Background::The hepatitis B virus (HBV) vaccine has been efficiently used for decades. However, hepatocellular carcinoma caused by HBV is still prevalent globally. We previously reported that interferon (IFN)-induced tripartite motif-containing 25 (TRIM25) inhibited HBV replication by increasing the IFN expression, and this study aimed to further clarify the anti-HBV mechanism of TRIM25.Methods::The TRIM25-mediated degradation of hepatitis B virus X (HBx) protein was determined by detecting the expression of HBx in TRIM25-overexpressed or knocked-out HepG2 or HepG2-NTCP cells via Western blotting. Co-immunoprecipitation was performed to confirm the interaction between TRIM25 and HBx, and colocalization of TRIM25 and HBx was identified via immunofluorescence; HBV e-antigen and HBV surface antigen were qualified by using an enzyme-linked immunosorbent assay (ELISA) kit from Kehua Biotech. TRIM25 mRNA, pregenomic RNA (pgRNA), and HBV DNA were detected by quantitative real-time polymerase chain reaction. The retinoic acid-inducible gene I (RIG-I) and pgRNA interaction was verified by RNA-binding protein immunoprecipitation assay.Results::We found that TRIM25 promoted HBx degradation, and confirmed that TRIM25 could enhance the K90-site ubiquitination of HBx as well as promote HBx degradation by the proteasome pathway. Interestingly, apart from the Really Interesting New Gene (RING) domain, the SPRY domain of TRIM25 was also indispensable for HBx degradation. In addition, we found that the expression of TRIM25 increased the recognition of HBV pgRNA by interacting with RIG-I, which further increased the IFN production, and SPRY, but not the RING domain is critical in this process.Conclusions::The study found that TRIM25 interacted with HBx and promoted HBx-K90-site ubiquitination, which led to HBx degradation. On the other hand, TRIM25 may function as an adaptor, which enhanced the recognition of pgRNA by RIG-I, thereby further promoting IFN production. Our study can contribute to a better understanding of host-virus interaction.

Background::Ischemic acute kidney injury (AKI) is a common syndrome associated with considerable mortality and healthcare costs. Up to now, the underlying pathogenesis of ischemic AKI remains incompletely understood, and specific strategies for early diagnosis and treatment of ischemic AKI are still lacking. Here, this study aimed to define the transcriptomic landscape of AKI patients through single-cell RNA sequencing (scRNA-seq) analysis in kidneys.Methods::In this study, scRNA-seq technology was applied to kidneys from two ischemic AKI patients, and three human public scRNA-seq datasets were collected as controls. Differentially expressed genes (DEGs) and cell clusters of kidneys were determined. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, as well as the ligand-receptor interaction between cells, were performed. We also validated several DEGs expression in kidneys from human ischemic AKI and ischemia/reperfusion (I/R) injury induced AKI mice through immunohistochemistry staining.Results::15 distinct cell clusters were determined in kidney from subjects of ischemic AKI and control. The injured proximal tubules (PT) displayed a proapoptotic and proinflammatory phenotype. PT cells of ischemic AKI had up-regulation of novel pro-apoptotic genes including USP47, RASSF4, EBAG9, IER3, SASH1, SEPTIN7, and NUB1, which have not been reported in ischemic AKI previously. Several hub genes were validated in kidneys from human AKI and renal I/R injury mice, respectively. Furthermore, PT highly expressed DEGs enriched in endoplasmic reticulum stress, autophagy, and retinoic acid-inducible gene I (RIG-I) signaling. DEGs overexpressed in other tubular cells were primarily enriched in nucleotide-binding and oligomerization domain (NOD)-like receptor signaling, estrogen signaling, interleukin (IL) -12 signaling, and IL-17 signaling. Overexpressed genes in kidney-resident immune cells including macrophages, natural killer T (NKT) cells, monocytes, and dendritic cells were associated with leukocyte activation, chemotaxis, cell adhesion, and complement activation. In addition, the ligand-receptor interactions analysis revealed prominent communications between macrophages and monocytes with other cells in the process of ischemic AKI. Conclusion::Together, this study reveals distinct cell-specific transcriptomic atlas of kidney in ischemic AKI patients, altered signaling pathways, and potential cell-cell crosstalk in the development of AKI. These data reveal new insights into the pathogenesis and potential therapeutic strategies in ischemic AKI.

本研究通过对呼吸道合胞病毒（RSV）毛细支气管炎患儿血白细胞进行RNA测序，探讨与发病机制及疾病严重度相关的生物学特征。本研究是一项病例对照研究，以2019年10月至2022年4月于温州医科大学附属第二医院儿童呼吸科住院临床诊断为毛细支气管炎，RSV抗原阳性和（或）RSV核酸阳性的87例患儿作为病例组，将病例组按病情分为轻、中、重三组，按有无特应征分为特应征组和无特应征组，选择同期40名健康体检儿童作为对照组，提取病例组和对照组儿童的全血白细胞RNA进行测序，分析数据得到差异表达基因（DEGs），再通过基因本体论（GO）注释、京都基因和基因组百科全书（KEGG）注释、蛋白质相互作用网络（PPI）构建筛选与发病机制、病情以及特应征相关的免疫生物学通路及基因。通过加权基因共表达网络分析（WGCNA）构建共表达网络，寻找与疾病严重度相关的潜在生物学指标。结果显示，病例组与对照组相比DEGs共有1 782个，其中上调基因1 586个，下调基因196个，这些DEGs的GO通路富集显示在分子功能中主要富集在过氧化物酶活性、氧化还原酶活性等过程，在细胞学组分中主要富集在细胞质囊泡腔、分泌颗粒腔中，在生物学过程中主要富集在中性粒细胞激活参与免疫反应、中性粒细胞脱颗粒、中性粒细胞激活等过程。DEGs的KEGG富集分析显示主要富集于病毒蛋白与细胞因子及其受体的相互作用信号通路。构建PPI网络后得到 CCL2、 IL-10、 MMP9和 JUN共4个处于核心位置的基因。不同病情组与对照组比较所得DEGs主要富集在逆行内源性大麻素信号转导和细胞凋亡通路上。WGCNA分析显示与血氧饱和度相关的棕色模块与病情关系最为密切，其基因主要富集在RNA解旋酶维甲酸诱导基因-I（RIG-I）样受体信号通路上。特应征组的特异性DEGs有230个，无特应征组的特异性DEGs有444个，KEGG富集分析结果显示两组均富集到NF-κB信号通路上，特应征不会导致RSV毛细支气管炎儿童免疫反应和转录组特征发生明显改变。综上，中性粒细胞激活、脱颗粒途径以及病毒蛋白与细胞因子及细胞因子受体的相互作用信号通路参与RSV毛细支气管炎宿主的免疫反应。 CCL2、 IL-10、 MMP9和 JUN基因可能与发病相关。在RIG-I样受体、细胞凋亡及内源性大麻素相关的信号通路中可能存在与疾病严重度相关的潜在生物学标志物。

目的:检测遗传学及生理学实验室蛋白2(LGP2)、维甲酸诱导基因I(RIG-I)和黑色素瘤分化相关基因5(MDA5)在手足口病(HFMD)患儿的表达水平，探索其在HFMD中可能的临床意义。方法:选择2020年5月至2021年5月西安交通大学第二附属医院、西安市儿童医院和西安市中心医院就诊的50例HFMD患儿作为研究对象，并以同期同年龄段体检儿童20例作为对照组。HFMD患儿按病原学检测结果分为肠道病毒71(EV-A71)型和柯萨奇病毒A6(CV-A6)型，再根据疾病轻重程度分为轻症、重症患儿。比较并分析各组LGP2、RIG-I和MDA5的mRNA相对表达量及三者相关性。结果:50例HFMD患儿中EV-A71型26例(轻症16例，重症10例)，CV-A6型24例(轻症17例，重症7例)。HFMD组患儿与对照组儿童的年龄、性别差异均无统计学意义( P>0.05)。LGP2 mRNA在EV-A71型和CV-A6型HFMD患儿中的相对表达量分别为2.37(1.78，3.25)%和1.88(1.35，3.13)%，均低于对照组2.97(2.61，3.55)%，CV-A6型HFMD患儿与对照组间差异有统计学意义( Z＝－2.310， P＝0.021)；RIG-I mRNA在EV-A71型和CV-A6型HFMD患儿中的相对表达量分别为9.95(7.79，14.62)%和9.78(7.04，15.83)%，均低于对照组18.47(13.00，21.07)%，差异均有统计学意义(均 P<0.05)；MDA5 mRNA在EV-A71型和CV-A6型HFMD患儿中的相对表达量分别为4.41(2.82，5.99)%和3.98(2.18，7.41)%，均低于对照组5.10(3.52，7.71)%，但差异均无统计学意义。LGP2、RIG-I和MDA5 mRNA在EV-A71型和CV-A6型HFMD患儿轻症、重症间表达水平差异均无统计学意义。LGP2、RIG-I和MDA5 mRNA表达量具有较高相关性(均 P<0.001)。 结论:LGP2、RIG-I和MDA5 mRNA相对表达量在HFMD患儿中呈不同程度降低，且三者具有相关性。

目的:探讨可诱导神经生长因子(VGF)过表达在进展期前列腺癌(PCa)患者组织中的作用及其可能的分子生物学机制。方法:应用肿瘤基因组图谱(TCGA)和GEO数据库下载PCa中VGF芯片数据进行分析；根据不同的临床病理参数评估VGF在PCa患者中的表达和甲基化水平；TCGA和GSE21032数据库下载VGF芯片数据并用Kaplan-Meier生存曲线分析其与PCa患者生存期的相关性；应用David及京都基金及基因组百科全书(KEGG)通路富集分析VGF在PCa中的作用机制，并用String系统构建PPI网络，应用Mcode plugin分析识别关键模块；PROMO系统预测VGF的潜在转录因子并进行回归分析。采用 t检验。 结果:分析发现VGF mRNA在PCa中的表达上调，在GSE55945数据库中，PCa与正常组织中VGF相对表达水平分别为29.8、24.5 ( t＝7.612， P<0.05)，TCGA数据库中PCa与正常组织中VGF相对表达水平分别为25.5、15.6( t＝4.712， P<0.05)，而且VGF表达与其甲基化水平呈显著负相关；VGF在晚期PCa中明显高表达，其甲基化水平在晚期PCa中明显降低；分析显示VGF低表达PCa患者无进展生存期明显延长，VGF低甲基化患者无进展生存期明显缩短；分析显示VGF在PCa中通过GnRH、RIG-I样受体和丝裂原活化蛋白激酶(MAPK)等多种途径发挥调控作用；回归分析显示SP1、MAZ、NR3C1和环磷酸腺苷反应元件结合蛋白1(CREB1)是PCa中VGF表达的调节因子。 结论:VGF在PCa组织中高表达且表达强度与PCa进展明显相关。

目的:探讨抑制树突状细胞相关RIG-1的表达从而抑制小鼠骨髓来源树突状细胞(BMDC)成熟并促进免疫耐受的作用及其机制。方法:体外培养BMDC，并于第5天转染慢病毒载体Lv-RIG-1-RNA干扰(RNAi)(MOI＝100)，将其分为未成熟DC组，DC-绿色荧光蛋白(DC-GFP)组和DC-RIG-1-RNAi组，检测树突状细胞(DC)转染前后细胞表型[白细胞分化抗原(CD)40、CD80、CD86和主要组织相容性复合体 (MHC)Ⅱ]和功能变化；建立小鼠同种异体胰岛移植模型，术前3 d和1 d受体分别通过尾静脉注射1×10 6个DC-RIG-1-RNAi、DC-绿色荧光蛋白(GFP)和100 μl磷酸盐缓冲液(PBS)，观察各组移植术后胰岛存活时间，于术后第7天收集并比较受体糖耐量、胰岛移植物的病理学改变、脾脏T细胞亚群含量的变化。所有数据使用SPSS 22.0软件进行统计学分析，多组间比较采用单因素方差分析，两组间比较采用 t检验。 结果:Lv-RIG-1-RNAi组DC中等水平表达CD80、CD86，低水平表达CD40、MHC Ⅱ，脂多糖(LPS) 刺激后白细胞介素(IL)-12分泌水平[(135.4±7.3) ng/L]低于imDC组[(823.0±30.9) ng/L， t＝68.483， P<0.05]和DC-GFP组[(896.4±47.7) ng/L， t＝49.870， P<0.05]，差异均有统计学意义，IL-10分泌水平[(556.6±50.4) ng/L]高于imDC组[(279.4±66.2) ng/L， t＝10.535， P<0.05]和DC-GFP组[(291.8±65.6) ng/L， t＝10.122， P<0.05]，差异均有统计学意义，并且其抗原提呈功能被有效抑制，具有一定程度的未成熟属性。DC-RIG-1-RNAi组胰岛生存时间[(38.5±3.8) d]高于对照组[(22.0±3.1) d， t＝8.241， P<0.05]和DC-GFP组[(9.5±2.1) d， t＝16.361， P<0.05]，差异均有统计学意义。术后第7天，DC-RIG-1-RNAi组胰岛移植受体葡萄糖耐量较其他两组好，病理学检测结果提示DC-RIG-1-RNAi组胰岛活性更好。DC-RIG-1-RNAi组辅助性T细胞17/调节性T细胞(Th17/Treg)比值(0.08±0.02)低于DC-GFP组(0.28±0.02， t＝11.670， P<0.01)和对照组(0.48±0.08， t＝20.110， P<0.01)，差异均有统计学意义。 结论:抑制RIG-1表达的DC表现出一定程度的未成熟表型和功能，保护小鼠胰岛移植物，诱导免疫耐受。

目的 探讨《中华医典》中治疗腰腿痛方剂的用药特点及其规律.方法 检索《中华医典》方书类古籍治疗腰腿痛的方剂,建立数据库,对中药四气、五味、归经和分类进行分析,使用采用IBM SPSS Statistics 25和IBM SPSS Modeler 18软件对筛选处方中的药物进行关联规则分析和聚类分析,选取频率最高的药对作为网络药理学分析对象,采用HERB平台构建相应靶点数据库,采用GeneCards数据库预测疾病相关靶点基因.使用维恩图取交集靶点,并导入STRING数据库构建蛋白质-蛋白质相互作用网络,并将蛋白质-蛋白质相互作用网络导入Cytoscape 3.10筛选核心靶点,通过微生信网站进行基因本体(GO)富集分析和京都基因和基因组数据库(KEGG)信号通路富集分析.结果 筛选后共纳入方剂490首,包含中药202种,总用药频次4 737次,药物四气五味以温、辛、苦为主,归经主要在肝、脾、肾;中药分类以祛风湿药、补阳药、活血化瘀药为主.关联性药物以当归-附子-肉桂出现频次最高.59种高频药物共聚为六类.中药关键核心靶点包括TNF、PTGS2、CXCL8、CASP3、MYC、BCL2、RELA、CASP8、IL-18、NFKBIA;GO富集分析结果可知,主要涉及生物过程包括对脂多糖的反应等;主要涉及的细胞组分为膜筏、膜微区、膜区、突触前膜的组成部分等;主要涉及的分子功能为蛋白质异二聚等.KEGG富集最显著的通路为RIG-I样受体通路和IL-17通路.结论《中华医典》中治疗腰腿痛处方古籍中治疗腰腿痛的药物具有温、通、补的特点,当归、附子、肉桂作为核心药物对TNF、PTGS2、CXCL8等相关靶点,通过调控RIG-I样受体、IL-17等通路治疗腰腿痛.

Objective:Anemoside B4(AB4),the most abundant triterpenoidal saponin isolated from Pulsatilla chinen-sis,inhibited influenza virus FM1 or Klebsiella pneumoniae-induced pneumonia.However,the anti-SARS-CoV-2 effect of AB4 has not been unraveled.Therefore,this study aimed to determine the antiviral activ-ity and potential mechanism of AB4 in inhibiting human coronavirus SARS-CoV-2 in vivo and in vitro.Methods:The cytotoxicity of AB4 was evaluated using the Cell Counting Kit-8(CCK8)assay.SARS-CoV-2 infected HEK293T,HPAEpiC,and Vero E6 cells were used for in vitro assays.The antiviral effect of AB4 in vivo was evaluated by SARS-CoV-2-infected hACE2-IRES-luc transgenic mouse model.Furthermore,label-free quantitative proteomics and bioinformatic analysis were performed to explore the potential antiviral mechanism of action of AB4.Type Ⅰ IFN signaling-associated proteins were assessed using Western blotting or immumohistochemical staining.Results:The data showed that AB4 reduced the propagation of SARS-CoV-2 along with the decreased Nucleocapsid protein(N),Spike protein(S),and 3C-like protease(3CLpro)in HEK293T cells.In vivo antivi-ral activity data revealed that AB4 inhibited viral replication and relieved pneumonia in a SARS-CoV-2 infected mouse model.We further disclosed that the antiviral activity of AB4 was associated with the enhanced interferon(IFN)-β response via the activation of retinoic acid-inducible gene I(RIG-1)like receptor(RLP)pathways.Additionally,label-free quantitative proteomic analyses discovered that 17 pro-teins were significantly altered by AB4 in the SARS-CoV-2 coronavirus infections cells.These proteins mainly clustered in RNA metabolism.Conclusion:Our results indicated that AB4 inhibited SARS-CoV-2 replication through the RLR pathways and moderated the RNA metabolism,suggesting that it would be a potential lead compound for the development of anti-SARS-CoV-2 drugs.

目的 基于生物信息学分析探索影响扩张型心肌病(DCM)干细胞移植疗效的差异表达微小RNA(miRNAs)及其靶基因.方法 本研究筛选术前基线特征相似的DCM患者,以干细胞移植术后左心室射血分数改善＞5％为标准,分为治疗良好组和无效组.通过对两组患者的骨髓间充质干细胞样本进行miRNAs高通量测序,运用edgeR筛选出两组间的差异表达miRNAs,并使用MiRanda、TargetScan和RNAhybrid数据库进行靶基因预测,对预测得到的靶基因进行GO、KEGG及Reactome富集分析.使用STRING数据库和Cytoscape软件,并参考紧密度、介数中心性、度三种拓扑分析方法和韦恩图筛选关键基因.两组间比较采用t检验.结果 本研究共筛选出44个差异表达miRNAs,其中23个上调,21个下调.上调的差异表达miRNAs靶基因有908个,下调的差异表达miRNAs靶基因835个.GO分析结果显示靶基因在生物过程中主要富集在细胞代谢过程的调节,分子功能主要富集在离子结合,细胞组分则主要与细胞膜相关.KEGG通路和Reactome分析发现靶基因主要与调节干细胞多能性的信号通路、RIG-I样受体信号通路和RNA聚合酶Ⅱ转录等相关.此外,韦恩图共筛选出4个关键基因,分别为 MED1、SMAD2、TRAF6、GSK3B,其中 GSK3B 和TRAF6分别为上调的 hsa-miR-1304-3p和下调的hsa-miR-146a-3p靶基因,MED1和SMAD2为下调的hsa-miR-1299靶基因.结论 本研究提供了hsa-miR-1304-3p、hsa-miR-1299、hsa-miR-146a-3p 3 个可能用于提示 DCM 患者干细胞移植预后的差异表达miRNAs,其调控的关键基因MED1、SMAD2、TRAF6、GSK3B及所在通路可能是提高干细胞移植治疗DCM的潜在作用靶点.

目的 探讨香雪抗病毒口服液对甲型流感病毒的活性及其在炎症免疫应答中的潜在作用机制.方法 采用噻唑蓝(MTT)染色法检测香雪抗病毒口服液对MDCK细胞和A549细胞的细胞毒性作用.采用细胞病变抑制法(CPE)及空斑减少实验对香雪抗病毒口服液进行抗病毒活性检测.采用免疫荧光法检测 NP 蛋白.实时荧光定量PCR测定炎症细胞因子的表达情况.采用蛋白印迹实验检测香雪抗病毒口服液对炎症相关信号通路的蛋白表达的影响.结果 香雪抗病毒口服液对甲型流感病毒 A/PR/8/34(H1N1)和 A/Aichi/2/68(H3N2)均有一定的抗病毒抑制作用,半数细胞毒性浓度(TC50)分别为42.12、79.36 mg/mL,半数抑制浓度(IC50)分别为 8.665、5.260 mg/mL,并可减少H1N1 病毒空斑形成.香雪抗病毒口服液能呈剂量相关性地降低H1N1诱导的A549 细胞炎症因子肿瘤坏死因子-α(TNF-α)、视黄酸(维甲酸)诱导基因蛋白I(RIG-I)、趋化因子(CC基序)配体 5(CCL5)、巨噬细胞炎症蛋白-1β(MIP-1 β)、干扰素诱导蛋白-10(IP-10)、白细胞介素-10(IL-10)、λ干扰素(IFN-λ)、β干扰素(IFN-β)、白细胞介素-8(IL-8)mRNA的表达(P＜0.05、0.01、0.001).在流感病毒单复制周期中,香雪抗病毒口服液在早期阶段 0～2 h干预,具有良好的抗流感病毒效果.香雪抗病毒口服液能显著抑制Toll样模式识别受体 3(TLR3)、RIG-I、黑色素瘤分化相关基因 5(MDA-5)受体的表达,下调核转录因子-κB亚基p65(NF-κB p65)、NF-κB抑制蛋白α(IκBα)、信号转导和转录激活因子 1(STAT1)、STAT2、STAT3 的磷酸化水平,且在 20 mg/mL浓度下明显下调这些蛋白的表达.结论 香雪抗病毒口服液不仅能有效抑制甲流感病毒在人宿主细胞中复制,降低流感病毒诱导的炎症因子表达,而且能显著抑制流感病毒诱导天然免疫信号通路的相关蛋白表达,表明香雪抗病毒口服液具有防治流感病毒的潜力.