细胞焦亡与糖尿病血管并发症关系的研究进展

糖尿病血管病变是主要的糖尿病慢性并发症,是导致患者生活质量下降和预期寿命缩短的最主要原因。细胞质内模式识别受体(PRRs)可感受多种刺激形成炎性小体,激活焦亡通路,加重糖尿病患者代谢紊乱,促进糖尿病血管并发症的发生发展。本文浅谈细胞焦亡对不同糖尿病血管并发症的促进作用,以及抑制焦亡通路可能为糖尿病血管并发症的早期诊治提...     

Salmonella infects B cells by macropinocytosis and formation of spacious phagosomes but does not induce pyroptosis in favor of its survival

We have previously reported that Salmonella infects B cells and survives within endosomal-lysosomal compartments. However, the mechanisms used by Salmonella to enter B cells remain unknown. In this study, we have shown that Salmonella induces its own entry by the induction of localized ruffling, macropinocytosis, and spacious phagosome formation. These events were associated with the rearrangement of actin and microtubule networks. The Salmonella pathogenesis island 1 (SPI-1) was necessary to invade B cells. In contrast to macrophages, B cells were highly resistant to cell death induced by Salmonella. These data demonstrate the ability of Salmonella to infect these non-professional phagocytic cells, where the bacterium can find an ideal intracellular niche to support persistence and the possible dissemination of infection.     

五味子丙素抑制脂多糖诱导心肌细胞的炎症反应与 细胞焦亡的作用及机制研究

目的:探究五味子丙素(SchC)对脂多糖(LPS)诱导心肌细胞HL-1炎症反应与细胞焦亡的影响。方法:培养小鼠心肌细胞HL-1,将其分为空白对照组、模型组(LPS)、LPS+SchC组。SchC预处理1 h后,分别以LPS刺激24 h或48 h,ELISA法提取细胞培养液上清液,检测细胞因子IL-1β、HMGB1和TNF-α的分泌,MTT法收集细胞检测细胞活力,Western Blot检测cleaved-Caspase1、GSDMD-N和NLRP3的表达;将HL-1细胞分为空白对照组、模型组(LPS)、LPS+siGSDMD(GSDMD siRNA)组、LPS+siGSDMD+SchC组,给药预处理1 h后,LPS刺激24 h,收集细胞培养液上清液,ELISA法检测IL-1β的分泌,MTT法检测细胞活力。结果:与LPS组比较,LPS+SchC组细胞活力得到改善(P<0.05),IL-1β、HMGB1和TNF-α的分泌均显著降低(P<0.05);Western Blot结果表明,与LPS组比较,LPS+SchC组的cleaved-Caspase1、GSDMD-N段和NLRP3蛋白水平显著降低(P<0.05);ELISA和MTT结果表明,与LPS组比较,LPS+siGSDMD组IL-1β的分泌显著降低(P<0.05)且细胞活力显著得到改善(P<0.01),然而LPS+siGSDMD组与LPS+siGSDMD+SchC组的IL-1β分泌水平和细胞活力并无显著性差异。结论:五味子丙素能有效缓解LPS诱导HL-1的炎症反应和细胞焦亡。     

程序性细胞坏死及细胞焦亡信号通路研究进展概述

细胞死亡是生命活动中一个非常重要的事件,真核生物可因物理损伤刺激导致细胞死亡,因特异信号通路介导的程序性细胞死亡近年来得到越来越多的关注。目前程序性细胞死亡主要存在以下3种方式:凋亡(apoptosis)、程序性坏死(necroptosis)、细胞焦亡(pyroptosis)。程序性坏死和细胞焦亡是近年来才发现的新的细胞程序性死亡方式,在细菌或病毒感染宿主细胞过程中起着关键作用。这两种细胞死亡都是细胞裂解型死亡,但其信号通路存在明显差异。本文就这两种程序性细胞死亡的形态学特征、信号转导通路及其在病原体感染过程中的作用等方面的研究进展作一综述。     

NLRP6 expressed in astrocytes aggravates neurons injury after OGD/R through activating the inflammasome and inducing pyroptosis

NLRP6, the nucleotide oligomerization domain-like receptor family pyrin domain containing 6, has a substantiable effect on inflammation and host defense against microorganisms. In our previous study, NLRP6 promotes inflammation after cerebral I/R injury in a MCAO model. However, the effect of NLRP6 in different nerve cells subjected to OGD/R needs to be further understood. Here, evidence shows that the expression of NLRP6 is increased in different nerve cells subjected to OGD/R, and mainly expressed in astrocytes. NLRP6 may up-regulate inflammation factors (IL-1β, Il-8) via the form of inflammasomes in astrocytes after OGD/R. Then, primary neuron-astrocyte co-culture model under OGD/R in vitro was performed, and we found that NLRP6 decreased the neurons viability and aggravated apoptosis of neurons. Mechanically, NLRP6 could induce pyroptosis to regulate the survival of neurons through activating caspase-1.     

电针足三里对功能性消化不良大鼠十二指肠半胱氨酸蛋白酶-1和消皮素D的影响

目的 探讨电针足三里对功能性消化不良（FD）大鼠十二指肠细胞焦亡的影响和可能的作用机制。 方法 将24只7日龄Sprague-Dawley（SD）大鼠按照随机数字表法分为空白组、模型组、电针组,每组8只。模型组和电针组采用多因素诱导法（碘乙酰胺灌胃法+夹尾刺激法）制备FD大鼠模型。造模成功后,电针组大鼠给予针刺“足三里”穴后连接韩式穴位神经刺激仪治疗2周,空白组和模型组不予任何干预。于基线期、造模后、电针干预7 d和14 d后记录3组大鼠的体重,于电针干预结束后检测3组大鼠的胃排空率和小肠推进率,并采用酶联免疫法（ELISA）检测其血清白介素-1β(IL-1β)和白介素-6（IL-6）的表达水平,采用实时荧光定量聚合酶链式反应检测3组大鼠十二指肠IL-1β和IL-6转录水平,采用免疫印迹法检测3组大鼠十二指肠半胱氨酸蛋白酶-1（caspase-1）p20和消皮素D（GSDMD）蛋白表达水平。 结果 造模成功后,模型组和电针组大鼠的平均体重与空白组造模成功后比较,差异均有统计学意义（P＜0.01）;电针干预7 d和14 d后,模型组和电针组大鼠的平均体重与空白组同时间点比较,差异均有统计学意义（P＜0.05）,且电针组电针干预7 d和14 d后的平均体重与模型组同时间点比较,差异均有统计学意义（P＜0.05）。电针干预结束后,电针组大鼠胃内残留率和小肠推进率与模型组比较,差异均有统计学意义(P＜0.01),而模型组大鼠胃内残留率和小肠推进率与空白组比较,差异均有统计学意义(P＜0.01)。电针干预结束后,模型组和电针组大鼠血清中IL-1β和IL-6表达水平、十二指肠组织IL-1β和IL-6 mRNA的表达水平和十二指肠组织GSDMD、caspase-1 p20蛋白表达水平与空白组比较,差异均有统计学意义(P＜0.01),且电针组大鼠血清中IL-1β和IL-6表达水平、十二指肠组织IL-1β和IL-6 mRNA的表达水平和十二指肠组织GSDMD、caspase-1 p20蛋白表达水平与模型组比较,差异均有统计学意义（P＜0.05）。 结论 电针足三里可显著降低FD大鼠十二指肠的细胞焦亡水平,改善十二指肠低度炎症,进而减轻FD临床症状,其作用机制可能与电针足三里可下调caspase-1 p20、GSDMD-N蛋白的表达水平,降低IL-1β和IL-6等炎性因子的表达水平,以及缓解FD大鼠十二指肠的低度炎症有关。     

大鼠脑冲击伤早期启动神经元焦亡的发生及可能机制

目的探讨大鼠脑冲击伤(bTBI)早期是否启动神经元焦亡的发生及可能机制。方法选取雄性SD大鼠52只,按随机数字表法分为空白对照组和bTBI组,bTBI组又根据观察时间分为1,6,12 h三个亚组,各组13只大鼠。bTBI组麻醉后将颈部以下保护,用BST-Ⅰ型激波管致头部冲击伤。空白对照组除不致伤外,其他操作与bTBI组一致。伤后6,12 h进行改良神经功能严重程度评分(mNSS)检测,观察大鼠神经功能障碍程度。伤后1,6,12 h行MRI T2WI扫描观察异常信号影;解剖大鼠头部进行大体观察;HE染色观察脑组织病理改变;电镜下观察神经元超微结构改变;ELISA检测炎症因子[白细胞介素(IL)-1β、IL-18]的表达水平;免疫荧光共染检测蛋白[凋亡相关微粒蛋白(ASC)、核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)]在神经元上的表达情况。结果(1)bTBI 6 h组mNSS[3(3.0,3.0)分]高于空白对照组[0(0,0)分](P<0.05),bTBI 12 h组mNSS[2(2.0,2.0)分]高于空白对照组[0(0,0)分](P<0.05);bTBI 6 h组mNSS与bTBI 12 h组比较,差异无统计学意义(P>0.05)。(2)与空白对照组比较,bTBI组MRI T2WI序列均发现脑组织体积增大,整体T2信号影增高,扣带回增宽变扁,大脑皮质、第三脑室较周围存在高信号影,但各bTBI亚组间无明显差异。(3)bTBI组大体观察发现脑组织出现不同程度的水肿和充血;HE染色显示各bTBI亚组脑组织皮质区部分细胞出现肿胀变形和血管管腔收缩,血管周围间隙增宽。(4)电镜下发现bTBI组神经元肿胀主要发生在线粒体,且在伤后12 h内肿胀程度逐步加深。(5)bTBI 1,6,12 h组IL-18水平分别为(4.12±0.42)pg/ml、(5.20±0.29)pg/ml、(6.82±0.61)pg/ml,较空白对照组的(2.94±0.49)pg/ml均明显升高(P均<0.01);bTBI 1,6,12 h组IL-1β水平分别为(2.48±0.15)pg/ml、(4.10±0.38)pg/ml、(5.04±0.28)pg/ml,较空白对照组的(1.86±0.32)pg/ml均明显升高(P均<0.01);各bTBI亚组血清中IL-18及IL-1β蛋白在伤后12 h内均呈不同程度的升高(P均<0.05)。(6)ASC和NLRP3蛋白在脑皮质神经元阳性细胞的胞质上表达,且随时间延长表达均呈上升趋势。结论大鼠在bTBI早期启动脑皮质神经元发生焦亡。bTBI大鼠神经功能的缺失可能是冲击波作用于头部导致神经元焦亡引起。     

Necroptosis,pyroptosis and apoptosis: an intricate game of cell death

Cell death is a fundamental physiological process in all living organisms. Its roles extend from embryonic development, organ maintenance, and aging to the coordination of immune responses and autoimmunity. In recent years, our understanding of the mechanisms orchestrating cellular death and its consequences on immunity and homeostasis has increased substantially. Different modalities of what has become known as ‘programmed cell death’ have been described, and some key players in these processes have been identified. We have learned more about the intricacies that fine tune the activity of common players and ultimately shape the different types of cell death. These studies have highlighted the complex mechanisms tipping the balance between different cell fates. Here, we summarize the latest discoveries in the three most well understood modalities of cell death, namely, apoptosis, necroptosis, and pyroptosis, highlighting common and unique pathways and their effect on the surrounding cells and the organism as a whole.     

Evidence that NLRC4 inflammasome mediates apoptotic and pyroptotic microglial death following ischemic stroke

Stroke is the second leading cause of death in the world and a major cause of long-term disability. Recent evidence has provided insight into a newly described inflammatory mechanism that contributes to neuronal and glial cell death, and impaired neurological outcome following ischemic stroke – a form of sterile inflammation involving innate immune complexes termed inflammasomes. It has been established that inflammasome activation following ischemic stroke contributes to neuronal cell death, but little is known about inflammasome function and cell death in activated microglial cells following cerebral ischemia. Microglia are considered the resident immune cells that function as the primary immune defense in the brain. This study has comprehensively investigated the expression and activation of NLRP1, NLRP3, NLRC4 and AIM2 inflammasomes in isolates of microglial cells subjected to simulated ischemic conditions and in the brain following ischemic stroke. Immunoblot analysis from culture media indicated microglial cells release inflammasome components and inflammasome activation-dependent pro-inflammatory cytokines following ischemic conditions. In addition, a functional role for NLRC4 inflammasomes was determined using siRNA knockdown of NLRC4 and pharmacological inhibitors of caspase-1 and -8 to target apoptotic and pyroptotic cell death in BV2 microglial cells under ischemic conditions. In summary, the present study provides evidence that the NLRC4 inflammasome complex mediates the inflammatory response, as well as apoptotic and pyroptotic cell death in microglial cells under in vitro and in vivo ischemic conditions.     

Gasdermin D蛋白对内毒素导致肺泡巨噬细胞焦亡的影响

目的探讨Gasdermin D(GSDMD)蛋白在内毒素导致肺泡巨噬细胞焦亡中的作用。 方法使用小干扰RNA技术建立GSDMD蛋白低表达的MH-S细胞株。将细胞分为野生型MH-S细胞组(WT)、转染空病毒载体的空白对照细胞组(NC)和GSDMD敲低MH-S细胞组(KD)，分别接受PBS、LPS+尼日利亚菌素和LPS+霍乱毒素B亚单位进行处理。通过测定其乳酸脱氢酶(LDH)和IL-1β，荧光显微镜下观察细胞PI染色情况，RT-qPCR和蛋白免疫印迹检测焦亡相关蛋白表达等指标，来判定MH-S细胞有无焦亡发生和炎症因子水平的差异。 结果在接受LPS+nigericin和LPS+CTB的处理后，三组细胞均出现了焦亡现象。其中KD组接受LPS+nigericin刺激后，细胞培养上清LDH水平和IL-1β浓度明显低于WT和NC组(P<0.05)；PI染色阳性百分比明显少于WT和NC组(P<0.05)；而GSDMD mRNA相对表达量和GSDMD-NT蛋白相对表达量也是三组中最低的(P<0.05)。而在经LPS+CTB处理后，WT和NC组细胞死亡发生率明显高于KD组(P<0.01)；细胞上清中IL-1β水平和PI染色阳性百分比增加幅度也是WT和NC组更大(P<0.05)。此外，KD组中GSDMD mRNA相对表达量和GSDMD-NT蛋白相对表达量相较于另两组也是更低的(P<0.05)。 结论下调MH-S细胞GSDMD蛋白表达可以起到抑制LPS导致的MH-S细胞焦亡和IL-1β分泌释放的作用。