.49±0.51,与C组比较表达均显著增加(P＜0.05).给予刺激1h时,L组p-p38MAPK、p-ERK1/2及p-JNK1/2的相对灰度值分别为11.78±0.75、0.58±0.05、3.31±0.55,与C组比较表达均显著增加(P＜0.05);P+L组p-ERK1/2的相对灰度值为0.14±0.02,与L组比较磷酸化水平显著降低(P＜0.05).给予刺激2h时,L组p-p38MAPK和p-JNK1/2的相对灰度值分别为15.60±0.96、8.33±0.70,与C组比较表达均显著增加(P＜0.05);P+L组p-p38MAPK和p-JNK1/2的相对灰度值分别为4.52±0.23、1.80±0.70,与L组比较磷酸化水平显著降低(P＜0.05).给予刺激6h时,L组p-p38MAPK及p-JNK1/2的相对灰度值分别为18.89±1.22、2.58±0.50,与C组比较表达均显著增加(P＜0.05);P+L组p-p38MAPK的相对灰度值为3.91±0.30,与L组比较磷酸化水平显著降低(P＜0.05).结论 丙泊酚抑制由LPS刺激THP-1细胞引起的p-p38MAPK、p-ERK1/2及p-JNK1/2表达增加,这可能是其抗炎的重要作用机制之一.     

活化血管内皮生长因子受体1诱导肝癌细胞株MHCC97-H细胞上皮-间叶表型转化的分子机制

目的 探讨活化血管内皮生长因子受体1(VEGFR-1)诱导肝癌细胞株MHCC97-H细胞上皮-间叶表型转化(EMT)的分子机制.方法 将MHCC97-H细胞分为对照组(1％胎牛血清的DMEM培养)、PP2组(10 μmol/L PP2培养)、PBS组(10 μmol/L PBS培养)、VEGF-B组(50 μg/L的VEGF-B培养)、PP2+ VEGF-B组(10 μmol/L PP2和50 μg/L的VEGF-B培养)、PBS+ VEGF-B组(10 μmol/L PBS和50 μg/L VEGF-B培养).Westem blot法检测各组上皮标志物E-钙黏蛋白、α-连环蛋白和间叶标志物波形蛋白、N-钙黏蛋白的表达水平;细胞免疫荧光检测上述蛋白的表达部位;细胞侵袭和迁移试验检测各组MHCC 97-H细胞的侵袭和运动能力.组间比较采用t检验.结果 对照组、PP2组、PBS组、VEGF-B组、PP2+ VEGF-B组、PBS+ VEGF-B组的MHCC97-H细胞上皮标志物E-钙黏蛋白的表达分别为3.23±0.76、4.18±0.32、2.83 +0.65、2.06±0.15、6.12±0.08、1.36±0.54;α-连环蛋白的表达分别为3.01 +0.25、3.29+0.11、3.03±0.27、2.84+0.76、5.45±0.37、1.26±0.45;波形蛋白的表达分别为3.01±0.22、4.85±0.36、1.37±0.24、5.79±0.38、3.36±0.42、4.05±0.17;N-钙黏蛋白的表达分别为2.63±0.40、3.02±0.52、2.98±0.36、5.54±0.28、3.26±0.13、1.05±0.33.PP2组、PP2+ VEGF-B组的MHCC97-H细胞E-钙黏蛋白和α-连环蛋白表达明显上调,PP2+ VEGF-B组与VEGF-B组比较,差异有统计学意义(t=7.625,9.931,P＜0.05).PP2+ VEGF-B组的波形蛋白和N-钙黏蛋白的表达显著低于VEGF-B组(t=12.001,11.910,P＜0.05).VEGF-B处理6h后,VEGF-B组、PP2+ VEGF-B组、PBS+ VEGF-B组的MHCC97-H细胞迁移数量分别为19±l、5±2和16±1,VEGF-B组MHCC97-H细胞迁移数量显著多于PP2+ VEGF-B组(t=13.566,P＜0.05),PP2+ VEGF-B组中MHCC97-H细胞穿过Matrigel包被的改良侵袭小室的数量为4±2,显著少于VEGF-B组的16±l(t=12.350,P＜0.05).结论 VEGFR-1活化诱导MHCC97-H细胞发生EMT是由c-Src激酶信号转导通路介导的,c-Src作为该信号通路的关键因子之一可能是干预肝细胞癌侵袭和转移的有效靶点.     

异丙酚预先给药对内毒素诱导大鼠肾小球血管内皮细胞通透性升高的影响

目的 探讨异丙酚预先给药对脂多糖(LPS)诱导大鼠肾小球血管内皮细胞通透性升高的影响.方法 分离、培养SD大鼠肾小球血管内皮细胞,以1×106/ml的密度接种于24孔培养板(200 μl/孔)和transwell小室(100 μl/室),采用随机数字表法,将其随机分为6组(n=10),正常对照组(C组)不作任何处理;脂肪乳对照组(I 组)加入10%脂肪乳4 μg/ml;异丙酚组(P组)加入异丙酚4μg/ml;LPS组(L组):加入LPS 10μg/ml;LPS+脂肪乳组(L+I组)加入10%脂肪乳4 μg/ml及LPS 10μg/ml;LPS+异丙酚组(L+P组)加入异丙酚4μg/ml 及LPS 10 μg/ml.于加入LPS前30 min加入脂肪乳或异丙酚,药物的浓度均为终浓度.加入LPS后6 h,收集细胞,采用逆转录-聚合酶链反应测定血管内皮细胞生长因子(VEGF)mRNA表达水平;收集上清液,采用酶联免疫吸附法测定VEGF浓度;测定血管内皮细胞通透性.结果 与C组比较,L组、L+I组和L+P组VEGF mRNA表达上调,上清液中VEGF浓度和血管内皮细胞通透性增加(P＜0.05),而I组和P组上述指标差异无统计学意义(P＞O.05).与L组比较,L+P组VEGF mRNA表达下凋,上清液中VEGF浓度和血管内皮细胞通透性降低(P＜0.05),L+I组上述指标差异无统计学意义(P＞0.05).上清液中VEGF浓度与血管内皮细胞通透性呈正相关(r=0.833,P＜0.05).结论 异丙酚预先给药可抑制LPS诱导大鼠肾小球血管内皮细胞通透性升高,其机制与下调VEGF表达有关.

Abstract：

Objective To investigate the influence of propofol pretreatment on the increased glomerular endothelial cell permeability induced by lipopolysaccharide (LPS) in rats.Methods Glomerular endothelial cells isolated from SD rats were cultured in 24-well plates(200 μl/well) and transwell filters (100 μl/filter) at 1×106/ml and assigned into 6 groups (n=10 each):control group (group C) , introlipid group (group I), propofol group (group P) , LPS group (group L), LPS+introlipid group (group L+I) and LPS+propofol group (group L +P). In group I, 10% introlipid 4 μg/ml was added. In group P, 4 μg/ml propofol was added. In group L, 10 μg/ml LPS was added. In group L+I, 10% introlipid 4 fig/ml combined with 10 μg/ml LPS was added. In group L+ P, 4 μg/ml propofol combined with LPS 10 μg/ml was added. Introlipid or propofol was added 30 min before the administration of LPS and the corresponding concentrations mentioned above were all final concentrations.After 6 h incubation with LPS, the cells were collected for measurement of vascular endothelial growth factor (VEGF) mRNA expression using RT-PCR. The supernatant was collected for determination of the VEGF concentration by ELJSA. The endothelial cell permeability was determined. Results Compared with group C, the expression of VEGF mRNA was up-regulated and the VEGF concentration and endothelial cell permeability were significantly increased in L, L+I and L + P groups (P＜0.05 ) ,but no significant change was found in the parameters mentioned above in I and P groups (P＞0.05). Compared with group L, the expression of VEGF mRNA was downregulated and the VEGF concentration and endothelial cell permeability were significantly decreased in L+P group (P＜0.05), but no significant change was found in the parameters mentioned above in group L+I(P＞0.05). A positive correlation existed between the concentration of VEGF and the permeability of endothelial cells(r= 0.833,P＜0.05).Conclusion Propofol pretreatment can decrease the increased glomerular endothelial cell permeability induced by LPS probably through down-regulation of VEGF expression.     

Effects of hypoxia-inducible factor-2α on the expression of tight junction proteins and permeability in rat glomerular endothelial cells under hypoxia condition

Objective To investigate the role of hypoxia-inducible factor-2α (HIF-2α) in the expression of tight junction proteins and permeability alterations in rat glomerular endothelial cells (rGENCs) under hypoxia condition. Methods The expressions of the HIF-2α and tight junction proteins such as occludin and ZO-1 of rGENCs were examined after exposed to 5% oxygen at different treatment time periods (0 h, 12 h, 24 h and 48 h). Then lentiviral transfection was used to knock down HIF-2α expression in rGENCs. The cells were split into four groups, including i) control group where rGENCs were cultured under normal oxygen conditions, ii) hypoxia group, iii) negative control group where rGENCs were infected with a negative vector, iv) HIF-2α lentivirus transfection group. Group ii, iii and iv were kept in hypoxic chamber (5% O2, 5% CO2 and 90% N2) for 24 h. The expressions of occludin, ZO-1 and HIF-2α were assessed by Western blotting. The permeability of rGENCs was measured using trans-epithelium electrical resistant (TEER) by Millicell? ERS voltohmmeter. Results With the elongation of hypoxia time, the expression of HIF-2α was increased gradually, while the occludin expression was decreased, there was statistically significance difference in each group (all P＜0.01). The expression of ZO-1 also decreased gradually under hypoxia circumstance, but no statistically significant was found between 24 h and 48 h groups (all P＞0.05). And a dramatic decrease in TEER of hypoxia cells was detected as compare with control cells (P＜0.01). After knockdown of HIF-2α expression, both expressions of occludin and ZO-1 were increased significantly compared with hypoxia cells (P＜0.01), and TEER elevated at the same time (P＜0.01). Above indexes had no statistical difference between hypoxia cells and negative control cells (all P＞0.05). Conclusion Hypoxia may promote HIF-2α expression, which could increase the permeability of rGENCs by reducing the expression of occludin and ZO-1.     

糖皮质激素受体对内毒素性急性肺损伤大鼠的保护作用及机制

目的 探讨糖皮质激素受体(glucocorticoid receptor,GR)在内毒素急性肺损伤(acute lung injury,ALI)中的作用及机制.方法 SD雄性大鼠84只,随机数字表法分为5组:Control组,仅注射生理盐水;脂多糖(lipopoIysaccharide,LPS)组,经尾静脉注射LPS 5 mg/kg;地塞米松(dexamethasone,DEX)+LPS组,注射LPS前30 min腹腔注射Dex 6 mg/kg;米非司酮(RU486)组,皮下注射GR拮抗剂RU486 20 mg/kg,90 min后经尾静脉注射生理盐水;RU486+Dex+LPS组,按照上述顺序分别注射RU486、Dex和LPS.Control组和RU486组6 h后,其余3组分别在1、3、6 h各时间点处死.检测各组大鼠支气管肺泡灌洗液(brobehoalveolar lavage fluid,BALF)中蛋白浓度,肺水系数(1ung index,LI),肺组织的病理变化,凋亡指数(apoptosis index,AI)以及肺组织中p38MAPK的活化状态及表达.结果 与Control组BALF中蛋白浓度(49±5)g/L、LI(2.36±0.14)、AI(12.0±1.7)%相比,LPS组分别为(77±9)g/L、5.93±0.44、(43.9±3.1)%(P＜0.05),HE染色显示肺组织炎症和损伤严重.与LPS组相比,Dex+LPS组分别为(54±4)g/L、3.77±0.48、(32.7±2.7)%(P＜0.05),且肺组织损伤程度减轻,应用GR抑制剂RU486后,Dex的肺保护作用消失.另外,LPS组肺组织中磷酸化p38MAPK(p-p38MAPK)的表达与Control组相比显著升高(P＜0.05);与LPS相比,Dex+LPS组p-p38MAPK表达下调(P＜0.05),而RU486+Dex+LPS组的表达上调(P＞0.05).结论 糖皮质激素受体在内毒素导致的急性肺损伤中发挥着重要的作用.激素活化的GR可能通过抑制p38MAPK的活化/磷酸化抑制肺组织细胞的凋亡,缓解肺损伤的程度.     

曲尼司特对环孢素A诱导的人肾小管上皮细胞向间充质转变的抑制作用

目的 研究曲尼司特(Tran)对环孢素A(CsA)诱导的人肾小管上皮细胞(HK-2)向间充质转变的影响,并探讨该药抗纤维化的机制.方法 所有用于实验的HK-2细胞株均为8～12代细胞,分为4组:(1)空白对照组,收获细胞,不做任何处理;(2)CsA组,加入4.2μmol/LCsA;(3)CsA+Tran组,预先加入100μmol/L Tran,作用2 h后再加入4.2 μmol/L CsA;(4)Tran组,仅加入100μmol/L Tran.72 h后于共聚焦显微镜下观察各组细胞形态学变化;用免疫荧光法以及免疫印迹法检测各组钙黏蛋白(E-cadherin)、平滑肌肌动蛋白α(α-SMA)和骨桥蛋白(OPN)的表达.结果 HK-2细胞在正常情况下表现为典型的"鹅卵石"样形态,细胞圆钝,且与邻近的细胞连接较为紧密;空白对照组和Tran组细胞表现为典型的HK-2细胞形态;CsA组细胞变狭长,甚至向周边伸出"伪足"样改变,细胞间连接较为稀疏;CsA+Tran组的细胞形态学改变有明显改善.CsA组细胞E-cadherin荧光表达强度明显弱于对照组,α-SMA、OPN荧光表达强于对照组;CsA+Tran组细胞E-cadherin荧光表达强于CsA组,α-SMA、OPN荧光表达弱于CsA组.免疫印迹检查中,CsA组细胞E-cadherin 的表达明显低于对照组,而α-SMA、OPN的表达明显高于对照组,CsA+Tran组细胞E-cadherin的表达高于CsA组,而α-SMA、OPN的表达低于CsA组.结论 曲尼司特能抑制CsA诱导的HK-2细胞由肾小管上皮向间充质细胞转化的过程,其机制可能与抑制OPN的表达有关.

Abstract：

Objective To study the effect of tranilast on cyclosporine A (CsA)-induced epithelial-to-mesenchymal transition in human renal tubular epithelial cells, and investigate the mechanism of its antifibrotic effect. Methods Cultured HK-2 cells were divided into four groups: (1)In the control group, cells were treated without any medicine; (2) The cell were treated with CsA (4. 2μmol/L) for 72 h; (3) The cells were treated with a combination of CsA (4. 2 μmol/L) and tranilast (100μmol/L); (4) The cells were treated with tranilast (100 μmol/L) alone for 72 h.Morphological changes of the cells were assessed by phase-contrast microscopy. The immunofluorescence and Western blotting were adopted to detect the expression of E-cadherin, α-SMA and OPN mRNA and proteins respectively. Results Tranilast could markedly ameliorate the morphological changes of HK-2 cells stimulated by CsA. The irmmunofluorescence staining revealed the expression of E-cadherin was markedly decreased in HK-2 cells stimulated with CsA for 72 as compared with the control group, while the expression of α-SMA and OPN was significantly higher in CsA group than the control group. The expression of E-cadherin in the CsA + Tranilast group was higher than the CsA group, while the expression of α-SMA and OPN in the CsA + Tranilast group was lower than the CsA group. Western blotting showed that protein expression level of E-cadherin in CsA group was dramatically lower than that in the control group (P＜0. 05), while that of α-SMA and OPN in CsA group was significantly higher than in the control group (P＜0.05). The protein expression level of E-cadherin in HK-2 cells in the CsA + Tranilast group was markedly higher than in the CsA group (P＜0.05), and that of α-SMA and OPN in CsA + Tranilast group was significantly lower than in the CsA group (P＜0. 05). Conclusion Tranilast can block the CsA-induced epithelialto-mesenchymal transition in HK-2 cells probably by suppressing the expression of OPN.     

毛蕊异黄酮对PI3K/Akt信号通路与PC-3细胞凋亡的作用

目的 探究毛蕊异黄酮促进前列腺癌细胞PC-3凋亡的机制.方法 选取0、25、50、100、200μmol/L的毛蕊异黄酮(Calycosin)处理PC-3细胞,并设置溶剂对照(DMSO,二甲基亚砜)组,采用MTT法检测48h后细胞增殖情况;流式细胞术检测0、25、50、100、200 μmol/L48h后细胞凋亡率;Western Blot法检测0μmoL/L组、200μmol/L毛蕊异黄酮组、200μmol/L毛蕊异黄酮+ PI3K特异性抑制剂(Wortmannin)共处理组细胞的Akt、磷酸化的Akt(p-Akt)、Bax、Bcl-2和Caspase-3蛋白的表达水平.结果 MTT实验显示毛蕊异黄酮能够抑制PC-3细胞的增殖,且呈现剂量依赖性(P＜0.05);流式细胞实验显示,0、25、50、100、200μmol/L浓度水平的毛蕊异黄酮处理PC-3细胞48h后的凋亡率分别为(0.1±0.04)％、(6.8±0.6)％、(9.2±0.2)％、(11.5±0.27)％、(59.2±0.36)％(P ＜0.05);Western blot法显示,与0μmol/L组相比,200μmol/L毛蕊异黄酮组p-Akt、Bcl-2表达水平下降(P＜0.05),加入Wortmannin后,二者水平再次下降;与0μmol/L组相比,200μmol/L毛蕊异黄酮组Bax、Caspsse-3表达水平增加(P＜0.05),加入Wortmannin后,二者水平再次增加.结论 毛蕊异黄酮可抑制PC-3细胞的增殖并诱导其凋亡,其机制是下调PI3K/Akt信号通路,启动线粒体诱导的凋亡途径.     

Effects of 12-lipoxygenase and angiotensinⅡ on p21, p27 and p57 in rat diabetic glomeruli

Objective To investigate the effects of 12-lipoxygenase (12-LO) and angiotensin Ⅱ(AngⅡ) on the CIP/KIP family of cyclin-dependent kinase inhibitors (CKIs) p21, p27 and p57 related to cell hypertrophy. Methods Mesangial cells were treated with high glucose for 24 hours and 48 hours respectively. 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] and AngⅡ were infused to rats by osmotic mini-pump for 1 week and 2 weeks respectively. Rats fed high fat diet were received low dose streptozotocin (STZ) to make type 2 diabetes (DN). The rats were divided into normal control group, DN group, DN+AngⅡ type 1 receptor blocker (ARB) group or 12-LO inhibitor (CDC) group. DN+ARB rats were treated by losartan for 6 weeks, and DN+CDC rats were treated for 8 weeks. Urine albumin and protein expressions of p21, p27 and p57 were detected by ELISA and Western blotting respectively. Glomeruli injury and expressions of p21 and p27 were detected by PAS staining and immunohistochemistry respectively. Results High glucose increased p21 and p27 protein expression in mesangial cells significantly compared with the relative control (all P＜0.05), but had no effect on p57. AngⅡ increased p27 protein expression in glomeruli significantly (P＜0.05), but had no effect on p21 and p57 protein expression. 12(S)-HETE increased both p21 and p27 protein expression in glomeruli significantly (all P＜0.05), but had no effect on p57 protein expression. Blood glucose, kidney/body weight, urinary protein, and glomerular p21 and p27 protein expressions were increased in DN group (all P＜0.05) compared with those in control group, with little change of p57 protein expression (P＜0.05). Moreover, glomerular hypertrophy and extra cellular matrix accumulation were observed in DN group. However, urine protein,kidney/body weight, renal injury, but not blood glucose, were decreased in DN+ARB group and DN+CDC group compared with DN group respectively (P＜0.05). Further DN+CDC rats had decreased both p21 and p27 protein expressions in glomeruli, but DN+ARB rats only had decreased p27 protein expression (all P＜0.05). Conclusions 12-LO may induce both p21 and p27 protein expression in DN glomeruli,but AngⅡ may induce only p27 expression.     

尿激酶型纤溶酶原激活物对高糖诱导大鼠系膜细胞增殖及表型转化的影响及其机制

目的 通过体外培养大鼠系膜细胞（MC）,观察尿激酶型纤溶酶原激活物（uPA）对高糖环境下MC增殖及表型转化的影响及其可能的信号转导机制。 方法 体外培养大鼠MC,分为4组：对照组、高糖组、高糖+渥曼青霉素组、高糖+uPA组。MTT法检测各组MC增殖情况。流式细胞仪分析各组MC细胞周期改变。Western印迹法检测各组MC表达CDK2与p27Kip1变化,并测定MC中信号蛋白Akt的活性。激光共聚焦显微镜检测各组MC中α-SMA表达方式及表达量变化。 结果 培养24 h后高糖组MC增殖程度较对照组显著增加（P < 0.01）,渥曼青霉素与uPA组可明显抑制细胞增殖（P < 0.01）。高糖刺激Akt活性,渥曼青霉素与uPA组Akt活性均较高糖组显著降低（均P < 0.01）。高糖组MC培养24 h后, p27Kip1蛋白表达较对照组显著减少（P < 0.01）;高糖+渥曼青霉素组、高糖+uPA组p27Kip1蛋白表达均较高糖组显著增加（均P < 0.01）;各组CDK2蛋白表达无明显变化。高糖组MC培养24 h后,胞质中α-SMA表达较对照组显著增加（P < 0.01）,并在核周出现聚集;高糖+渥曼青霉素组、高糖+uPA组α-SMA表达量均较高糖组显著减少（均P < 0.01）,其分布与对照组无显著差异。 结论 uPA可能通过抑制Akt信号分子活性,上调p27Kip1表达,拮抗高糖所致MC增殖与表型转化。     

雷公藤甲素对内毒素致大鼠急性肺损伤的影响

目的 评价雷公藤甲素对内毒素(LPS)致大鼠急性肺损伤的影响.方法 雄性SD大鼠65只,体重200 ～ 250 g,采用随机数字表法,将其随机分为5组,对照组(C组,n=5):尾静脉注射生理盐水,同时腹腔注射1％二甲基亚砜(DMSO);LPS组(L组,n=15)和不同剂量雷公藤甲素组(TP1～3组,n=15):尾静脉注射LPS 5 mg/kg,同时腹腔分别注射1％ DMSO和雷公藤甲素25、50、100μg/kg.于给药前1h和给药后1、3、6、12 h时行动脉血气分析;给药后12 h时心脏采血后处死大鼠,取肺组织,收集支气管肺泡灌洗液(BALF),采用ELISA法测定血清和BALF中TNF-α浓度;测定肺组织湿重(W)和干重(D),计算W/D比;光镜下观察肺组织病理学结果,并进行弥漫性肺泡损伤评分(DAD评分);采用荧光定量PCR法测定肺组织Toll样变体4(TLR4) mRNA表达;采用Western blot法测定肺组织TLR4蛋白表达.结果 与C组相比,L组和TP1～3组给药后3、6和12 h时PaO2下降,DAD评分及W/D比升高,L组、TP1组和TP2组血清和BALF中TNF-α浓度升高,肺组织TLR4 mRNA及其蛋白表达上调,TP3组血清和BALF中TNF-α浓度降低,肺组织TLR4mRNA及其蛋白表达下调(P＜0.05).与L组和TP1组相比,TP2组和TP3组给药后6和12 h时PaO2升高,DAD评分、W/D比、血清和BALF中TNF-α浓度降低,肺组织TLR4 mRNA及其蛋白表达下调(P＜0.05).与TP2组相比,TP3组血清和BALF中TNF-α浓度降低,肺组织TLR4 mRNA及其蛋白表达下调(P＜0.05).L组和TP1组间、TP2组和TP3组间血气指标、DAD评分及W/D比较差异无统计学意义(P＞0.05).TP1～3组肺组织病理学损伤较L组减轻.结论 雷公藤甲素可减轻LPS诱发的大鼠急性肺损伤,且与剂量有关,其机制与抑制TLR4表达的上调,减少TNF-α的释放有关.     

Effects of KIM-1 on high glucose induced the expression of MCP-1 and FN in rat tubular epithelial cells

Objective To evaluate the effects of KIM-1 on high glucose induced the expression of MCP-1 and FN in rat tubular epithelial cells and to explore the possible mechanisms of KIM-1 involved in renal interstitial fibrosis of DN. Methods The rat renal tubular epithelial cells (NRK52E) were cultured in vitro and divided into five groups: Normal control group (D-glucose 5.6 mmol/L), Hypertonic group (D-glucose 5.6 mmol/L＋D-mannitol 24.4 mmol/L), High glucose group (D-glucose 30 mmol/L), Control siRNA group,KIM-1 siRNA group. ELISA assay was used to assess the levels of MCP-1 and FN in the cells supernatant; Western blotting was used to detect the protein expression of KIM-1; RT-PCR was used to detect mRNA expression of KIM-1, MCP-1 and FN. Results Compared with the control group, the protein and mRNA expression of KIM-1 in the high glucose group were increased at 12 h (P＜0.05), and reached the peak at 48 h (P＜0.05); the protein and mRNA expression of MCP-1 and FN in high glucose group were increased at 24 h significantly (P＜0.05), and peaked at 48 h (P＜0.05). Compared with the high glucose group, the protein and mRNA expressions of MCP-1 and FN in KIM-1 siRNA group were decreased (P＜0.05). Conclusions Down-regulating the expression of KIM-1 can significantly inhibit the expression of MCP-1 and FN, which suggests that KIM-1 may be involved in renal interstitial fibrosis of DN by regulating expression of MCP-1 and FN.