Drp1介导线粒体能量代谢拮抗大鼠缺血再灌注肾损伤

目的　探讨线粒体动力相关蛋白1（Drp1）介导线粒体能量代谢参与缺血再灌注肾损伤的分子机制。方法　实验时间2020年10月14日。8～10周龄雄性Wistar大鼠随机分为正常对照组、假手术组、模型组和Drp1抑制剂组,每组5只,每只体质量为0.25～0.30 kg。通过手术建立大鼠肾脏缺血再灌注急性肾损伤模型,Drp1抑制剂组大鼠腹腔注射线粒体分裂抑制剂（mitochondrial division inhibitor 1,Mdivi-1）20 mg/kg体质量,其余各组大鼠注射等体积生理盐水,造模24 h留取血清及肾脏组织。比较组间大鼠血肌酐水平、肾组织病理学改变、肾组织细胞凋亡情况、线粒体超微结构、线粒体ATP酶活力,并建立体外HK-2细胞缺氧复氧模型,经处理后JC-1染色法检测细胞线粒体膜电位变化。采用方差分析。结果　与假手术组比较,模型组大鼠血肌酐明显升高［（41.12±1.895）µmol/L比（48.68±2.065）µmol/L］,肾小管损伤评分升高［（1.29±0.426）分比（6.50±0.577）分］,每高倍镜视野下大鼠肾组织细胞凋亡数量增加［（2.40±0.547）个比（10.20±1.095）个］,电镜下线粒体损伤更明显,线粒体ATP酶活力降低［（6.38±0.321）U/mg prot比（4.18±0.198）U/mg prot］,HK-2细胞缺氧复氧模型中,模型组较假手术组线粒体膜电位降低的细胞比例增多［（9.81±0.251）%比（4.24±0.598）%］,差异均有统计学意义（均P<0.05）。与模型组相比,Drp1抑制剂组大鼠血肌酐下降［（48.68±2.065）µmol/L比（43.28±0.895）µmol/L］,肾小管损伤评分降低［（6.50±0.577）分比（4.50±0.578）分］,每高倍镜视野下大鼠肾组织细胞凋亡数量减少［（10.20±1.095）个比（6.60±1.140）个］,线粒体结构损伤减轻,细胞线粒体ATP酶活力增加［（4.18±0.198）U/mg prot比（5.16±0.628）U/mg prot］,HK-2细胞缺氧复氧模型中,Drp1抑制剂组较模型组线粒体膜电位降低的细胞比例减少［（5.90±0.360）%比（9.81±0.251）%］,差异均有统计学意义（均P<0.05）。结论　选择性抑制Drp1可通过抑制线粒体分裂,有效改善能量代谢障碍,减少细胞凋亡,减轻肾缺血再灌注损伤。     

Exendin-4对h IAPP诱导的胰岛β细胞凋亡的保护作用

目的 探究Exendin-4对人源胰岛淀粉样多肽（h IAPP）诱导的胰岛β细胞凋亡的保护作用及其可能的作用机制。方法 体外培养胰岛β细胞INS-1E，加入2.5、5、10、20μmol/L的hIAPP培养48 h，建立hIAPP诱导细胞凋亡模型。20、50、100 nmol/L Exendin-4预处理24 h,CCK-8法检测细胞活力，筛选有效的药物剂量。随后实验分为空白组、Exendin-4组、h IAPP模型组、h IAPP+Exendin-4组以及阳性对照组。LDH释放检测法分析膜通透性变化；化学发光法测定细胞内腺苷三磷酸（ATP）水平；JC-1荧光探针法检测线粒体膜电位的改变；Western blot检测线粒体凋亡相关蛋白Bcl-2、Bax以及Bad的表达。结果 20μmol/L的hIAPP可以明显抑制INS-1E细胞增殖，增加LDH的释放，增加细胞内ATP的消耗，增加去极化线粒体膜电位比例，下调抗凋亡蛋白Bcl-2的表达，上调促凋亡蛋白Bax和Bad的表达（均P<0.05）。与hIAPP组相比，h IAPP+Exendin-4组能够显著提高细胞活力，减少LDH释放，...     

异丙酚对内毒素诱导人单核细胞系THP-1细胞TREM-1表达的影响

【摘要】目的 探讨异丙酚对内毒素诱导人单核细胞系THP-1细胞髓样细胞触发性受体-1（TREM-1）表达的影响。方法 培养THP-1细胞,分成7组,分别对应：Control组,单纯无血清培养基培养;内毒素（LPS）组,单纯LPS100μg/L刺激;Pro组,单纯加入异丙酚100μmol/L;P1组,LPS+异丙12.5μmol/L;P2组,LPS+异丙酚25μmol/L;P3组,LPS+异丙酚50μmol/L;P4组,LPS+异丙酚100μmol/L。观察16h后收集细胞培养上清液采用ELISA测定肿瘤坏死因子（TNF）-α水平;收集细胞采用流式细胞术检测TREM-1蛋白表达水平;收集细胞采用实时定量PCR检测TREM-1mRNA表达水平。结果 与LPS组相比,P3组与P4组的细胞表面TREM-1蛋白表达水平明显增高（P<0.05）,细胞上清液中TNF-α水平明显下降（P<0.05）;与Control组比较,LPS组TREM-1mRNA表达明显增加（P<0.05）;与LPS组比较,P4组细胞TREM-1mRNA表达水平降低（P<0.05）。结论 异丙酚可使LPS诱导的THP-1细胞表面TREM-1蛋白表达水平增加,细胞TNF-α的释放减少,TREM-1mRNA的表达量下降。     

番石榴叶水提物对四氧嘧啶诱导体外胰岛INS-1细胞凋亡的保护作用及其机制

目的 探讨番石榴叶(PGL)水提物对四氧嘧啶(ALX)诱导的体外INS-1细胞凋亡的保护作用及其可能的机制。方法 以大鼠胰岛细胞瘤细胞株INS-1建立体外模型,利用CCK-8法测定药物对INS-1细胞活力的影响;采用化学自发光法测定各组细胞的细胞内ATP含量;采用Hoechst33342法测定凋亡形态学;采用Annexin V-FITC法测定细胞凋亡;采用DCFH-DA法测定细胞内活性氧含量;采用Rhodamin 123法检测细胞线粒体膜电位;采用Western blot法测定Bcl-2、Bax、Cleaved-Caspase 3、GRP78、CHOP、Nrf2、HO-1蛋白的表达量。结果 PGL水提物各剂量组的细胞活力、细胞内ATP含量、线粒体膜电位及Bcl-2、Nrf2及HO-1等凋亡及抗氧化应激相关蛋白的表达量和Nrf2的核转位程度均高于ALX20组,差异有统计学意义(P<0.05);PGL水提物各剂量组的细胞核浓染、核固缩及凋亡细胞的比例、细胞内活性氧含量以及Bax、Cleaved-Caspase 3、GRP78及CHOP等凋亡及内质网应激相关蛋白的表达均低于ALX组,差...     

右美托咪定对高氧诱导的人肺泡上皮细胞线粒体损伤及HIF-1α/ERK通路的影响

摘要：目的:观察右美托咪定（Dex）对高氧诱导的人肺泡上皮细胞HPAEpiC线粒体损伤及缺氧诱导因子-1α（HIF-1α）/细胞外信号调节激酶（ERK）通路的影响。方法:培养HPAEpiC细胞,分为对照（Control）组、高氧模型（Hyperoxia）组、高氧和药物处理（Hyperoxia+Dex）组、高氧和HIF-1α激动剂处理（Hyperoxia+DMOG）组以及高氧和药物、HIF-1α抑制剂处理（Hyperoxia+Dex+YC-1）组。采用活性氧（ROS）检测试剂盒和MitoSOXTM?Red荧光染色分别检测细胞中和线粒体中ROS含量;线粒体膜电位检测试剂盒（JC-1法）检测线粒体膜电位;流式细胞仪检测细胞凋亡率;Western blot检测细胞中HIF-1α/ERK通路相关蛋白及核呼吸因子-1（NRF-1）蛋白水平。结果:与Control组比较,Hyperoxia组HPAEpiC细胞中和线粒体中ROS含量升高,线粒体膜电位降低,细胞凋亡率增高,细胞中HIF-1α、p-ERK和NRF-1蛋白水平降低（P<0.05）。与Hyperoxia组比较,Hyperoxia+Dex组和Hyperoxia+DMOG组HPAEpiC细胞中和线粒体中ROS含量降低,线粒体膜电位升高,细胞凋亡率降低,细胞中HIF-1α、p-ERK和NRF-1蛋白水平升高（P<0.05）。与Hyperoxia+Dex组比较,Hyperoxia+DMOG组和Hyperoxia+Dex+YC-1组HPAEpiC细胞中和线粒体中ROS含量升高,线粒体膜电位降低,细胞凋亡率增高,细胞中HIF-1α、p-ERK和NRF-1蛋白水平降低（P<0.05）。结论:Dex可能通过激活HIF-1α/ERK通路减轻高氧诱导的人肺泡上皮细胞线粒体损伤。     

去氢骆驼蓬碱诱发PC12细胞凋亡时对线粒体融合分裂的影响

目的 探讨去氢骆驼蓬碱(HM)对PC12细胞线粒体功能损伤和线粒体融合分裂相关蛋白表达水平的影响。方法 PC12细胞分为细胞对照组、HM组、线粒体分裂抑制剂Mdivi-1组、HM+Mdivi-1组、线粒体裂变激动剂WY14643组、HM+WY14643组,药物浓度均为1、10、25、50、100μmol·L^-1,处理24 h,噻唑蓝(MTT)法检测细胞存活率,显微镜观察细胞形态;MitoTracker Red探针染色观察线粒体形态和纵横轴长度比值,JC-1染色检测线粒体膜电位,试剂盒检测ROS、ATP水平和乳酸脱氢酶(LDH)活性,免疫荧光染色法和Western blotting检测胱天蛋白酶3(caspase-3)、促凋亡蛋白(Bax)、细胞色素C(cyt-c)和线粒体融合蛋白(Mfn2)、线粒体分裂蛋白(Drp-1)的表达水平;电穿孔法转染Drp1的干扰序列,筛选转染效果好的siRNA序列,协同药物干预,通过荧光法、MTT法、免疫印迹法检测相关指标。结果 MTT结果显示,与细胞对照组比较,HM组、Mdivi-1组、HM+Mdivi-1组、WY14643组和HM...     

大黄素诱导人肝癌HepG2细胞线粒体凋亡作用研究

目的 研究大黄素对人肝癌HepG2细胞线粒体凋亡的影响。方法 培养人肝癌HepG2细胞,与5、10、20、40、60、80、100 μmol/L的大黄素作用24、48 h,MTS法检测细胞增殖;40、80、160 μmol/L大黄素作用HepG2细胞24 h,AO/EB双荧光染色法观察细胞凋亡的形态学改变;Annexin V/PI染色经流式细胞仪检测细胞凋亡;分光光度法检测caspase 3活性;ATP试剂盒检测细胞ATP含量,不同荧光探针加载后流式细胞仪测定大黄素对HepG2细胞内活性氧（ROS）含量、Ca²⁺浓度、线粒体膜电位（MMP）变化的影响。结果 大黄素抑制HepG2细胞生长,且呈时间、浓度相关性,半数抑制浓度（IC₅₀）为（77.42±1.25）μmol/L;随着大黄素浓度升高,AO/EB双染观察到细胞核浓缩、碎裂、凋亡小体等凋亡形态;与对照组比较,大黄素40、80、160 μmol/L作用于HepG2细胞24 h后细胞凋亡率显著增加,caspase 3活性显著增强,ROS水平、Ca²⁺浓度明显增加（P<0.05、0.01、0.001）,80、160 μmol/L组线粒体膜电位明显降低,ATP含量显著下降（P<0.05、0.01、0.001）。结论 大黄素造成HepG2细胞内ROS堆积,ATP合成功能障碍,线粒体膜电位明显下降,进而诱导线粒体通透转运孔开放,导致钙离子和细胞色素C外流,活化caspase蛋白家族,导致细胞凋亡。     

烟酰胺核糖在视神经节细胞中的线粒体保护作用

目的 探究烟酰胺核糖（NR）在羰基氰化物间氯苯腙（CCCP）所诱导的R28细胞氧化应激损伤模型中的保护作用。方法 使用4μmol/L CCCP诱导R28细胞产生氧化应激,并使用400 nmol/L NR进行干预,CCK-8实验检测细胞活力,Annexin-V/PI双染流式细胞术检测细胞凋亡,蛋白质印记法检测细胞色素C、半胱氨酸蛋白酶-3、半胱氨酸蛋白酶-9表达水平以评估细胞凋亡;同时用四甲基罗丹明乙酯检测线粒体膜电位（MMP）,MitoSOX检测线粒体活性氧（mtROS）水平,三磷酸腺苷（ATP）试剂盒检测ATP生成能力以评价线粒体功能。结果 CCCP处理R28细胞后,细胞活力下降,凋亡蛋白水平和凋亡率上升,MMP下降,mtROS生成增加（P <0.05）;NR预处理后,细胞活力上升,凋亡蛋白水平和细胞凋亡率下降,MMP上升,mtROS生成减少（P <0.05）。结论 NR通过抑制氧化应激反应,保护线粒体功能,从而增强细胞活性,降低凋亡蛋白的表达,最终减少视网膜神经节细胞凋亡。     

五味子乙素对HK-2细胞缺氧损伤的保护作用

卢爱龙 1, 谭小月 2, 张勉之 3△, 吴银娜摘要： 目的 探讨五味子乙素 （Sch B） 对氯化钴 （CoCl2） 诱导的人类近端肾小管上皮 （HK-2） 细胞缺氧损伤的保护作用及其可能机制。方法 取离体培养 HK-2 细胞, 随机分为 4 组。对照 （C） 组： 细胞未经任何处理。CoCl2组 （化学乏氧组）： 加入 600 μmol/L 的 CoCl2 处理 24 h。Sch B 预保护（CoCl2+ Sch B）组： 分别加入终浓度为 1 μmol/L 和 10 μmol/L Sch B 预处理 2 h 后, 其余操作同 CoCl2 组。Sch B 组： 分别加入终浓度 1 μmol/L 和 10 μmol/L Sch B 处理 2 h。CCK-8 试剂盒检测各组细胞活性; AnnexinV-FITC/PI 双标记流式细胞仪检测各组细胞凋亡率; Western Blot 检测各组缺氧诱导因子-1α（HIF-1α）蛋白表达; RT-PCR 检测各组 HIF-1α和诱导型一氧化氮合酶（iNOS） mRNA 表达。结果 与对照组相比, CoCl2组细胞活性明显降低, 细胞凋亡率、 HIF-1α蛋白表达量和 iNOS mRNA 表达量显著增加, HIF-1α mRNA 表达量差异无统计学意义; Sch B 预保护组较 CoCl2组细胞活性显著增加, 细胞凋亡率、 HIF-1α蛋白表达量、 HIF-1α及 iNOS mRNA 表达量均显著减少; Sch B 组与对照组细胞活性、 细胞凋亡率差异无统计学意义, Sch B 组几乎不表达 HIF-1α蛋白。结论 Sch B 可能通过抑制 HIF-1α蛋白和 iNOS mRNA 的表达减少 HK-2 细胞的凋亡, 从而对 HK-2 细胞缺氧损伤起保护作用。     

注射用益气复脉(冻干)对阿霉素诱导H9c2(2-1)心肌细胞毒性的保护作用

目的 探讨注射用益气复脉（冻干,YQFM）对阿霉素（doxorubicin,DOX）诱导H9c2（2-1）心肌细胞毒性的保护作用。方法 H9c2（2-1）心肌细胞随机分为对照组,模型组（终浓度为0.3 μmol/L的DOX处理细胞48 h）,YQFM低、中、高剂量（125、625、3 125 μg/mL）组（提前2 h加入药物预处理,然后加入终浓度为0.3 μmol/L的DOX处理48 h）,采用CCK-8法检测细胞活力;使用乳酸脱氢酶（LDH）和ATP试剂盒检测细胞LDH和ATP水平;应用Hoechst 33258染色法检测细胞凋亡;JC-1法检测细胞线粒体膜电位;Western blotting法检测caspase-3蛋白的表达水平。结果 与模型组比较,YQFM中、高剂量组显著增加细胞存活率（P<0.05、0.01）,低、高剂量组明显改善细胞凋亡;低、中、高剂量组LDH活性显著降低（P<0.05、0.01）,ATP含量显著增加（P<0.05、0.01）,线粒体膜电位明显恢复。结论 YQFM抑制DOX诱导H9c2（2-1）的细胞毒性,其作用机制可能与抑制线粒体凋亡信号通路的激活有关。     

Carbon monoxide-releasing molecules (CO-RMs) attenuate the inflammatory response elicited by lipopolysaccharide in RAW264.7 murine macrophages

The enzyme heme oxygenase-1 (HO-1) is a cytoprotective and anti-inflammatory protein that degrades heme to produce biliverdin/bilirubin, ferrous iron and carbon monoxide (CO). The anti-inflammatory properties of HO-1 are related to inhibition of adhesion molecule expression and reduction of oxidative stress, while exogenous CO gas treatment decreases the production of inflammatory mediators such as cytokines and nitric oxide (NO). CO-releasing molecules (CO-RMs) are a novel group of substances identified by our group that are capable of modulating physiological functions via the liberation of CO. We aimed in this study to examine the potential anti-inflammatory characteristics of CORM-2 and CORM-3 in an in vitro model of lipopolysaccharide (LPS)-stimulated murine macrophages. Stimulation of RAW264.7 macrophages with LPS resulted in increased expression of inducible NO synthase (iNOS) and production of nitrite. CORM-2 or CORM-3 (10-100 microM) reduced nitrite generation in a concentration-dependent manner but did not affect the protein levels of iNOS. CORM-3 also decreased nitrite levels when added 3 or 6 h after LPS exposure. CORM-2 or CORM-3 did not cause any evident cytotoxicity and produced an increase in HO-1 expression and heme oxygenase activity; this effect was completely prevented by the thiol donor N-acetylcysteine. CORM-3 also considerably reduced the levels of tumor necrosis factor-alpha, another mediator of the inflammatory response. The inhibitory effects of CORM-2 and CORM-3 were not observed when the inactive compounds, which do not release CO, were coincubated with LPS. These results indicate that CO liberated by CORM-2 and CORM-3 significantly suppresses the inflammatory response elicited by LPS in cultured macrophages and suggest that CO carriers can be used as an effective strategy to modulate inflammation.     

CO from enhanced HO activity or from CORM-2 inhibits both O2- and NO production and downregulates HO-1 expression in LPS-stimulated macrophages

Carbon monoxide (CO) arising from heme degradation, catalyzed particularly by the stress-inducible heme oxygenase-1 (HO-1), has recently been demonstrated to provide cytoprotection against cell death in macrophages stimulated with bacterial lipopolysaccharide (LPS). In the present study, we determined the effects of CO on the production of reactive oxygen species (ROS) and nitric oxide (NO) by the LPS-stimulated RAW 264.7 macrophages. In addition, effect of CO-exposure on the production of superoxide (O(2)(-)) in the phorbol myristate acetate (PMA)-stimulated PLB-985 neutrophils was determined. Production of ROS by the LPS-stimulated macrophages pretreated with 50microM [Ru(CO)(3)Cl(2)](2), a CO-releasing molecule (CORM-2), was abolished and the production of O(2)(-) by the PMA-stimulated neutrophils pretreated with the CORM-2 was decreased markedly. The CORM-2 (50microM) was not cytotoxic to both the unstimulated and LPS-stimulated macrophages when determined by employing mitochondrial reductase function test (MTT assay). In macrophages pretreated with increasing doses of CORM-2, both the LPS-derived upregulations of iNOS (NO production) and HO-1 expression (CO production) were suppressed in a dose-dependent manner. Alternatively, when the macrophages were treated with LPS and CO-donor together, the LPS-derived increase in NO production was decreased. Conversely, when the control and LPS-stimulated macrophages were treated with zinc protoporphyrin IX (ZnPP) to inhibit the HO activity blocking endogenous production of CO (basal and enhanced), macrophages died extensively. Interestingly, production of NO in the LPS-stimulated macrophages increased significantly following the ZnPP treatment. Addition of CORM-2 to the LPS-treated cells that were being treated additionally with ZnPP did not prevent the cell death. However, endogenous overproduction of CO by super-induction of HO-1 (obtained by pretreatment of macrophages with either buthionine sulfoximine or hemin) decreased the LPS-derived iNOS expression without affecting cell survival. Combined, these results indicated that enhanced HO activity is essential for the survival of LPS-stimulated macrophages. Thus, upregulation of HO-1 and overproduction of CO may allow the survival of LPS-stimulated macrophages; first, by eliminating the free heme to prevent Fenton reaction, second, by limiting the availability of free heme required for induction of NO-producing heme enzyme (i.e., iNOS), third, by limiting additional production of O(2)(-) and NO via CO-derived inhibition on the activities of heme enzymes like NADPH oxidase and iNOS, respectively. CO may allow the LPS-activated macrophages to return back to the normal quiet state insensitive to additional stimuli causing oxidative stress.     

The role of dynamin-related protein 1 in cancer growth: a promising therapeutic target?

Mitochondrial functions are altered in many human diseases including cancer. Development of mitochondria-targeted therapies, either through restoring normal mitochondrial function or promoting mitochondrial-induced cell death, is one of the attractive strategies to improve the outcome of cancer treatment. Recent advances have revealed the important functional involvement of mitochondrial dynamics in cancer biology. Dynamin-related protein 1 (Drp1), a member of the dynamin family of GTPases required for mitochondrial fission, has been found upregulated in certain types of cancers, such as lung and breast cancers. In addition, the roles of Drp1 in cell cycle progression, genome instability, cell migration and apoptosis in cancer cells have also been recently uncovered. These findings raise the possibility of targeting Drp1-mediated mitochondrial fission as an effective therapy for treating cancer. This article explores the function of Drp1 in cancer cells and discusses the theoretical basis for the development of potential targeted therapy.     

瑞舒伐他汀通过调控UCP2-SIRT3通路减轻脑缺血/再灌注对神经元线粒体的损伤

目的探讨瑞舒伐他汀(rosuvastatin,RS)通过UCP2-SIRT3信号通路在脑缺血/再灌注(CIR)神经元线粒体损伤中的作用及其机制。方法建立SH-SY5Y细胞的脑梗死再灌注模型(OGD/R),给予不同浓度RS(40和2.5μmol·L^-1)分别处理,观察两组中细胞增殖和凋亡的变化及UCP2和SIRT3分子的表达;构建UCP2沉默细胞系,研究不同浓度RS对UCP2沉默前后细胞形态和线粒体膜电位的影响、以及SIRT3分子、线粒体外膜异位酶20(TOMM20)和线粒体合成相关蛋白(Drp1、Opa1和PGC1)的表达变化。结果RS能提高OGD/R细胞的存活率、抑制细胞凋亡、改变细胞形态、稳定细胞线粒体膜电位;增加OGD/R细胞中UCP2、SIRT3分子和TOMM20蛋白表达,并诱导Drp1和Opa1 mRNA的表达,抑制PGC1 mRNA的表达;沉默UCP2后能明显降低OGD/R细胞的存活率及TOMM20蛋白的表达,降低Drp1和Opa1 mRNA的表达,使PGC1 mRNA的表达增加。结论RS通过调控UCP2-SIRT3通路减轻CIR对神经元线粒体的损伤,发挥神经细胞保护作用。     

Drp1-dependent mitochondrial fission in cardiovascular disease

Mitochondria are highly dynamic organelles undergoing cycles of fusion and fission to modulate their morphology, distribution, and function, which are referred as ‘mitochondrial dynamics’. Dynamin-related protein 1 (Drp1) is known as the major pro-fission protein whose activity is tightly regulated to clear the damaged mitochondria via mitophagy, ensuring a strict control over the intricate process of cellular and organ dynamics in heart. Various posttranslational modifications (PTMs) of Drp1 have been identified including phosphorylation, SUMOylation, palmitoylation, ubiquitination, S-nitrosylation, and O-GlcNAcylation, which implicate a role in the regulation of mitochondrial dynamics. An intact mitochondrial homeostasis is critical for heart to fuel contractile function and cardiomyocyte metabolism, while defects in mitochondrial dynamics constitute an essential part of the pathophysiology underlying various cardiovascular diseases (CVDs). In this review, we summarize current knowledge on the critical role of Drp1 in the pathogenesis of CVDs including endothelial dysfunction, smooth muscle remodeling, cardiac hypertrophy, pulmonary arterial hypertension, myocardial ischemia–reperfusion, and myocardial infarction. We also highlight how the targeting of Drp1 could potentially contribute to CVDs treatments.     

白细胞介素-4负调控NF-κB通路抑制炎症反应的机制研究

摘要：目的 探讨白细胞介素（IL）-4 对于脂多糖（LPS）诱导的 Ana-1 细胞髓样分化因子 88（MyD88）/核因子 （NF）-κB信号通路的影响。方法 将小鼠巨噬细胞Ana-1分为LPS组（给予50 μg/L LPS刺激）和LPS+IL-4组（10 μg/L IL-4预培养1 h后,给予LPS 刺激）。在0、0.5、1和2 h时收集细胞培养上清液。采用RT-PCR 检测MyD88和 NF-κB mRNA的相对表达水平;Western blot法检测MyD88、NF-κB总蛋白、NF-κB p65蛋白表达水平;ELISA法检测 NF-κB p65胞核/胞浆比例,以及细胞培养上清液中IL-6和肿瘤坏死因子（TNF）-α含量。结果 随着细胞培养时间 的延长,LPS组MyD88、NF-κB mRNA和蛋白表达水平,NF-κB p65胞核/胞浆比例,IL-6和TNF-α水平均呈逐渐升高 的趋势（P＜0.05）;而 LPS+IL-4 组 MyD88 mRNA 表达水平无明显变化（P＞0.05）,MyD88 蛋白表达水平、NF-κB mRNA和蛋白表达水平、NF-κB p65胞核/胞浆比例、IL-6和TNF-α水平则均呈先升高后降低的趋势（P＜0.05）;LPS+ IL-4组MyD88 mRNA和蛋白表达水平、NF-κB p65胞核/胞浆比例、IL-6和TNF-α水平在1 h和2 h时显著低于LPS组 （P＜0.05）,而2组不同时间点NF-κB mRNA和蛋白表达水平比较差异均无统计学意义（P＞0.05）。结论 IL-4发挥 抗炎作用可能与抑制MyD88/NF-κB信号通路活化有关,IL-4可下调促炎细胞因子IL-6和TNF-α的表达。     

Cell damage following carbon monoxide releasing molecule exposure: implications for therapeutic applications

The cytoprotective properties of carbon monoxide (CO) gas and CO-releasing molecules (CORMs) are well established. Despite promising pre-clinical results, little attention has been paid to the toxicological profile of CORMs. The effects of CORM-2 and its CO-depleted molecule (iCORM-2) (20-400 μM) were compared in primary rat cardiomyocytes and two cell lines [human embryonic kidney (HeK) and Madine-Darby canine kidney Cells (MDCK)]. Cells were assessed for cell viability, apoptosis, necrosis, cytology, mitochondrial energetics, oxidative stress and cell cycle arrest markers. In separate experiments, the anti-apoptotic effects of CORM-2 and i-CORM-2 treatment were compared against CO gas treatment in HeK and MDCK lines. H(2)O(2) -induced cellular damage, measured by lactate dehydrogenase (LDH) release from primary cardiomyocytes, was reduced by 20 μM CORM-2; LDH activity, however, was directly inhibited by 400 μM CORM-2. Both CORM-2/iCORM-2 and CO gas decreased cisplatin-induced caspase-3 activity in MDCK and HeK cells suggesting an anti-apoptotic effect. Conversely, both CORM-2 and iCORM-2 induced significant cellular toxicity in the form of decreased cell viability, abnormal cell cytology, increased apoptosis and necrosis, cell cycle arrest and reduced mitochondrial enzyme activity. Comparison of these markers after CO gas administration to MDCK cells found significantly less cellular toxicity than in 100 μM CORM-2/iCORM-2-treated cells. CO gas did not have an adverse effect on mitochondrial energetics and integrity. Release of CO by low concentrations of intact CORM-2 molecules provides cytoprotective effects. These results show, however, that the ruthenium-based CORM by-product, iCORM-2, is cytotoxic and suggest that the accumulation of iCORM-2 would seriously limit any clinical application of the ruthenium-based CORMs.     

Exogenous carbon monoxide suppresses Escherichia coli vitality and improves survival in an Escherichia coli-induced murine sepsis model

Aim:

Endogenous carbon monoxide (CO) has been shown to modulate inflammation and inhibit cytokine production both in vivo and in vitro. The aim of this study was to examine whether exogenous carbon monoxide could suppress the vitality of Escherichia coli (E coli) and improve the survival rate in an E coli-induced murine sepsis model.

Methods:

ICR mice were infected with E coli, and immediately injected intravenously with carbon monoxide releasing molecule-2 (CORM-2, 8 mg/kg) or inactive CORM-2 (8 mg/kg). The survival rate was monitored 6 times daily for up to 36 h. The blood samples, liver and lung tissues were collected at 6 h after the infection. Bacteria in peritoneal lavage fluid, blood and tissues were enumerated following culture. Tissue iNOS mRNA expression was detected using RT-PCR. NF-κB expression was detected with Western blotting.

Results:

Addition of CORM-2 (200 and 400 μmol/L) into culture medium concentration-dependently suppressed the growth of E coli and decreased the colony numbers, but inactive CORM-2 had no effect. Treatment of the infected mice with CORM-2 significantly increased the survival rate to 55%, while all the infected mice treated with inactive CORM-2 died within 36 h. E coli infection caused severe pathological changes in liver and lungs, and significantly increased serum transaminases, lipopolysaccharide, TNF-α and IL-1β levels, as well as myeloperoxidase activity, TNF-α and IL-1β levels in the major organs. Meanwhile, E coli infection significantly increased the number of colonies and the expression of iNOS mRNA and NF-κB in the major organs. All these abnormalities were significantly attenuated by CORM-2 treatment, while inactive CORM-2 was ineffective.

Conclusion:

In addition directly suppressing E coli, CORM-2 protects the liver and lungs against E coli-induced sepsis in mice, thus improving their survival.