Overview of necroptosis and pyroptosis signaling pathways / 中华微生物学和免疫学杂志

Cell death is an important event in the life cycle. Physical injury can cause cell death in eukaryotes. Besides, specific signaling pathway-mediated programmed cell death has attracted increasing attention. Currently, programmed cell death mainly includes apoptosis, programmed necrosis (necroptosis) and pyroptosis. Necroptosis and pyroptosis, as two new ways of programmed cell death, have been found to play a key role in the process of pathogen infection. Both necroptosis and pyroptosis have the characteristics of programmed lytic cell death, but the signaling pathways involved in them have significant differences. This review focused on the morphological characteristics, signal transduction pathways and the role played in the process of pathogen infection of necroptosis and pyroptosis.

Abnormal Effective Connectivity of the Anterior Forebrain Regions in Disorders of Consciousness / 神经科学通报·英文版

A number of studies have indicated that disorders of consciousness result from multifocal injuries as well as from the impaired functional and anatomical connectivity between various anterior forebrain regions. However, the specific causal mechanism linking these regions remains unclear. In this study, we used spectral dynamic causal modeling to assess how the effective connections (ECs) between various regions differ between individuals. Next, we used connectome-based predictive modeling to evaluate the performance of the ECs in predicting the clinical scores of DOC patients. We found increased ECs from the striatum to the globus pallidus as well as from the globus pallidus to the posterior cingulate cortex, and decreased ECs from the globus pallidus to the thalamus and from the medial prefrontal cortex to the striatum in DOC patients as compared to healthy controls. Prediction of the patients' outcome was effective using the negative ECs as features. In summary, the present study highlights a key role of the thalamo-basal ganglia-cortical loop in DOCs and supports the anterior forebrain mesocircuit hypothesis. Furthermore, EC could be potentially used to assess the consciousness level.

Host-cell death pathways in L929 cells induced by Chlamydia muridarum infection / 中华微生物学和免疫学杂志

Objective To identify the host-cell death pathways (apoptosis, autophagy or necrosis) in L929 cells at the time point of 48 hours post infection (h.p.i.) with Chlamydia muridarum.Methods L929 cells were infected with Chlamydia muridarum at a multiplicity of infection (MOI) of 0.85 for 48 hours.Nuclear fragmentation was observed under fluorescence microscopy following staining L929 cells with DAPI (4′,6-diamidino-2-phenylindole).L929 cells were stained with propidium iodide (PI) plus Annexin Ⅴ and then analyzed by fluorescence-activated cell sorting (FACS) to clarify whether apoptosis or necrosis occurred after Chlamydia muridarum infection.L929 cells were transiently transfected with GFP-LC3 and observed under fluorescent microscopy to analyze cell autophagy.Western blot assay was performed to detect LC3 protein for further analysis of autophagy.Results Apoptosis was not induced in L929 cells by Chlamydia muridarum infection at 48 h.p.i.as no significant nuclear fragmentation was observed.Results of FACS showed that most cells died due to necrosis.Moreover, fluorescent dots of GFP-LC3 formed after infecting transfected L929 cells with Chlamydia muridarum.An increased ratio of LC3Ⅰ to LC3Ⅱ in the L929 cells infected with Chlamydia muridarum was detected by Western blot assay, indicating that autophagy occurred during Chlamydia muridarum infection.Conclusion Necrosis and autophagy rather than apoptosis are induced in L929 cells 48 hours after infection with Chlamydia muridarum.

Establishment of a flow cytometry-based test for the detection of pyroptosis of BMDM / 中华微生物学和免疫学杂志

Objective To establish a flow cytometry-based test for the detection of pyroptosis of murine bone marrow-derived macrophages (BMDM). Methods Bone marrow cells were isolated from wild type (WT) C57BL/ 6 mice and/ or caspase-1-/ - C57BL/ 6 mice and then stimulated with macrophage colony-stimulating factor (M-CSF) to differentiate into murine BMDM. PBS, LPS and LPS+adenosine triphosphate (ATP) were respectively used to stimulate the BMDM. Western blot assay was performed to detect the cleav-age of IL-1β and caspase-1. The levels of IL-1β in the supernatants of cell culture were measured by ELISA. Lactate dehydrogenase (LDH) released in the culture media was detected by using LDH kit. The pyroptosis of murine BMDM was detected by using flow cytometry analysis after double-staining with TMR red+caspase-1, AnnexinⅤ+caspase-1 and propidium iodide (PI)+caspase-1. Results IL-1β was detected in the culture medium of BMDM treated with LPS+ATP and the cleavage of IL-1β and caspase-1 was confirmed by Western blot assay, which indicated that the NLRP3 inflammasome was activated by LPS+ATP treatment. Compared with the caspase-1-/ - mice group, higher levels of LDH were detected in the culture medium of BMDM isolated form the WT mice. Results of the flow cytometry analysis after staining BMDM with caspase-1 plus AnnexinⅤ or PI showed that more cells undergoing pyroptosis were detected in the LPS+ATP treat-ment group than that in LPS or PBS treatment group, which were consistent with the results of the reported flow cytometry with caspase-1+TMR red staining. Conclusion The flow cytometry-based test with double-staining of caspase-1 plus AnnexinⅤ or PI could be used for the detection of pyroptosis of murine BMDM.

A brief review on the progress of mouse models of liver cancer / 中国实验动物学报

Liver cancer remains one of the leading cause of cancer death in the world.Animal models, especially mouse models, are important tools for studying the biological characteristics, pathogenesis, new drug screening and therapy of liver cancer.Up to now, although the development of various animal models accelerates the research of liver cancer, all the existing models have their own disadvantages.Lacking of economical and applicable animal models that can mimic the human liver cancer seriously restrict the further study of liver cancer.With the development of genetically modified technologies, it provides a fast, easy and reliable method to establish liver cancer models.In this review, we describe the different types of mouse models used in liver cancer research, with emphasis on genetically engineered mice used in this field, which may open an avenue for functional cancer genomics and generation of liver cancer models by using gene editing technologies.

Inhibitory effects of eplerenone on cell proliferation via down-regulated SGK-1 pathway in rats with unilateral ureteral obstruction / 中国药理学通报

Aim To observe the effect of mineralocor-ticoid receptor blockade eplerenone on cell proliferation in obstructed kidney of rats. Methods Renal intersti-tial fibrotic animals were made with unilateral ureteral obstruction (UUO) and treated with eplerenone100 mg · kg - 1 · d - 1 . The kidneys were harvested on the 10th day and proliferating cell nuclear antigen ( PC-NA ), serum and glucocorticoid induced kinase-1 (SGK-1 ) and transforming growth factor-β1 ( TGF-β1 ) were detected with immunohistochemistry and Western blot. Results Renal histopathology showed large quantities extracellular matrix (ECM) accumula-tion in kidney with UUO, large numbers of inflammato-ry cells infiltrated in renal interstitium, renal tubular expansion and exfoliation of epithelial cells . The cell proliferation and ECM accumulation were inhibited in eplerenone treated rats significantly. Immunohisto-chemistry and Western blot showed that expressions of PCNA,SGK-1 and TGF-β1 were significantly up-regu-lated with UUO and down-regulated by eplerenone. Conclusion Eplerenone plays the role in inhibiting the cell proliferation and reducing ECM accumulation by down-regulating expression of SGK-1 pathway in rats with unilateral ureteral obstruction.

The establishment of the uPA inducible expression system / 中国比较医学杂志

Objective To establish uPA inducible expression system using recombinant retroviral system for the further construction of inducible uPA-SCID animal model .Methods The Inducible expression system need to construct two plasmids:pLNHXO1O2-Alb-GLUC-FMN2A -rtTA and pLNHXO5O6-TRE2-uPA-IRES-ZsGreen respectively. Both plasmids were based on retroviral vector pLNHX , Albumin promoter gene ( Alb) and rtTA gene or uPA gene and ZsGreen were obtained by PCR reaction and inserted into pLNHX .The Gaussia enzyme fluorescent element ( GLUC) was used to monitor rtTA expression in pLNHXO1O2-Alb-GLUC-FMN2A-rtTA, and the ZsGreen for uPA expression monitoring in pLNHXO5O6-TRE2-uPA-IRES-ZsGreen.The correct constructed plasmids were transfected into packaging cell line GP 2-293 to gain recombinant viral particles .NIH/3T3 cells were infected with these viral particles and selected with G 418.Gene expression in the surviving cells was confirmed by the PCR method .Results The recombinant retroviral vectors harbouring target genes were successfully cloned .The rtTA gene in pLNHXO1O2-Alb-GLUC-FMN2A-rtTA was expressed, and uPA can be induced to express in pLNHXO5O6-TRE2-uPA-IRES-ZsGreen by doxycycline (Dox) when the plasmid transfected into the HepG-Tet-on cell.The constructed recombinant two retroviral vectors were transfected into GP 2-293 packaging cells respectively to gain infectious viral particles .Then,NIH/3T3 cells were infected with these viral particles and single-cell clones which stably expressed the transgenes were successfully established .Conclusion This study primarily established uPA inducible expression system , it laid a foundation for the murine model of inducible liver damage , and provided a novel technical platform for further building the liver humanised murine models for viral hepatitis studying .