自噬对氧化应激引起的脊髓神经元细胞损伤的作用及其机制

氧化应激是脊髓损伤(spinal cord injury,SCI)后脊髓神经元细胞继发性损伤的重要机制,但是如何缓解氧化应激目前仍然不明确。该文培养SD(Sprague Dawley)大鼠原代脊髓神经元细胞,使用不同浓度的H-2O_2作用于神经元细胞12 h后,Western blot检测LC3-II、Beclin-1和P62蛋白质水平变化,分析自噬水平,电镜和绿色荧光蛋白标的记微管相关蛋白轻链3(green fluorescent proteinmicrotubule-associated protein 1 light chain 3,GFP-LC3)转染观察自噬的数量。ATG5 si RNA转染抑制自噬和雷帕霉素(rapamycin)促进细胞自噬,并使用CCK-8和TUNEL(terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling)染色分析自噬对氧化应激下神经元细胞的作用。结果显示,随着H-2O_2浓度增加,LC3-II和Beclin-1蛋白质水平显著升高,而P62蛋白质水平显著下降(P0.05)。透射电镜和共聚焦观察发现,10、50μmol/L H-2O_2可以增加神经元细胞中自噬体的数量。与对照组比较,H-2O_2明显抑制神经元细胞的活力(P0.05),ATG5 si RNA抑制自噬水平后,H-2O_2作用下的细胞活力进一步下降,而雷帕霉素促进自噬后却可以提高细胞的活力(P0.05)。TUNEL和膜联蛋白V/PI(annexin V/PI)染色结果发现,雷帕霉素可以抑制H-2O_2引起的神经元细胞凋亡(P0.05)。该研究结果提示,脊髓神经元细胞处于氧化应激状态下时,自噬代偿性激活并保护神经元细胞。     

紫檀芪通过激活自噬缓解脊髓神经元细胞氧化应激损伤的作用及其机制

自噬在保护脊髓神经元细胞氧化应激损伤中具有重要的作用。紫檀芪(pterostilbene,PTE)是具有抗氧化作用的天然植物的提取物,但其对神经元细胞的作用及其机制尚不清楚。该文采用CCK-8分析PTE对大鼠原代脊髓神经元细胞的细胞毒性;不同浓度PTE作用神经元细胞24 h和48 h,透射电镜和Western blot检测微管相关蛋白轻链3-II(microtubule-associated protein 1 light chain3-II,MAP1LC3-II)、Beclin-1和P62蛋白质水平并分析自噬水平。PTE处理H2O2作用下的神经元细胞24 h,Western blot检测LC3-II水平,GFP-LC3转染观察自噬的数量。2′,7′-二氯二氢荧光素乙酰乙酸(2′,7′-dichloro-djhydrofl uorescein diacetate,DCFDA)和Mito SOX染色分析细胞活性氧(reactive oxygen species,ROS)水平,自噬相关基因5(autophagy related gene 5,ATG5)si RNA转染分析自噬在其中的作用。结果显示,20μmmol/L PTE对于神经元细胞无细胞毒性,PTE作用下神经元细胞中LC3-II、Beclin-1蛋白质水平呈剂量依懒性升高,而P62则呈剂量依懒性下降(P0.05)。PTE增加H2O2作用下的神经元细胞中LC3-II蛋白质水平(P0.05),自噬体数量增加,PTE可提高神经元细胞中自噬体数量。PTE明显降低神经元细胞中ROS水平,但ATG5 si RNA转染抑制自噬后显著逆转PTE的保护作用。该研究结果提示,PTE可能通过提高氧化应激状态下的脊髓神经元细胞自噬水平来抑制细胞ROS的产生。     

塞内卡病毒A结构蛋白VP2诱导PK-15细胞自噬的研究

本研究在塞内卡病毒A(Senecavirus A,SVA)诱导自噬的基础上,着重探究VP2蛋白在细胞自噬过程中的作用。构建VP2基因真核表达载体pcDNA3.1-VP2,将其转染至PK-15细胞,通过检测自噬蛋白和相关基因的表达情况,明确VP2蛋白对细胞自噬的影响。结果显示,本研究成功构建了pcDNA3.1-VP2真核表达载体,且SVA VP2基因在PK-15细胞中正常表达;与对照组相比,VP2蛋白显著上调LC3蛋白的表达水平（P<0.01）;同时,自噬基因LC3、Beclin-1和ATG5转录水平均显著提高（P<0.01）。综上所述,本研究证实SVA VP2蛋白可诱导PK-15细胞自噬,且VP2蛋白与自噬蛋白和基因表达水平呈正相关,为进一步研究病毒感染与致病机制打下基础。     

Pravastatin激活自噬抑制糖皮质激素引起的髓核细胞凋亡

地塞米松注射可以缓解椎间盘退变引起的腰痛症状,但是具有一定的副作用。普伐他汀(Pravastatin)被发现可以缓解骨关节炎的炎症和症状,但是其对髓核细胞及椎间盘退变的作用及其机制尚不清楚。该文培养SD大鼠原代髓核细胞,用不同浓度地塞米松(dexamethasone,DXM)作用髓核细胞48 h后, DCFH-DA和MitoSOX Red染色分析细胞总活性氧(reactive oxygen species, ROS)和线粒体ROS水平, Annexin V/PI流式和DAPI染色分析细胞凋亡水平, N-acetyl-Lcysteine(NAC)抑制ROS水平。Western blot检测LC3-II、Beclin-1和P62等自噬相关蛋白质水平,ATG5 siRNA转染抑制自噬。结果显示,随着DXM处理浓度的增加,髓核细胞内总ROS和线粒体ROS水平及凋亡率升高(P0.05)。Pravastatin增加DXM处理下髓核细胞中LC3-II和Beclin-1蛋白质水平,降低P62蛋白质水平(P0.05)。Pravastatin可以抑制DXM诱导的髓核细胞中的ROS产生和细胞凋亡,而ATG5 siRNA抑制自噬后,显著逆转Pravastatin对细胞的保护作用(P0.05)。该研究结果提示, Pravastatin可能通过激活髓核细胞自噬抑制DXM诱导的ROS产生从而减少细胞凋亡。     

小鼠缺血性脑损伤模型急性期海马SIRT3与自噬相关蛋白表达水平的相关性分析

摘要 目的：观察小鼠缺血性脑损伤模型急性期海马区域沉默信息因子3(SIRT3)和自噬相关蛋白的表达,并探讨两者的相关性。方法：选择C57BL/6J小鼠,采用大脑中动脉阻塞(MCAO)法建立缺血性脑损伤模型,并将小鼠随机分为假手术组(sham)和模型组(MCAO)。缺血性脑损伤急性期(6h),采用间接免疫荧光法观察小鼠海马CA1、CA3和DG区域SIRT3的表达,应用蛋白印迹法检测SIRT3、自噬相关蛋白LC3 I/II和Beclin-1的表达,而后用Spearman相关性分析明确SIRT3和LC3-II、Beclin-1表达的相关性。结果：海马各区域SIRT3阳性细胞数量在损伤后明显增多(P<0.05),且SIRT3蛋白表达也相对上调(P<0.05);损伤后自噬相关蛋白LC3-II和Beclin-1表达亦增高(P<0.05);Spearman相关性分析发现SIRT3与自噬相关蛋白LC3-II、Beclin-1表达均呈显著正相关(P<0.05)。结论：小鼠缺血性脑损伤模型急性期海马区域SIRT3和自噬相关蛋白的表达具有显著相关性,SIRT3对海马区域自噬的调节可能有重要作用。     

抑郁症大鼠海马神经元凋亡率和自噬相关蛋白的改变

目的 观察抑郁症模型大鼠海马神经元形态结构改变及凋亡、自噬的变化,探讨抑郁症海马体积异常的机制.方法 选用雄性成年SD大鼠,随机分为正常对照组和模型组,通过给予不可预见慢性温和应激建立抑郁症模型;采用尼氏染色、透射电镜技术观察海马神经元形态变化,流式细胞术检测海马神经元凋亡,采用透射电镜观察海马神经元自噬体,Western blott检测自噬相关蛋白LC-3和Beclin 1.结果 与对照组比较,模型组海马神经元体积萎缩,数量减少,细胞凋亡率增高（P〈0.05）.模型组海马神经元胞质内可见自噬体;与对照组相比,模型组LC-3Ⅱ蛋白和LC-3Ⅱ/LC-3Ⅰ比值增高,Beclin-1相对表达增高（P〈0.05）.结论 抑郁症大鼠海马神经元存在体积萎缩现象,可能与神经元凋亡和自噬增强有关.     

甲状旁腺激素通过激活自噬调节糖皮质激素引起的髓核细胞衰老及其机制

骨质疏松与椎间盘退变密切相关,甲状旁腺素(parathyroid hormone,PTH)被认为可以缓解伴有骨松的椎间盘退变,但其对髓核细胞及椎间盘退变的作用及其机制尚不清楚。该文培养原代SD大鼠髓核细胞并进行表型鉴定,不同浓度的地塞米松(dexamethasone,DXM)作用髓核细胞48 h,细胞表面积大小测量及β-半乳糖苷酶染色分析DXM对髓核细胞衰老的影响。Western blot检测LC3-II、Beclin-1、P62、p-m TOR和p-p70S6k蛋白质水平,分析PTH对DXM处理下髓核细胞自噬水平以及m TOR信号通路的调节作用。ATG5 si RNA转染后,分析PTH对髓核细胞衰老的调控及自噬在其中的作用机制。结果显示,随着DXM的处理浓度增加,髓核细胞体积增大,β-半乳糖苷酶阳性率增加(P0.05);PTH提高DXM处理下髓核细胞中LC3-II和Beclin-1蛋白质水平,降低P62蛋白质水平,并且降低p-m TOR和p-p70S6k蛋白质水平(P0.05);PTH可以抑制DXM引起的髓核细胞的体积增大和β-半乳糖苷酶阳性率升高,但是ATG5 si RNA转染抑制自噬后却显著逆转PTH对细胞衰老的保护作用(P0.05)。该研究结果提示,PTH可能通过m TOR信号通路激活髓核细胞自噬来缓解DXM引起的髓核细胞衰老。     

自噬在熊果酸诱导前列腺癌PC3细胞凋亡中的作用机制研究

为了探讨自噬在熊果酸抑制前列腺癌PC3细胞凋亡中的作用机制,PC3细胞培养至对数生长期后,以无糖、无氨基酸培养液代替原培养液培养细胞,并用不同浓度的熊果酸进行干预。72 h后收集细胞,采用透射电镜和免疫荧光技术观察PC3细胞自噬情况;Western Blot检测ATG5和Beclin-1蛋白表达;Elisa法测定Caspase-3、Caspase-8和Caspase-9含量;流式细胞技术检测PC3细胞凋亡情况。结果表明,PC3细胞饥饿72 h后,细胞自噬明显增强。与对照组比较,熊果酸作用后的细胞自噬程度明显减弱;ATG5和Beclin-1蛋白表达显著减少(P0.05);Caspase-3、Caspase-8和Caspase-9含量显著增加(P0.05);PC3细胞凋亡率极显著升高(P0.01)。实验初步揭示,熊果酸可通过抑制饥饿状态的前列腺癌PC3细胞自噬,并进一步通过促进凋亡因子的分泌诱导其凋亡。     

青藏高原黑果枸杞花青素对人肝癌细胞增殖和自噬的影响

探究黑果枸杞花青素在体外对人肝癌HepG2细胞增殖和自噬的影响。利用CCK-8法测定细胞活力,EdU和细胞划痕试验检测细胞增殖和迁移效果,RT-PCR和Western blot检测增殖和自噬相关基因的mRNA和蛋白表达。结果显示,黑果枸杞花青素可有效抑制人肝癌HepG2细胞的增殖和迁移;上调增殖因子(LATS1、LATS2和MOB1)和自噬因子(Beclin-1、LC3-Ⅱ和AMPK),并下调增殖因子YAP的mRNA水平;下调自噬因子p-mTOR和细胞周期因子CDK4,并上调自噬因子p-AMPK和LC3-Ⅱ的蛋白表达。由此推测黑果枸杞花青素可在体外抑制人肝癌HepG2细胞增殖和迁移,并促进人肝癌HepG2细胞发生自噬。     

不同生长阶段的烟曲霉对果蝇自噬水平的影响

目的探究不同生长阶段的烟曲霉对果蝇自噬水平的影响。方法用沙保弱平板培养烟曲霉,收获静息孢子;用沙保弱液体培养基震荡培养烟曲霉,在不同时间收获膨胀孢子、菌丝。以3种形态的烟曲霉分组处理转基因果蝇,组织切片观察烟曲霉形态,Rt-PCR(逆转录PCR)检测自噬相关蛋白LC3BⅡ、Beclin-1mRNA的转录水平,Western blot检测LC3BⅡ、Beclin-1的表达量。结果组织切片观察菌的形态与注入时的形态相符,Rt-PCR检测LC3BⅡ、Beclin-1mRNA的转录水平升高,同时,感染膨胀烟曲霉孢子及菌丝组的果蝇LC3BⅡ、Beclin-1蛋白的表达水平升高。结论烟曲霉膨胀孢子及菌丝能显著提高果蝇自噬水平,而静息孢子不能引起果蝇自噬应答。     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

Enterovirus 71 3C proteolytically processes the histone H3 N-terminal tail during infection

Highlights
1. The N-terminal tail of histone H3 is specifically cleaved during EV71 infection.
2. Viral protease 3C is identified as a protease responsible for proteolytically processing the N-terminal H3 tail.
3. Our finding reveals a new epigenetic regulatory mechanism for Enterovirus 71 in virus-host interactions.     

Detection of EBV and HHV6 in the Brain Tissue of Patients with Rasmussen’s Encephalitis

Rasmussen’s encephalitis (RE) is a rare pediatric neurological disorder, and the exact etiology is not clear. Viral infection may be involved in the pathogenesis of RE, but conflicting results have reported. In this study, we evaluated the expression of both Epstein-Barr virus (EBV) and human herpes virus (HHV) 6 antigens in brain sections from 30 patients with RE and 16 control individuals by immunohistochemistry. In the RE group, EBV and HHV6 antigens were detected in 56.7% (17/30) and 50% (15/30) of individuals, respectively. In contrast, no detectable EBV and HHV6 antigen expression was found in brain tissues of the control group. The co-expression of EBV and HHV6 was detected in 20.0% (6/30) of individuals. In particular, a 4-year-old boy had a typical clinical course, including a medical history of viral encephalitis, intractable epilepsy, and hemispheric atrophy. The co-expression of EBV and HHV6 was detected in neurons and astrocytes in the brain tissue, accompanied by a high frequency of CD8⁺ T cells. Our results suggest that EBV and HHV6 infection and the activation of CD8⁺ T cells are involved in the pathogenesis of RE.     

Perinatal Vertical Transmission of Chikungunya Virus in Ruili,a Town on the Border between China and Myanmar

正Dear Editor,Chikungunya virus (CHIKV), an arbovirus in the family of Togaviridae, genus Alphavirus, is transmitted by the A.aegyptii or A. albopictus mosquito, and causes disease in humans characterized by fever, rash, and arthralgia (Silva and Dermody 2017; Suhrbier 2019). It was first reported in 1953 in Tanzania, and caused only a few outbreaks and sporadic cases in Africa and Asia in last century. However, in the epidemic in 2004, CHIKV acquired mutations that conferred enhanced transmission by the A. albopictus mosquito(Schuffenecker et al. 2006). Since then, it has successively caused outbreaks in Africa, the Indian Ocean, South East Asia, the South America, and Europe (Zeller et al. 2016).     

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

In conclusion, the novel visual RT-LAMP assay is a simple, rapid, and sensitive approach for detection of SARS-CoV-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current SARS-CoV-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. Furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of SARS-CoV-2 from bats to humans.