NLRP3/caspase-1/IL-1β信号通路在 HK-2细胞高糖缺氧复氧损伤中的作用

目的 探讨Nod样受体蛋3炎性体(nod-like receptor protein-3, NLRP3)/半胱天冬酶-1(caspase-1)/白介素-1β(interleukin-1β, IL-1β)信号通路介导的高糖缺氧复氧诱导人肾小管上皮细胞(human renal proximal tubular cells,HK-2)的损伤。方法 采用数字表法将人肾小管上皮细胞(HK-2)随机分为5组(n=5),即低糖组(NG组)、低糖缺氧复氧组(NHR组)、高糖组(HG组)、高糖缺氧复氧组(HHR组)和高糖缺氧复氧+NLRP3抑制剂BAY11-7082(5μmol/L)组(HHR-BAY组)。采用高糖(30mmol/L)刺激72h建立高糖模型,缺氧4h复氧2h建立缺氧复氧模型。CCK-8检测细胞存活率;酶标仪测定超氧化物歧化酶(superoxide dismutase, SOD)活性;荧光探针DCFH-DA法检测细胞内活性氧自由基(reactive oxygen species, ROS)含量;ELISA法检测IL-1β含量和caspase-1活性;免疫印迹法和免疫荧光法检测细胞NLRP3蛋白的表达。结果 与NG组比较,NHR组与HG组ROS和IL-1β含量,caspase-1活性,NLRP3表达升高,细胞存活率,SOD活性降低(P均<0.05);分别与HG组和NHR组比较,HHR组ROS和IL-1β含量,caspase-1活性,NLRP3表达升高,细胞存活率,SOD活性降低(P均<0.05);而NLRP3抑制剂BAY11-7082预处理可显著抑制细胞损伤和氧化应激水平,下调NLRP3蛋白水平,caspase-1活性和IL-1β含量(P均<0.05)。结论 NLRP3/caspase-1/IL-1β信号通路参与了高糖缺氧复氧诱导的HK-2细胞损伤过程。     

参附注射液对糖尿病大鼠心肌缺血再灌注时PTEN和P13 K表达的影响

Objective To investigate the effect of Shenfu injectio (SFI) on the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and phosphatidylinositol 3-kinase (PI3K) during myocardial ischemia-reperfusion (IR) in diabetic rats.Methods Thirty adult male diabetic SD rats weighing 220-280 g were used in this study. Diabetes mellitus was induced with intraperitoneal streptozotocin 60 mg/kg and confirmed by fasting blood glucose > 16.7 mmol/L. The animals were randomly divided into 3 groups ( n = 10 each): group I sham operation (group S); group II IR and group Ⅲ SFI. Myocardial IR was produced by occlusion of left anterior descending branch (LAD) of coronary artery for 30 min followed by 120 min reperfusion in group IR and SFI. LAD was exposed but not occluded in group S.SFI was infused iv at 10 ml·kg-1 ·h-1 before opening the thoracic cavity and at 3ml·kg-1·h-1 after opening the thoracic cavity in group SFI until the end of operation. Equal volume of Lactated Ringer's solution and hydroxyethyl starch was infused instead of SFI in group S and IR. The rats were killed and hearts removed at 120 min of reperfusion for microscopic examination and determination of cardiomyocyte apoptosis (by TUNEL)and expression of PTEN and PDK (by immunohistochemical method). PTEN/PI3K ratio, myocardial infarct size of left ventricle and apoptosis index were calculated.Correlation between apoptosis index and PTEN/PI3K ratio was analyzed. Results Myocardial infarct size and apoptosis index were significantly increased, while expression of PTEN and PI3K was up-regulated in group IR and SFI as compared with group S ( P < 0.05 or 0.01) . PTEN/PI3K ratio was significantly decreased in group SFI as compared with group S (P< 0.05). Myocardial infarct size and apoptosis index were significantly decreased, PTEN expression was down-regulated, PI3K expression was up-regulated and PTEN/PI3K ratio was significantly decreased in group SFI as compared with group IR( P < 0.05) . Myocardial pathological damage was attenuated in group SFI as compared with group IR. Apoptosis index was positively correlated to PTEN/PI3K ratio (r =0.452,P <0.05) .Conclusion SFI can attenuate myocardial IR injury via down-regulating the expression of PTEN,up-regulating the expression of PBK and activating PI3K/Akt signal pathway in diabetic rats.     

参附注射液对糖尿病大鼠心肌缺血／再灌注损期间PTEN表达的影响

目的 观察参附注射液(SFI)对心肌缺血/再灌注期间糖尿病大鼠PTEN表达的影响,探讨其对糖尿病心肌缺血/再灌注损伤保护作用分子机制.方法 健康雄性SD大鼠,腹腔注射链尿佐菌素60 mg/kg制备糖尿病模型.造模成功的30只大鼠再喂养8周,随机分为三组(n=10):假手术组(S组)、缺血/再灌注组(I/R组)、SFI防治组(SFI组).S组只穿线包绕冠状动脉左前降支,I/R组结扎冠状动脉左前降支30 min后松开制备心肌缺血/再灌注模型,SFI组开胸前持续泵注SFI 10 mL·kg-1·h-1,开胸后以3 mL·kg-1·h-1持续输注至手术结束,S组和I/R组开胸前持续泵注晶胶液(晶胶比为3∶ 1)10 mL·kg-1·h-1,开胸后以3 mL·kg-1·h-1持续输注至手术结束.再灌注120 min时处死大鼠计算左心室心肌梗死面积、检测心肌凋亡百分比和PTEN表达,并观察心肌组织病理变化.结果 与S组比较,I/R组、SFI组心肌梗死面积,心肌细胞凋亡百分比均增加,PTEN表达增强(P＜0.05或0.01);与I/R组比较,SFI组心肌梗死面积,心肌细胞凋亡百分比和PTEN表达均降低(P＜0.05或0.01).SFI组心肌组织病理损伤程度明显轻于I/R组.结论 SFI能减轻糖尿病心肌缺血/再灌注损伤,其机制与其抑制心肌细胞PTEN的表达、减少心肌细胞凋亡有关.     

参附注射液对糖尿病大鼠心肌缺血／再灌注期间磷脂酰肌醇3激酶表达的影响

目的 探讨参附注射液(SFI)对糖尿病大鼠心肌缺血/再灌注(I/R)损伤的保护作用及其分子机制.方法 采用腹腔注射链脲佐菌素(STZ)法建立糖尿病大鼠模型.取制模成功的SD大鼠30只,喂养8周后随机分成假手术(sham)组、I/R组、SFI组,每组10只.sham组大鼠对冠状动脉(冠脉)只穿线不结扎;其余两组大鼠通过结扎心脏左冠脉前降支30 min、再灌注120 min制备I/R模型.SFI组于开胸前持续泵注SFI 10 ml·kg-1·h-1,开胸后为3 ml·kg-1·h-1;其余两组泵注等量晶胶液.光镜下进行心肌组织病理学观察,测定各组心肌梗死面积;原位末端缺刻标记法(TUNEL)检测心肌细胞凋亡,计算细胞凋亡指数;免疫组化染色检测心肌磷脂酰肌醇3激酶(PI-3K)表达.结果 SFI组心肌梗死面积较I/R组明显减少[(36.32±2.72)%比(47.26±3.48)%,P<0.05].I/R组凋亡细胞显著增多;SFI组凋亡细胞较I/R组显著减少,而多于sham组(P均<0.05).与sham组和I/R组比较,SFI组的心肌PI-3K表达明显升高(P均<0.05).结论 SFI能明显减轻糖尿病时心肌I/R损伤,其机制可能与激活PI-3K/Akt信号通路而发挥心肌保护作用有关.     

参附注射液对糖尿病大鼠心肌缺血再灌注时PTEN和P13 K表达的影响

Objective To investigate the effect of Shenfu injectio (SFI) on the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and phosphatidylinositol 3-kinase (PI3K) during myocardial ischemia-reperfusion (IR) in diabetic rats.Methods Thirty adult male diabetic SD rats weighing 220-280 g were used in this study. Diabetes mellitus was induced with intraperitoneal streptozotocin 60 mg/kg and confirmed by fasting blood glucose > 16.7 mmol/L. The animals were randomly divided into 3 groups ( n = 10 each): group I sham operation (group S); group II IR and group Ⅲ SFI. Myocardial IR was produced by occlusion of left anterior descending branch (LAD) of coronary artery for 30 min followed by 120 min reperfusion in group IR and SFI. LAD was exposed but not occluded in group S.SFI was infused iv at 10 ml·kg-1 ·h-1 before opening the thoracic cavity and at 3ml·kg-1·h-1 after opening the thoracic cavity in group SFI until the end of operation. Equal volume of Lactated Ringer's solution and hydroxyethyl starch was infused instead of SFI in group S and IR. The rats were killed and hearts removed at 120 min of reperfusion for microscopic examination and determination of cardiomyocyte apoptosis (by TUNEL)and expression of PTEN and PDK (by immunohistochemical method). PTEN/PI3K ratio, myocardial infarct size of left ventricle and apoptosis index were calculated.Correlation between apoptosis index and PTEN/PI3K ratio was analyzed. Results Myocardial infarct size and apoptosis index were significantly increased, while expression of PTEN and PI3K was up-regulated in group IR and SFI as compared with group S ( P < 0.05 or 0.01) . PTEN/PI3K ratio was significantly decreased in group SFI as compared with group S (P< 0.05). Myocardial infarct size and apoptosis index were significantly decreased, PTEN expression was down-regulated, PI3K expression was up-regulated and PTEN/PI3K ratio was significantly decreased in group SFI as compared with group IR( P < 0.05) . Myocardial pathological damage was attenuated in group SFI as compared with group IR. Apoptosis index was positively correlated to PTEN/PI3K ratio (r =0.452,P <0.05) .Conclusion SFI can attenuate myocardial IR injury via down-regulating the expression of PTEN,up-regulating the expression of PBK and activating PI3K/Akt signal pathway in diabetic rats.     

瑞芬太尼持续泵注用于肾移植手术麻醉14例

目的 探讨瑞芬太尼持续微量泵输注用于肾移植手术麻醉的可行性。方法 肾移植手术24例，按麻醉维持用药不同分为A组14例，B组10例，两组麻醉诱导药物相同。A组持续泵注瑞芬太尼（0.1～0.2 μg·kg^-1·min^-1），持续吸入异氟醚（1%）并根据手术需要调节瑞芬太尼用量，手术结束前15 min停吸入麻醉药、5 min停止瑞芬太尼输入。B组持续吸入异氟醚（1%～3%）。记录两组各时段血流动力学变化、术中生命体征、麻醉恢复情况（睁眼时间、呼吸满意、拔管时间）。结果 术中两组生命体征维持稳定，应激指标无明显差异；A组麻醉苏醒时间及拔管时间明显短于B组（P＜0.05），且A组苏醒平稳和耐受气管导管情况良好，无呼吸遗忘及呼吸抑制。结论 瑞芬太尼持续微量泵输注复合异氟醚用于肾移植手术麻醉安全、有效，是较理想的麻醉方法。     

硫氧还蛋白相互作用蛋白在大鼠急性肾损伤中的作用

目的 探讨硫氧还蛋白相互作用蛋白（TXNIP）在大鼠急性肾损伤中的作用。方法 16 只雄性Sprague Dawley 大鼠随机分为假手术组（S）,缺血再灌注组（IR）,每组8 只。采用夹闭双侧肾蒂25 min,再灌注48 h 复制大鼠急性肾损伤模型。取大鼠肾脏HE 染色观察病理学结果,血标本测定血尿素氮（BUN）和血肌酐（Scr）水平,比色法测定超氧化物歧化酶（SOD）活性及丙二醛（MDA）含量,末端脱氧核苷酸转移酶介导的dUTP 缺口末端标记法检测细胞凋亡指数,Western blot 检测TXNIP 和炎性体3 蛋白（NLRP3）的表达,酶联免疫吸附测定法检测白细胞介素1β（IL-1β）。结果 与S 组比较,IR 组肾小管肿胀,间质水肿,刷状缘丢失,空泡变性坏死;与S 组比较,IR 组BUN,Scr,MDA,TXNIP,NLRP3 及IL-1β 表达增高（P <0.05）,凋亡指数增高（P <0.05）,SOD 活性降低（P <0.05）。结论 TXNIP 可能通过激活NLRP3/IL-1β 炎症通路参与大鼠急性肾损伤。     

参附注射液对糖尿病大鼠心肌缺血再灌注时PTEN和P13 K表达的影响

Objective To investigate the effect of Shenfu injectio (SFI) on the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and phosphatidylinositol 3-kinase (PI3K) during myocardial ischemia-reperfusion (IR) in diabetic rats.Methods Thirty adult male diabetic SD rats weighing 220-280 g were used in this study. Diabetes mellitus was induced with intraperitoneal streptozotocin 60 mg/kg and confirmed by fasting blood glucose > 16.7 mmol/L. The animals were randomly divided into 3 groups ( n = 10 each): group I sham operation (group S); group II IR and group Ⅲ SFI. Myocardial IR was produced by occlusion of left anterior descending branch (LAD) of coronary artery for 30 min followed by 120 min reperfusion in group IR and SFI. LAD was exposed but not occluded in group S.SFI was infused iv at 10 ml·kg-1 ·h-1 before opening the thoracic cavity and at 3ml·kg-1·h-1 after opening the thoracic cavity in group SFI until the end of operation. Equal volume of Lactated Ringer's solution and hydroxyethyl starch was infused instead of SFI in group S and IR. The rats were killed and hearts removed at 120 min of reperfusion for microscopic examination and determination of cardiomyocyte apoptosis (by TUNEL)and expression of PTEN and PDK (by immunohistochemical method). PTEN/PI3K ratio, myocardial infarct size of left ventricle and apoptosis index were calculated.Correlation between apoptosis index and PTEN/PI3K ratio was analyzed. Results Myocardial infarct size and apoptosis index were significantly increased, while expression of PTEN and PI3K was up-regulated in group IR and SFI as compared with group S ( P < 0.05 or 0.01) . PTEN/PI3K ratio was significantly decreased in group SFI as compared with group S (P< 0.05). Myocardial infarct size and apoptosis index were significantly decreased, PTEN expression was down-regulated, PI3K expression was up-regulated and PTEN/PI3K ratio was significantly decreased in group SFI as compared with group IR( P < 0.05) . Myocardial pathological damage was attenuated in group SFI as compared with group IR. Apoptosis index was positively correlated to PTEN/PI3K ratio (r =0.452,P <0.05) .Conclusion SFI can attenuate myocardial IR injury via down-regulating the expression of PTEN,up-regulating the expression of PBK and activating PI3K/Akt signal pathway in diabetic rats.     

参附注射液对糖尿病大鼠心肌缺血再灌注时PTEN和P13 K表达的影响

目的 探讨参附注射液(SFI)对糖尿病大鼠心肌缺血再灌注时第10染色体同源丢失性磷酸酶-张力蛋白酶基因(PTEN)和磷脂酰肌醇-3激酶(PI3K)表达的影响.方法 健康成年雄性SD大鼠,体重220-280 g,单次腹腔注射1%链脲佐菌素.柠檬酸盐缓冲液60 m~Ckg制备糖尿病模型,取糖尿病模型制备成功的大鼠30只,随机分为3组(n=10):假手术组(S组)、缺血再灌注组(IR组)和SH组.IR组和SFI组采用结扎冠状动脉左前降支30 min,再灌注120 min的方法建立心肌缺血再灌注模型,S组只穿线不结扎;SFI组开胸前以10ml·kg-1·h-1 的速率静脉输注SFI,开胸后以3 ml·kg-1·h-1的速率静脉输注至术毕,S组和IR组均静脉输注等容量乳酸钠林格氏液和羟乙基淀粉.于再灌注120 min时处死大鼠,取左心室心肌组织,计算心肌梗死面积.采用TUNEL法检测心肌细胞凋亡情况,计算细胞凋亡指数.采用免疫组织化学法测定心肌组织PTEN和PI3K的表达水平,并计算其比值(PTEN/PI3K).观察心肌组织病理学结果.细胞凋亡指数与PTEN/PI3K行直线相关分析.结果 与S组比较,IR组和SFI组心肌梗死面积增加,细胞凋亡指数升高,PTEN和PI3K的表达上调(P<0.05或0.01),SFI组PTEN/PI3K降低(P<0.05);与IR组比较,SFI组心肌梗死面积减小,细胞凋亡指数降低,FTEN表达下调,PI3K表达上调,PTEN/PI3K降低(P<0.05);SFI组心肌损伤程度比IR组减轻.细胞凋亡指数与PTEN/PI3K呈正相关(r=0.452,P<0.05).结论 SFI减轻糖尿病大鼠心肌缺血再灌注损伤的机制与下调心肌组织FTEN表达,上调PI3K表达,从而激活PI3K/Akt信号通路有关.     

参附注射液对糖尿病大鼠心肌缺血再灌注时PTEN和P13 K表达的影响

Objective To investigate the effect of Shenfu injectio (SFI) on the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and phosphatidylinositol 3-kinase (PI3K) during myocardial ischemia-reperfusion (IR) in diabetic rats.Methods Thirty adult male diabetic SD rats weighing 220-280 g were used in this study. Diabetes mellitus was induced with intraperitoneal streptozotocin 60 mg/kg and confirmed by fasting blood glucose > 16.7 mmol/L. The animals were randomly divided into 3 groups ( n = 10 each): group I sham operation (group S); group II IR and group Ⅲ SFI. Myocardial IR was produced by occlusion of left anterior descending branch (LAD) of coronary artery for 30 min followed by 120 min reperfusion in group IR and SFI. LAD was exposed but not occluded in group S.SFI was infused iv at 10 ml·kg-1 ·h-1 before opening the thoracic cavity and at 3ml·kg-1·h-1 after opening the thoracic cavity in group SFI until the end of operation. Equal volume of Lactated Ringer's solution and hydroxyethyl starch was infused instead of SFI in group S and IR. The rats were killed and hearts removed at 120 min of reperfusion for microscopic examination and determination of cardiomyocyte apoptosis (by TUNEL)and expression of PTEN and PDK (by immunohistochemical method). PTEN/PI3K ratio, myocardial infarct size of left ventricle and apoptosis index were calculated.Correlation between apoptosis index and PTEN/PI3K ratio was analyzed. Results Myocardial infarct size and apoptosis index were significantly increased, while expression of PTEN and PI3K was up-regulated in group IR and SFI as compared with group S ( P < 0.05 or 0.01) . PTEN/PI3K ratio was significantly decreased in group SFI as compared with group S (P< 0.05). Myocardial infarct size and apoptosis index were significantly decreased, PTEN expression was down-regulated, PI3K expression was up-regulated and PTEN/PI3K ratio was significantly decreased in group SFI as compared with group IR( P < 0.05) . Myocardial pathological damage was attenuated in group SFI as compared with group IR. Apoptosis index was positively correlated to PTEN/PI3K ratio (r =0.452,P <0.05) .Conclusion SFI can attenuate myocardial IR injury via down-regulating the expression of PTEN,up-regulating the expression of PBK and activating PI3K/Akt signal pathway in diabetic rats.