RIP3/MLKL-mediated neuronal necroptosis induced by methamphetamine at 39℃

Methamphetamine is one of the most prevalent drugs abused in the world.Methamphetamine abusers usually present with hyperpyrexia (39℃),hallucination and other psychiatric symptoms.However,the detailed mechanism underlying its neurotoxic action remains elusive.This study investigated the effects of methamphetamine + 39℃ on primary cortical neurons from the cortex of embryonic Sprague-Dawley rats.Primary cortex neurons were exposed to 1 mM methamphetamine + 39℃.Propidium iodide staining and lactate dehydrogenase release detection showed that methamphetamine + 39℃ triggered obvious necrosis-like death in cultured primary cortical neurons,which could be partially inhibited by receptor-interacting protein-1 (RIP1) inhibitor Necrostatin-1 partially.Western blot assay results showed that there were increases in the expressions of receptor-interacting protein-3 (RIP3) and mixed lineage kinase domain-like protein (MLKL) in the primary cortical neurons treated with 1 mM methamphetamine + 39℃ for 3 hours.After pre-treatment with RIP3 inhibitor GSK’872,propidium iodide staining and lactate dehydrogenase release detection showed that neuronal necrosis rate was significantly decreased;RIP3 and MLKL protein expression significantly decreased.Immunohistochemistry staining results also showed that the expressions of RIP3 and MLKL were up-regulated in brain specimens from humans who had died of methamphetamine abuse.Taken together,the above results suggest that methamphetamine + 39℃ can induce RIP3/MLKL regulated necroptosis,thereby resulting in neurotoxicity.The study protocol was approved by the Medical Ethics Committee of the Third Xiangya Hospital of Central South University,China (approval numbers: 2017-S026 and 2017-S033) on March 7,2017.     

羟基红花黄色素A通过抑制程序性坏死减轻小鼠重症中暑引起的急性肺损伤

目的 研究羟基红花黄色素A（HSYA）是否在重症中暑肺损伤中起保护作用及其可能的作用机制。方法 使用不同浓度（1.125、2.25、4.5 mg/kg）HSYA腹腔注射预处理小鼠，建立重症中暑（sHS）小鼠模型，分为低、中、高剂量HSYA中暑组、单纯中暑组及正常对照组，12只/组。初步观察及比较各组热耐受的情况，以确定HSYA最佳治疗剂量；后使用中剂量HSYA及RIP1活化抑制剂Nec-1预处理小鼠，分组为HSYA+HS组、Nec-1+HS组、HS组及正常对照组，8只/组，观察72 h恢复期核心体温变化特征，比较热耐受情况及生存情况。给予相同处理因素处理小鼠分组为正常对照组，HS组，HSYA+HS组及Nec-1+HS组，正常对照组6只，其余18只/组，分别于重症中暑恢复期不同阶段（0、2、6、12、24 h）处死小鼠，每个时间点处死3只小鼠，收集小鼠的肺组织、肺泡灌洗液及血液样本，取肺组织行HE染色，并进行病理评分，检测肺湿干重比，肺含水量，肺泡灌洗液中白细胞、中性粒细胞、蛋白含量；ELISA法检测肺泡灌洗液中HMGB1水平及血清中TNF-α、IL-6及HMGB1水平；Western blotting检测恢复期（2、6、12 h）肺组织中RIP1、RIP3、MLKL-s358、MLKL表达水平，及经HSYA预处理后MLKL-s358蛋白水平。结果 中剂量及高剂量HSYA预处理可明显改善小鼠热耐受能力，中剂量与高剂量无显著差异，后续药物预处理以中剂量（2.25 mg/kg）作为标准剂量；与HS组相比，HSYA+HS组和Nec-1+HS组小鼠热耐受程度均增加（P<0.05），HSYA+HS组和Nec-1+HS组无明显差异。HSYA及Nec-1预处理组小鼠生存率增加（P<0.05），肺组织病理评分、TNF-α、IL-6及HMGB1水平降低（P<0.05），肺湿干重比，肺含水量，肺泡灌洗液中白细胞、中性粒细胞、蛋白含量及HMGB1水平降低（P<0.05），HS小鼠恢复期肺组织RIP1水平及MLKL-s358磷酸化水平升高（P<0.05），与HS组相比，HSYA+HS组MLKL-s358磷酸化水平降低。结论 重症中暑小鼠肺组织可发生程序性坏死，HSYA可通过抑制程序性坏死发挥肺保护作用。     

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??Objective    To study condyle chondrocyte cell death under circumstances of high TNF-α concentration. Methods    Primary human condyle chondrocytes were cultured in vitro and then 20 μg/L TNF-α was added to induce cell death ??T group??. The rate of apoptotic and necrotic cell death were detected by flow cytometry. Before application of TNF-α??chondrocytes were pre-treated with pan-caspase inhibitor Z-VAD-FMK ??ZT Group???? or specific programmed necrosis inhibitors Nec-1 ??NT group???? or the combination of these two inhibitors ??ZNT group??. CM-H2DCFDA staining was used to determine ROS levels. Cell death rates and ROS levels were respectively compared and statistically analyzed. Results    T group showed exacerbated cell death and increased ROS level in remaining living cells ??while cell death and ROS levels of ZT?? NT and ZNT groups were significantly alleviated compared to T group ??P < 0.05??. Amongst these three groups?? ZNT group achieved the lowest cell death rate and ROS levels. Conclusion    Under circumstances of high TNF-α concentration?? condylar chondrocytes could go through both apoptosis and necropotosis?? and inhibiting both forms of programmed cell death pathways can significantly improve cell survival rate.     

Recent Advances in Pathology: the 2019 Annual Review Issue of The Journal of Pathology

In this Annual Review Issue of The Journal of Pathology, we present 15 invited reviews on topical aspects of pathology, ranging from the impacts of the microbiome in human disease through mechanisms of cell death and autophagy to recent advances in immunity and the uses of genomics for understanding, classifying and treating human cancers. Each of the reviews is authored by experts in their fields and our intention is to provide comprehensive updates in specific areas of pathology in which there has been considerable recent progress. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

程序性细胞坏死参与急慢性损伤脑内神经元死亡的研究进展

 程序性细胞坏死是一种新发现的细胞死亡形式。这类死亡细胞形态类似坏死,而其死亡过程受胞内主动机制的调节,由此改变了坏死细胞不受内在机制调控的经典概念。当细胞膜上死亡受体与其配体结合,细胞内凋亡因子caspase-8被抑制时,激活受体相互作用蛋白（receptor interaction protein,RIPK1/RIPK3）激酶及关键底物——混合谱系激酶结构域样蛋白（mixed lineage kinase domain-like protein,MLKL）的信号通路,从而触发细胞发生程序性细胞坏死。后者参与多种疾病的病理过程,如肿瘤、免疫炎症、神经退行性疾病、脑缺血损伤等。本文就程序性细胞坏死的信号通路、重要调控分子及在神经损伤相关疾病致病机制进行综述,并对研究方法和分析工具药物进行了归纳总结。     

Hydrogen peroxide induces programmed necrosis in rat nucleus pulposus cells through the RIP1/RIP3‐PARP‐AIF pathway

This study aimed to systematically investigate whether programmed necrosis contributes to H₂O₂‐induced nucleus pulposus (NP) cells death and to further explore the underlying mechanism involved. Rat NP cells were subjected to different concentrations of H₂O₂ for various time periods. The cell viability was measured using a cell counting kit‐8, and the death rate was detected by Hoechst 33258/propidium iodide (PI) staining. The programmed necrosis‐related molecules receptor‐interacting protein 1 (RIP1), receptor‐interacting protein 3 (RIP3), poly (ADP‐ribose) polymerase (PARP), and apoptosis inducing factor (AIF) were determined by real‐time polymerase chain reaction and Western blotting, respectively. The morphologic and ultrastructural changes were examined by phasecontrast microscopy and transmission electron microscopy (TEM). In addition, the necroptosis inhibitor Necrostatin‐1 (Nec‐1), the PARP inhibitor diphenyl‐benzoquinone (DPQ) and small interfering RNA (siRNA) technology were used to indirectly evaluate programmed necrosis. Our results indicated that H₂O₂ induced necrotic morphologic and ultrastructural changes and an elevated PI positive rate in NP cells; these effects were mediated by the upregulation of RIP1 and RIP3, hyperactivation of PARP, and translocation of AIF from mitochondria to nucleus. Additionally, NP cells necrosis was significantly attenuated by Nec‐1, DPQ pretreatment and knockdown of RIP3 and AIF, while knockdown of RIP1 produced the opposite effects. In conclusion, these results suggested that under oxidative stress, RIP1/RIP3‐mediated programmed necrosis, executed through the PARP‐AIF pathway, played an important role in NP cell death. Protective strategies aiming to regulate programmed necrosis may exert a beneficial effect for NP cells survival, and ultimately retard intervertebral disc (IVD) degeneration. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1269–1282, 2018.

     

感染性眼内炎患者玻璃体液中坏死性凋亡相关蛋白 MCP-1的检测

目的：探讨感染性眼内炎患者玻璃体液中坏死性凋亡相关蛋白 MCP-1的表达及其与视力预后的关系,为预测疾病预后及进一步的眼内药物干预,保护视功能提供实验室依据。方法收集17例感染性眼内炎患者的玻璃体液,检测坏死性凋亡相关蛋白单核细胞趋化蛋白1(MCP-1)的表达,分析临床治疗效果。结果实验组感染性眼内炎患者玻璃体液中 MCP-1的浓度显著高于对照组 MCP-1的浓度(P <0．05);实验组的 MCP-1浓度与最佳矫正视力呈负相关性(P<0.05)。结论感染性眼内炎患者眼内有坏死性凋亡的发生,与患者视功能损害有关,这为进一步干预坏死性凋亡,保护患者视功能提供了依据。