单肺通气吸入不同氧分量对兔肺组织NF-κB的影响

Objective To study the effects of fraction of inspired oxygen on nuclear factor-κB (NF-κB) of lung tissue in rabbit one-lung ventilation(OLV) models.Methods 12 white Japanese rabbits were randomly divided into 2 groups(n=6).Bronchial intubation was performed with artificial double-lumen tube,and right OLV was conducted for 2 h and then tollowed tow-lung ventilation(TLV) for 1 h.Fraction of inspired oxygen was set as 1.0(group A) or 0.6(group B).NF-κB in lung tissue was detected,and arterial blood gases were analyzed before OLV,at 30 min of OLV and 30 min after TLV reversion.Pathology of lung tissue was examined and wet/dry(W/D) of lung weight was measured.Results After TLV restore,the oxygenation index was higher and lower than 300 in group B and group A,respectively.The W/D,the activation and the level of NF-κB in left lung tissue was less in group B than in group A (P＜0.01),and pathological change in left lung tissue was lighter in group B than in group A.Conclusion OLV with 60% oxygen may attenuate lung injury by decreasing the activation and the level of NF-κB in lung tissue.     

乌司他丁对大鼠肢体缺血再灌注时肺组织NF-κB表达的影响

目的 探讨乌司他丁对大鼠肢体缺血再灌注时肺组织NF-κB表达的影响.方法 雄性SD大鼠48只,体重230～260 g,采用随机数字表,将其随机分为3组(n=16):假手术组(S组)、肢体缺血再灌注组(I/R组)和乌司他丁组(U组).I/R组和U组采用夹闭双侧股动脉2 h再开放的方法 制备后肢缺血再灌注诱发肺损伤模型.分别于再灌注2、4 h时处死8只大鼠,取肺组织,计算肺组织湿/干重比,采用免疫组化法测定NF-κB表达水平,光镜下观察病理学结果.结果 与S组比较,I/R组肺组织湿/干重比升高,肺组织NF-κB表达上调(P＜0.05);与I/R组比较,U组肺组织湿/干重比降低,肺组织NF-κB表达下调(P＜0.05).U组肺组织损伤程度轻于I/R组.结论 乌司他丁可下调肺组织NFκB表达,从而减轻大鼠肢体缺血再灌注诱发肺损伤.

Abstract：

Objective To investigate the effects of ulinastatin on the expression of NF-κB in lung tissues during limb ischemia-reperfusion(I/R) in rats.Methods Forty-eight male SD rats weighing 230-260 g were randomly divided into 3 groups (n=16 each):sham operation group (group S);I/R group; ulinastatin group (group U).A rat model of lung injury induced by I/R of hind limbs which was produced by occlusion of the bilateral femoral arteries for 2 h followed by reperfusion was established.The rats were sacrificed at 2 and 4 h of reperfusion(8 rats at each time point) and the lung tissues removed for determination of NF-κB expression (by immuno-histochemistry) and microscopic examination.W/D lung weight ratio was calculated.Results W/D lung weight ratio was significantly increased and NF-κB expression was up-regulated in group I/R compared with group S(P＜ 0.05). W/D lung weight ratio was significantly decreased and NF-κB expression was down-regulated in group U compared with group I/R (P＜0.05). The lung injury induced by I/R was reduced in group U compared with group I/R. Conclusion Ulinastatin can reduce the lung injury induced by limb I/R by down-regulating the expression of NF-κB in rat lung tissues.     

联合内皮祖细胞移植对小鼠骨髓移植预处理中肝脏内皮损伤的修复作用

目的 研究联合内皮祖细胞(EPC)移植对小鼠骨髓移植预处理中肝脏内皮损伤的修复作用.方法 将C57BL/6小鼠分为4组,每组10只.(1)正常对照组:小鼠不做任何处理,仅作为正常对照;(2)单纯照射组:单次给予全身照射(TBI)预处理,不进行骨髓移植;(3)单纯移植组:给予单纯照射组相同的TBI预处理,TBI后4 h内经小鼠尾静脉输注C57BL/6小鼠骨髓单个核细胞5×106/只;(4)联合移植组,小鼠的处理方式与单纯移植组相同,仅在骨髓移植的同时经尾静脉输注C57BL/6小鼠EPC 5×105/只.TBI后第2、4、7、14、21天,检测各组小鼠肝脏重量的变化情况,并于TBI后第4、7、14、21天对各组小鼠肝脏进行组织病理学检查.结果 单纯照射组、单纯移植组和联合移植组小鼠肝脏重量均于TBI后第2天开始明显增加,于第14天达到高峰,峰值分别为正常对照组的(1.65±0.15)倍(P＜0.05)、(1.61±0.06)倍(P＜0.05)和(1.11±0.4(0)倍(P＜0.05);以后均呈下降趋势,第21天时单纯照射组和单纯移植组肝脏重量仍明显高于正常对照组(P＜0.05),但联合移植组小鼠肝脏重量已完全恢复正常.组织病理学检查显示,单纯照射组小鼠肝窦内皮损伤明显,肝细胞水肿及严重的炎症细胞浸润,第7天时肝细胞水肿、坏死较前明显加重,几乎无存活的肝血窦内皮细胞;第14天时单纯移植组小鼠肝窦内皮损伤较前有所减轻,但到第21天时仍未恢复正常;联合移植组小鼠第7天时肝窦内皮及肝细胞水肿、坏死程度均较轻,到第14天时已基本恢复正常.结论 造血干细胞移植前的预处理会造成受者肝脏内皮损伤,且此损伤持续存在;移植时联合输注EPC能修复肝窦内皮的损伤.

Abstract：

Objective To study the repair function of united endothelial progenitor cells (EPC)transplantation on injured liver endothelium by bone marrow transplantation (BMT) conditioning.Methods C57BL/6 mice were divided into four groups randomly: normal control group, without any treatment; irradiation alone group, administered a total body irradiation(TBI) pretreatment, without BMT; (3) BMT alone group: C57BL/6 mice were infused with bone marrow mononuclearcells (MNC) 5 × 106/only through caudal vein not more than 4 h after the same TBI pretreatment as the irradiation alone group; united transplantation group: receiving the same way as the BMT alone group, but C57BL/6 mice were infused with EPC 5 × 105/only at the same time. Two, 4, 7, 14, and 21 days after the TBI, the changes of the liver weight were observed regularly. The histopathological examination of liver was done at the 4th, 7th, 14th, and 21st day after the TBI. Results In irradiation alone group, BMT alone group and united transplantation group the liver weight began to increase significantly on the day 2 and peaked at 14th day after the TBI, and the peaks were respectively (1.65±0. 15) times (P＜0. 05), (1.61 ±0.06) times (P＜0.05), and (1.11 ±0.40)times (P＜0. 05) of those in normal control group. At the day 14, the liver weight in irradiation alone group, BMT alone group and united transplantation group began to decrease, and on the day 21 the liver weight in united transplantation group had been completely restored to normal level, however the liver weight in irradiation alone group and BMT alone group were still significantly heavier than that in normal control group (P＜0. 05). Liver histopathological examination revealed that there were obvious sinusoidal endothelial cells (SEC) injury, hepatocyte edema and severe inflammatory cell infiltration in irradiation alone group, and on the day 7 the hepatocyte edema and necrosis were significantly worse than before, and almost no alive SEC were found. On the day 14 the injury of SEC in BMT alone group was lighter than before, but on the day 21 the injury had not returned to normal. On the day 7 the injury of SEC, hepatocyte edema and necrosis were alleviated in united transplantation group as compared with irradiation alone group and BMT alone group, and on the day 14 the injury had returned to normal basically. Conclusion The transplantation conditioning could damage recipient liver endothelium and the injury would persist, and united EPC infusion could repair the injured SEC following BMT.     

艾拉莫德、吡咯烷二硫代氨基甲酸盐单用和联合治疗恶病质

目的 观察艾拉莫德(T614)、吡咯烷二硫代氨基甲酸盐(PDTC)联合应用对小鼠癌症恶病质的治疗作用.方法 于雄性BALB/C小鼠接种小鼠结肠腺癌Colon26(C26)细胞,9 d后恶病质模型基本建立.用药7 d后,记录小鼠左侧腓肠肌重量和去瘤体质量,检测血生化指标、血清白细胞介素6(IL-6)、肿瘤坏死因子-α(TNF-α)水平并用免疫组织化学法检测肿瘤组织核因子-κB(NF-κB)表达.结果 恶病质组小鼠血清IL-6、TNF-α浓度为(93.72 ±11.20)、(128.70±33.41)ng/L.T614组、PDTC组、T614+PDTC组IL-6浓度为(81.11±+11.08)、(79.25±9.91)、(53.60±10.5)ng/L,TNF-α浓度为(90.16±11.57)、(99.51±15.25)、(75.45±12.48)ng/L,均低于恶病质组(P＜0.05),各生化指标亦有不同程度的改善(P＜0.05).结论 通过抑制NF-κB可以改善恶病质.T614和PDTC联合应用治疗效果优于单种药物.

Abstract：

Objective To observe the effect of the combined use of iguratimod (T614) and pyrrolidine dithiocarbamate (PDTC) on animal cancer cachexia model. Methods Male BALB/C mice bearing colon 26 adenocarcinoma for 9 days served as models of cancer cachexia. Seven days after the treatment,body weight and gastrocnemius muscle were documented. Biochemical indicators, serum interleukin-6 (IL-6) , and tumor necrosis factor-α (TNF-α) levels were evaluated. The expression of nuclear factor-κB ( NF-κB ) in tumor was detected by using immunohistochemistry. Results The IL-6 levels in cachexia group, T614 group, PDTC group and T614 + PDTC group were (93. 72 ± 11. 20), (81.11 ± +11.08),(79. 25 ±9. 91) and (53. 60 ± 10. 5) ng/L, and those of TNF-a were (128. 70 ± 33. 41) , (90. 16 ±11. 57) , (99. 51 ± 15. 25) and (75. 45 ± 12. 48) ng/L, respectively. The IL-6 and TNF-α levels in T614 group, PDTC group and T614 + PDTC group were significantly lower than in cachexia group ( all P ＜0. 05). The expression of NF-kB was significantly down-regulated in T614 group, PDTC group and T614 +PDTC group as compared with cachexia group (P ＜ 0. 05). Conclusion Inhibition of NF-kB can attenuate the development of cachexia in colon 26 tumor bearing mice. These findings suggest that a therapeutic approach combining T614 with PDTC does have complementary effect in improving outcomes of cancer cachexia.     

脑死亡时猪肝脏功能及组织学改变的研究

Objective To observe how brain death affects the hepatic morphology and function of pigs and explore the roles of NF-κB. Methods Under general anaesthesia twelve healthy pigs were allocated randomly to two groups:control group(6 pigs),with non-inflacted Foley balloon catheter placed in the cerebral ventricle for 24 h,and brain death group,6 pigs,with estabhshment of brain death for 24 h.The serum and hepatic tissues in the same locus were taken at 6 h,12 h,and 24 h after the initial conformation of brain death.AST and ALT were determined by automatic biochemistry analyzer.IL-1βwas determined by ELISA.The NF-κB mRNA was determined by Real-time PCR and the NF-κB p65 by immunohistochemistry. Results The AST,ALT,IL-1β in serum,the NF-κB mRNA and the NF-κB p65 in hepatic tissues in brain death group were higher than those in control group and they all increased with the time(P<0.05).In brain death group,hepatocytes were edematous lightly after 12 hours,and the swelling progressively deteriorated after 24 hours,but there were no necrosis. Conclusion The activated NF-κB by brain death promoted the synthesis and release of inflammatory mediators,resulting in the hepatic dysfunction.     

脑死亡时猪肝脏功能及组织学改变的研究

Objective To observe how brain death affects the hepatic morphology and function of pigs and explore the roles of NF-κB. Methods Under general anaesthesia twelve healthy pigs were allocated randomly to two groups:control group(6 pigs),with non-inflacted Foley balloon catheter placed in the cerebral ventricle for 24 h,and brain death group,6 pigs,with estabhshment of brain death for 24 h.The serum and hepatic tissues in the same locus were taken at 6 h,12 h,and 24 h after the initial conformation of brain death.AST and ALT were determined by automatic biochemistry analyzer.IL-1βwas determined by ELISA.The NF-κB mRNA was determined by Real-time PCR and the NF-κB p65 by immunohistochemistry. Results The AST,ALT,IL-1β in serum,the NF-κB mRNA and the NF-κB p65 in hepatic tissues in brain death group were higher than those in control group and they all increased with the time(P<0.05).In brain death group,hepatocytes were edematous lightly after 12 hours,and the swelling progressively deteriorated after 24 hours,but there were no necrosis. Conclusion The activated NF-κB by brain death promoted the synthesis and release of inflammatory mediators,resulting in the hepatic dysfunction.     

艾拉莫德、吡咯烷二硫代氨基甲酸盐单用和联合治疗恶病质

Objective To observe the effect of the combined use of iguratimod (T614) and pyrrolidine dithiocarbamate (PDTC) on animal cancer cachexia model. Methods Male BALB/C mice bearing colon 26 adenocarcinoma for 9 days served as models of cancer cachexia. Seven days after the treatment,body weight and gastrocnemius muscle were documented. Biochemical indicators, serum interleukin-6 (IL-6) , and tumor necrosis factor-α (TNF-α) levels were evaluated. The expression of nuclear factor-κB ( NF-κB ) in tumor was detected by using immunohistochemistry. Results The IL-6 levels in cachexia group, T614 group, PDTC group and T614 + PDTC group were (93. 72 ± 11. 20), (81.11 ± +11.08),(79. 25 ±9. 91) and (53. 60 ± 10. 5) ng/L, and those of TNF-a were (128. 70 ± 33. 41) , (90. 16 ±11. 57) , (99. 51 ± 15. 25) and (75. 45 ± 12. 48) ng/L, respectively. The IL-6 and TNF-α levels in T614 group, PDTC group and T614 + PDTC group were significantly lower than in cachexia group ( all P ＜0. 05). The expression of NF-kB was significantly down-regulated in T614 group, PDTC group and T614 +PDTC group as compared with cachexia group (P ＜ 0. 05). Conclusion Inhibition of NF-kB can attenuate the development of cachexia in colon 26 tumor bearing mice. These findings suggest that a therapeutic approach combining T614 with PDTC does have complementary effect in improving outcomes of cancer cachexia.     

艾拉莫德、吡咯烷二硫代氨基甲酸盐单用和联合治疗恶病质

Objective To observe the effect of the combined use of iguratimod (T614) and pyrrolidine dithiocarbamate (PDTC) on animal cancer cachexia model. Methods Male BALB/C mice bearing colon 26 adenocarcinoma for 9 days served as models of cancer cachexia. Seven days after the treatment,body weight and gastrocnemius muscle were documented. Biochemical indicators, serum interleukin-6 (IL-6) , and tumor necrosis factor-α (TNF-α) levels were evaluated. The expression of nuclear factor-κB ( NF-κB ) in tumor was detected by using immunohistochemistry. Results The IL-6 levels in cachexia group, T614 group, PDTC group and T614 + PDTC group were (93. 72 ± 11. 20), (81.11 ± +11.08),(79. 25 ±9. 91) and (53. 60 ± 10. 5) ng/L, and those of TNF-a were (128. 70 ± 33. 41) , (90. 16 ±11. 57) , (99. 51 ± 15. 25) and (75. 45 ± 12. 48) ng/L, respectively. The IL-6 and TNF-α levels in T614 group, PDTC group and T614 + PDTC group were significantly lower than in cachexia group ( all P ＜0. 05). The expression of NF-kB was significantly down-regulated in T614 group, PDTC group and T614 +PDTC group as compared with cachexia group (P ＜ 0. 05). Conclusion Inhibition of NF-kB can attenuate the development of cachexia in colon 26 tumor bearing mice. These findings suggest that a therapeutic approach combining T614 with PDTC does have complementary effect in improving outcomes of cancer cachexia.     

延迟并连续小鼠异基因骨髓移植减轻移植物抗宿主病的探讨

目的 探讨延迟并连续小鼠异基因骨髓移植减轻急性移植物抗宿主病(aGVHD)的作用及其机制.方法 选择BALB/c(H-2d)/b鼠为受者,C57BL/6(H-2b)小鼠为供者.受者接受60Coγ射线全身照射(TBI)后,建立骨髓移植模型.实验分为5组.对照组:TBI后4 h经受者尾静脉输注RPMI 1640液0.4 ml;经典移植组:TBI后4 h经受者尾静脉输注供者的骨髓细胞(BMC)和脾细胞(SC)各1×107个/0.2 ml;连续移植组:TBI后4 h、1 d、2 d和3 d分4次经受者尾静脉输注供者的BMC和SC,每次输注量各为2.5 × 106个/0.05 ml;延迟移植组:TBI后第4天经受者尾静脉输注供者的BMC和SC各1× 107个/0.2 ml;延迟并连续移植组:TBI后第4、5、6和7天分4次经受者尾静脉输注供者的BMC和SC,每次输注量各为2.5×106个/0.05 ml.移植后观察受者的临床表现、病理学改变及存活时间,采用酶联免疫吸附试验(ELISA)测定受者外周血中白细胞介素(IL)-2、IL-4、IL-10及γ干扰素(IFN-γ)的水平,应用流式细胞仪测定受者H-2b细胞、T淋巴细胞亚群及自然杀伤(NK)细胞的百分率.结果 对照组和经典移植组受者均在TBI后3周内因造血功能衰竭和/或aGVHD死亡;连续移植组和延迟移植组受者TBI后60 d存活率分别为30%和50%;延迟并连续移植组受者TBI后60 d的存活率为70%,高于其它4组(P＜0.05).延迟并连续移植组IL-4、IL-10的表达高于经典移植组(P＜0.05);IL-2和IFN-γ的表达低于经典移植组(P＜0.05).延迟并连续移植组TBI后20 d时外周血白细胞计数恢复正常,30 d时外周血T淋巴细胞亚群和NK细胞的表达恢复正常,60 d时H-2b细胞的百分率为(98.13±1.11)%.结论 延迟并连续小鼠异基因骨髓移植能减轻移植后的aGVHD.其作用机制主要是避开了TBI后炎症冈子表达的高峰期,下调了炎症因子和Th1类细胞因子的表达,促进了Th2类细胞因子的表达.     

雌激素对大鼠肝切除肝缺血再灌注损伤中核因子-κB/IκB传导通路的影响及保护作用

目的 观察雌激素对大鼠肝切除肝缺血再灌注损伤中核因子-κB(NF-κB)/抑制蛋白(IκB)传导通路影响.方法 制作肝切除肝缺血再灌注损伤动物模型,雄性SD大鼠随机分为3组:假手术组(Sham组);肝切除肝缺血再灌注组(I/R组);肝切除肝缺血再灌注+雌激素组(I/R+ E2 组).分别在缺血再灌注后1、3、6h光镜下观察肝组织病理学改变,检测血清天冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)的水平和肝组织丙二醛(MDA)的含量及超氧化物岐化酶(SOD)的活性,免疫组织化学法测定肝组织NF-κB的表达,Western blot检测NF-κB抑制蛋白(IκB-α)和细胞间黏附分子-1(ICAM-1)表达,流式细胞仪检测细胞凋亡率.结果 再灌注后I/R组在各时相血清ALT、AST均显著高于I/R +E2组,并于6h达到峰值(P＜0.05).与I/R+E2组和Sham比较,I/R组肝细胞凋亡率显著升高(P＜0.01);肝组织中IκB-α表达降低,而NF-κB表达增高(P＜0.05);ICAM-1 和MDA的结果变化和NF-κB表达水平变化类似,SOD呈相反变化.在光镜下观察,I/R组肝小叶结构紊乱,肝窦淤血,肝细胞水肿变性,肝细胞片状坏死,在Sham组和I/R +E2组上述病理学变化明显改善.结论 雌激素对肝切除肝脏缺血再灌注损伤有显著保护作用,其作用机制可能与雌激素影响NF-κB/IκB传导通路、减轻脂质过氧化反应、减少炎症介质释放及抑制细胞凋亡有关.     

蛋白酶体抑制剂bortezomib对胆道梗阻大鼠肝脏的保护作用

目的探讨蛋白酶体抑制剂bortezomib对胆道梗阻大鼠肝脏的保护作用.方法将30只大鼠随机分为假手术组(SO组)、胆道梗阻对照组(Con组)和bortezomib实验组(Bor组).Con组通过胆总管结扎建立大鼠胆道梗阻模型,Bor组同法结扎胆总管并于术前1 d、术后第3天腹腔注射bortezomib,假手术组仅行剖腹和胆总管游离.所有大鼠均于术后7 d处死,处死前采集血清测定血清丙氨酸转氨酶(ALT),总胆红素(TB)和总胆汁酸(TBA)水平.免疫组化染色测定肝组织NF-κBp65含量.逆转录-聚合酶联反应测定肝脏组织中TNF-α mRNA水平.结果Con组与Bor组的TB和TBA水平并差异无统计学意义(P＞0.05),而Bor组的ALT水平[(92.4±21.4)μmo1/L]明显低于Con组[(145.7±33.5)μmol/L],P＜0.05.Bor组的NF-κB p65亚基阳性染色率(11.6%±2.7%)明显低于Con组(15.5%±4.3%),P＜0.05.而逆转录-聚合酶联反应发现,Bor组TNF-αmRNA相对表达量(1.0±0.2)明显低于Con组(1.3±0.4),P＜0.05.结论bortezomib可以通过抑制NF-κB的活化减少炎症反应的发生,从而减轻因胆道梗阻引起的肝脏损害.     

Mice with focal segmental glomerular sclerosis are induced by adriamycin and its mechanism

Objective To establish adriamycin-induced focal segmental glomerular sclerosis (FSGS) mice model, and observe the expressions of and relation between oxidative stress and p38 MAPK signal pathway in renal injury. Methods Eight-week-old male Balb/c mice were randomly divided into FSGS group (n=20) and control group (n=20). In FSGS group mice were intravenously injected with a single dose of adriamycin (0.01 mg/g), and mice in control group were received saline with the same dose. At day 3, 7, 14, 22 and 32, urine protein-to-urine creatinine ratio (P/C) was detected. At day 22 and 32, serum creatinine, blood urea nitrogen, nitric oxide (NO) and reactive oxygen species (ROS) in blood and urine, and ROS in kidney tissues were detected; changes of pathological morphology in renal tissue were analyzed by HE stain; the expressions of NF-κB, CD36, IL-13, BAX and Bcl-2 mRNA were detected by real time quantitative PCR; the expressions of NF-κB, p-p38 and p-ERK1/2 protein were detected by Western blotting. Results Compared with that in control group, P/C was gradually increasing in FSGS group, and peaked at day 22 (P＜0.05). At day 22 and 32, mice had higher creatinine, serum creatinine, urea nitrogen, ROS and NO in FSGS group than those in control group (all P＜0.05). There were mild hyperplasia of mesangial cells and mesangial matrix, segment with moderate exacerbations, podocytes with significant proliferation, and the capillary loops of the stenosed in the glomerular in FSGS group at day 32. Compared with those in control group, the mRNA expression of NF-κB, BAX, IL-13 and CD36, and the protein expressions of NF-κB and p-p38 MAPK were gradually increased in FSGS group, all showed statistical differences at day 32 (all P＜0.05); the expression of p-ERK1/2 was increased at day 22 (P＜0.05) but was reduced at day 32 (P＜0.05). Conclusions Adriamycin has induced FSGS in mice successfully, which may through oxidative stress activating p38, up-regulating NF-κB, increasing the inflammatory cytokines and inducing apoptosis pathways.     

Effect of high salt diet on the renal medullary cyclooxygenase 2 expression

Objective To examine the effect and mechanism of high salt diet on the renal medullary cyclooxygenase 2 (COX2) expression and urinary sodium excretion. Methods Thirty male C57BL/6j mice were divided into four groups: (1) normal salt diet group (0.4%NaCl, n=5); (2) high salt diet group (8% NaCl, n=5); (3) Bortezomib+normal salt diet group (n=10); (4) Bortezomib+high salt diet group (n=10). The different groups were pre-treated with saline or bortezomib，followed by normal salt diet or high salt diet for three days. All the mice were maintained on metabolic cage at the last day and allowed free access to water. Twenty-four hours urine was collected. Body weight, urine volumes were documented. At the end of experiments, mice were sacrificed under anesthesia and the kidneys were harvested for Western blotting and immunohistochemistry. The transgenic mice carrying a luciferase reporter driven by an NF-κB response promoter, HIV long terminal repeat (LTR) (HLL mice) were used to explore the effect of high salt diet on renal medullary NF-κB activity. HLL mice were fed with normal salt diet or high salt diet for 3 days, after which renal medullary luciferase activity was determined using a commercial luciferase assay kit. Luciferase activity was quantified with a luminometer and adjusted for the total amount of proteins. The cellular location of NF-κB was examined using immunohistochemistry of NF-κB p65 staining. Results (1) Western blotting results showed high salt diet significantly increased the COX2 expression in the renal medulla of C57BL/6j mice (P﹤0.05). (2) High salt diet significantly increased NF-κB luciferase reporter activity in the HLL mice renal medullary tissues when compared to normal salt diet (P﹤0.05). The immunohistochemistry of NF-κB p65 showed the expression of NF-κB was mainly in the renal interstitial cells. (3) Western blotting results showed bortezomib inhibited the renal medullar COX2 expression induced by high salt diet (P﹤0.05). (4) Bortezomib decreased the urinary sodium excretion of high salt diet mice (P﹤0.05), but had no change on urine volume. Conclusions High salt diet induce renal medullary COX2 over-expression and activate the activation of NF-κB in renal medullary. Bortezmoib can inhibit the renal medullar COX2 expression induced by high salt diet. The NF-κB pathway activation may involve in the regulation of renal medullar COX2 expression by high salt diet.     

Effects of 1,25-dihydroxyvitamin D3 on macrophage chemo taxis inhibitory factor and nuclear factor-κB/P65 in obstructive nephropathy rats

Objective To investigate the expression of macrophage migration inhibitory factor(MIF) and nuclear factor-κB/P65 (NF-κB/P65) in the kidneys of unilateral ureteral occlusion (UUO) model rats and the effect of 1,25-dihydroxyvitamin D3 on the expression. Method The cell model of obstructive nephropathy was established by renal tubular epithelial cells treated with TGF- beta 1. Thirty healthy adult male SD rats were randomly divided into 3 groups: sham operation group (n=10), UUO group (n=10) and 1,25-dihydroxyvitamin D3 group (n=10, UUO rats treated with 1,25-dihydroxyvitamin D3 by lavage 2 days before operation)．The rats in sham group and UUO group were treated with equal normal saline by lavage. Serum creatinine (Scr) and histopathological changes were tested at week 2. The expressions of collagen Ⅳ (ColⅣ), macrophage marker antigen ED-1, MIF and NF-κB/P65 in renal issue were measured by immunohistochemistry. The MIF mRNA was detected by real-time PCR and the protein expressions of MIF, NF-κB inhibitor α (IKBα) and p-IKBα were measured by Western blotting. In renal tubular epithelial cells (NRK52E) the expressions of MIF and NF-κB were detected by immunocytochemistry, and the the protein expression of MIF and the activation of IKBα were teasted by Western blotting. Results Compared with those in sham group, in model group rats had increaced Scr, tubulointerstitial damage area and expressions of ED-1 and ColⅣ, and up-regulated mRNA and protein expressions of MIF (all P＜0.05). Moreover, the amount of NF-κB/P65 nuclear positive cells and p-IKBα expression were significantly increased while the expression of IKBα decreased in model group (all P＜0.05). NRK52E cells had higher expressions of MIF, NF-κB and p-IKBα, and lower IKBα in model group than those in control group (all P＜0.05). After the application of 1,25-dihydroxyvitamin D3, those above effects were inhibited (all P＜0.05). The results of cell model and animal model were in agreement. Conclusions The expressions of MIF and the activation of NF-κB/P65 in UUO rats increased significantly. 1,25-dihydroxyvitamin D3 may ameliorate the progression of renal tubulointerstitial inflammation and renal fibrosis by intervening the expression of MIF, inducing phosphorylation of IKBα and decreasing the activation of NF-κB/P65.     

利用脊柱骨来源骨髓细胞建立急性移植物抗宿主病模型

目的探讨利用脊柱骨来源骨髓细胞建立小鼠异基因造血干细胞移植(allogeneic hematopoietic stem cell transplantation,Allo-HSCT)急性移植物抗宿主病(aGVHD)模型的可行性。方法选择C57BL/6(H-2b)雄性小鼠为供体鼠,BALB/c(H-2d)雌性小鼠为受体鼠。制备供体鼠的脾细胞和脊柱骨来源骨髓细胞悬液。受体鼠采用药物加小剂量辐照的预处理方式,于移植前8d～移植前4d腹腔注射氟达拉滨(200mg/kg),接着移植前3d～移植前1d腹腔注射环磷酰胺(60mg/kg),最后在移植前进行全身照射(total-body irradiation,TBI),照射剂量为4Gy(戈瑞)。18只受体鼠经预处理后随机分为3组,每组6只:(1)骨髓移植组,只输入1×107个脊柱骨来源骨髓细胞;(2)aGVHD组,输注1×107个脊柱骨来源骨髓细胞和5×106个脾细胞,建立aGVHD模型;(3)空白对照组,不输入任何细胞。观察3组小鼠生存状态及存活率,取aGVHD组与骨髓移植组存活21d的受体鼠进行病理学检查,取aGVHD组移植后21～28d存活的小鼠的脾脏进行流式细胞术检测骨髓细胞嵌合度。结果骨髓移植组小鼠全部存活,可重建造血,单纯输注骨髓细胞不会诱发aGVHD。aGVHD组小鼠出现aGVHD表现,100%发生aGVHD相关死亡,中位生存期为18d;病理检查结果显示符合aGVHD病理表现,移植后21～28d存活的小鼠诊断为供受体混合嵌合状态,符合aGVHD诊断标准。结论用脊柱骨来源骨髓建立的aGVHD模型完全符合标准,且更加经济,适合大规模建模。     

盐酸戊乙奎醚预先给药对内毒素性急性肺损伤大鼠肺组织NF-κB mRNA表达及SOD活性的影响

目的 探讨盐酸戊乙奎醚预先给药对内毒素性急性肺损伤大鼠肺组织NF-κB mRNA表达及SOD活性的影响.方法 健康雄性SD大鼠32只,月龄2月,体重230～280 g,随机分为4组(n=8),对照组(C组)腹腔和尾静脉均注射生理盐水1 ml/kg;急性肺损伤组(ALI组):腹腔注射生理盐水1 ml/kg,30 min后经尾静脉注射LPS 5 mg/kg;盐酸戊乙奎醚低剂量组(LP组)、高剂量组(HP组)分别腹腔注射盐酸戊乙奎醚0.3和1 mg/kg,30 min后经尾静脉注射LPS 5 mg/kg.静脉注射生理盐水或LPS后6 h时,取肺组织,检测NF-κB mRNA的表达、TNF-α和MDA的含量和SOD活性,计算肺组织湿/干重比(W/D)及含水量,观察肺组织病理学结果.结果 与C组比较,ALI组、LP组和HP组肺组织NF-κB mRNA表达上调,TNF-α及MDA含量升高,SOD活性降低,W/D和肺组织含水量升高(P＜0.05);与ALI组比较,LP组和HP组肺组织NF-κB mRNA表达下调,TNF-α及MDA含量降低,SOD活性升高,W/D和肺组织含水量降低(P＜0.05);与LP组比较,HP组肺组织NF-κB mRNA表达下调,TNF-α及MDA含量降低,SOD活性升高,W/D和肺组织含水量降低(P＜0.05).LP组和HP组肺组织病理学损伤较ALI组减轻.结论 盐酸戊乙奎醚预先给药减轻大鼠内毒素性急性肺损伤的机制可能与下调肺组织NF-κB mRNA表达,降低肺局部炎性反应,增强机体抗氧化能力有关.     

右美托咪定对老年患者全身麻醉术后认知功能障碍及炎症反应的影响

目的 观察右美托咪定(dexmedetomidine,Dex)对全身麻醉术后炎症反应及术后认知功能障碍(postoperative cognitive dysfunction,POCD)的影响. 方法 选择行择期腹部手术全身麻醉患者60例,年龄60～75岁,采用随机数字表法分为Dex组(D组)和对照组(C组),每组30例,两组患者均采用咪达唑仑0.03～0.05 mg/kg、芬太尼2～3 μg/kg、丙泊酚0.5～1.5 mg/kg、顺式阿曲库铵0.15 mg/kg静脉注射进行麻醉诱导.D组在诱导前10 min内静脉泵入1μg/kg Dex,随后以0.2～0.7 μg·kg-1·h-1维持泵注,根据患者HR、BP等变化及时调整输注速率;C组则与D组相同时间和途径注入相同容积的生理盐水.分别于麻醉后手术前(T1)、手术结束后即刻(T2)、手术结束后24 h(T3)抽取静脉血测定血浆IL-6含量及外周血中性粒细胞NF-κB表达水平.术前1d或2d及术后第1、3、7天用简易智能量表(mini-mental state examination,MMSE)测定认知功能,使用简易精神测定表(abbreviated mental test,AMT)评定术后谵妄情况. 结果 13例患者出现POCD(21.67％),其中D组3例,C组10例,两组间差异有统计学意义(P＜0.05).两组T2、T3时点外周血中性粒细胞NF-κB表达均较T1时增加(P＜0.05),但D组表达水平低于C组(P＜0.05).两组T2时点血浆IL-6水平均较T1时点明显升高(P＜0.01),且C组显著高于D组(P＜0.01);两组T3时点血浆IL-6水平较T2时点明显下降,但仍高于T1时点(P＜0.05),T3时点两组间差异无统计学意义(P＞0.05).D组外周血中性粒细胞NF-κB表达水平与血浆IL-6有良好的相关性(r=0.65,P＜0.01). 结论 POCD的发生可能与氧化应激反应有关,抑制NF-κB的激活可减少术后炎症反应及POCD的发生.     

慢性肾功能衰竭大鼠主动脉核因子κB活化及泛素通路的调节作用

目的　探讨慢性肾功能衰竭大鼠主动脉泛素（Ub）-NF-κB通路的表达变化规律。 方法　将大鼠随机分为假手术对照组（CON）、肾功能衰竭组（CRF）、肾功能衰竭加 MG-132干预组（CRF+M），观察6个月。采用部分肾动脉结扎加对侧肾切除法复制慢性肾功能衰竭大鼠模型。ELISA法测定血液中炎性因子白细胞介素1β（IL-1β）和肿瘤坏死因子α (TNF-α)的含量。RT-PCR半定量测定主动脉中NF-κB及Ub mRNA表达。免疫组织化学方法检测主动脉中NF-κB及Ub蛋白表达。Western印迹法测定主动脉泛素化蛋白的含量。凝胶电泳迁移率（EMSA）法测定动脉中NF-κB的活化情况。 结果　与CON组比较，CRF组大鼠术后4～6个月时血清中IL-1β[ (9.02±1.29)比(2.74±0.96) mg/L]和TNF-α[(50.02±9.52)比(14.04±1.29) mg/L]水平显著升高（P < 0.01）；主动脉NF-κB、Ub的 mRNA表达升高1.38倍和1.29倍（P < 0.01）；NF-κB、Ub的蛋白质表达升高 3.75倍和20.5倍（P < 0.01）；NF-κB活性增强1.82倍（P < 0.01），术后6个月各指标均有进一步上升趋势。与CRF组比较，CRF+M组大鼠干预治疗4～6个月后，大鼠血清中IL-1β[(2.94±0.33) mg/L]和TNF-α[(12.80±2.12) mg/L]含量显著下降（P < 0.01）；NF-κB 、Ub的mRNA及蛋白质表达显著减少（P < 0.01）；NF-κB的活性显著降低（P < 0.01），与CON组比较，差异已无统计学意义，而主动脉中泛素化蛋白含量显著升高（P < 0.01）。 结论 慢性肾功能衰竭大鼠主动脉泛素-NF-κB炎性反应信号明显活化，抑制蛋白酶体活性可能是降低主动脉NF-κB活化的重要药物靶点。