Effect and mechanism of reactive oxygen species-mediated NOD-like receptor family pyrin domain-containing 3 inflammasome activation in hepatic alveolar echinococcosis

BACKGROUND The NOD-like receptor family pyrin domain-containing 3(NLRP3)inflammasome is a significant component of the innate immune system that plays a vital role in the development of various parasitic diseases.However,its role in hepatic alveolar echinococcosis(HAE)remains unclear.AIM To investigate the NLRP3 inflammasome and its mechanism of activation in HAE.METHODS We assessed the expression of NLRP3,caspase-1,interleukin(IL)-1β,and IL-18 in the marginal zone and corresponding normal liver of 60 patients with HAE.A rat model of HAE was employed to investigate the role of the NLRP3 inflammasome in the marginal zone of HAE.Transwell experiments were conducted to investigate the effect of Echinococcus multilocularis(E.multilocularis)in stimulating Kupffer cells and hepatocytes.Furthermore,immunohistochemistry,Western blotting,and enzyme-linked immunosorbent assay were used to evaluate NLRP3,caspase-1,IL-1β,and IL-18 expression;flow cytometry was used to detect apoptosis and reactive oxygen species(ROS).RESULTS NLRP3 inflammasome activation was significantly associated with ROS.Inhibition of ROS production decreased NLRP3-caspase-1-IL-1βpathway activation and mitigated hepatocyte damage and inflammation.CONCLUSION E.multilocularis induces hepatocyte damage and inflammation by activating the ROS-mediated NLRP3-caspase-1-IL-1βpathway in Kupffer cells,indicating that ROS may serve as a potential target for the treatment of HAE.

Yemazhui(Herba Eupatorii Lindleyani)ameliorates lipopolysaccharide-induced acute lung injury via modulation of the toll-like receptor 4/nuclear factor kappa-B/nod-like receptor family pyrin domain-containing 3 protein signaling pathway and intestinal flor

OBJECTIVE:To investigate the impact of Yemazhui(Herba Eupatorii Lindleyani,HEL)against lipopolysaccharide(LPS)-induced acute lung injury(ALI)and explore its underlying mechanism in vivo.METHODS:The chemical constituents of HEL were analyzed by ultra-high performance liquid chromatographyquadrupole time-of-flight mass spectrometry method.Then,HEL was found to suppress LPS-induced ALI in vivo.Six-week-old male Sprague-Dawley rats were randomly divided into 6 groups:control,LPS,Dexamethasone(Dex),HEL low dose 6 g/kg(HEL-L),HEL medium dose 18 g/kg(HEL-M)and HEL high dose 54 g/kg(HEL-H)groups.The model rats were intratracheally injected with 3 mg/kg LPS to establish an ALI model.Leukocyte counts,lung wet/dry weight ratio,as well as myeloperoxidase(MPO)activity were determined followed by the detection with hematoxylin and eosin staining,enzyme linked immunosorbent assay,quantitative real time polymerase chain reaction,western blotting,immunohistochemistry,and immunofluorescence.Besides,to explore the effect of HEL on ALI-mediated intestinal flora,we performed 16s rRNA sequencing analysis of intestinal contents.RESULTS:HEL attenuated LPS-induced inflammation in lung tissue and intestinal flora disturbance.Mechanism study indicated that HEL suppressed the lung coefficient and wet/dry weight ratio of LPS-induced ALI in rats,inhibited leukocytes exudation and MPO activity,and improved the pathological injury of lung tissue.In addition,HEL reduced the expression of tumor necrosis factoralpha,interleukin-1beta(IL-1β)and interleukin-6(IL-6)in bronchoalveolar lavage fluid and serum,and inhibited nuclear displacement of nuclear factor kappa-B p65(NF-κBp65).And 18 g/kg HEL also reduced the expression levels of toll-like receptor 4(TLR4),myeloid differentiation factor 88,NF-κBp65,phosphorylated inhibitor kappa B alpha(phospho-IκBα),nod-like receptor family pyrin domain-containing 3 protein(NLRP3),IL-1β,and interleukin-18(IL-18)in lung tissue,and regulated intestinal flora disturbance.CONCLUSIONS:In summary,our findings revealed that HEL has a protective effect on LPS-induced ALI in rats,and its mechanism may be related to inhibiting TLR4/NF-κB/NLRP3 signaling pathway and improving intestinal flora disturbance.

膜蛋白ATAD3A在线粒体质量控制中的作用

线粒体质量控制对于线粒体网络的稳态和线粒体功能的正常发挥具有重要意义。三磷酸腺苷酶家族蛋白3A(ATAD3A)是同时参与调节线粒体结构功能、线粒体动力学和线粒体自噬等重要生物学过程的线粒体膜蛋白之一。近期研究表明,ATAD3A既可与Mic60/Mitofilin和线粒体转录因子A(TFAM)等因子相互作用以维持线粒体嵴的形态和氧化磷酸化功能,又能与发动蛋白相关蛋白1(Drp1)结合而正性/负性调节线粒体分裂,还可作为线粒体外膜转位酶(TOM)复合物和线粒体内膜转位酶(TIM)复合物之间的桥接因子而介导PTEN诱导激酶(PINK1)输入线粒体进行加工,显示出促自噬或抗自噬活性。本文对ATAD3A在调控线粒体质量控制中的作用及其机制进行了综述。

Qingchi San(青赤散)treats ulcerative colitis in mice by inhibiting the nuclear factor-kappa B signaling pathway and Nucleotide-binding oligomerization domain,leucine-rich repeat and pyrin domain-containing 3 inflammasome formation

OBJECTIVE:To investigate the efficacy of Qingchi San(青赤散,QCS),a preparation of Traditional Chinese Medicine,on ulcerative colitis(UC)in mice by inhibiting the nuclearfactor-kappa B(NF-κB)signaling pathway and nucleotide-binding oligomerization domain,leucine-rich repeat and pyrin domain-containing 3(NLRP3)inflammasome formation.METHODS:The UC model was established with male C57BL/6J as the animal model.Bodyweight,Disease Activity Index(DAI),colon length and weight were detected.Furthermore,colonic histology was performed by hematoxylin-eosin(HE)staining.interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),myeloperoxidase(MPO)and superoxide dismutase(SOD)were performed by enzyme-linked immunosorbent assay.Cyclooxygenase 2(COX2)and inducible nitric oxide synthase(iNOS)mRNA expression were conducted by real-time quantitative polymerase chain reaction(RT-qPCR).NF-κB,inhibitor of NF-κBα(iκBα),Phosphorylated inhibitor of NF-κBα(p-iκBα),caspase-1,NLRP3 and Apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC)protein expression were conducted by Western blotting.RESULTS:Compared with UC model group,Bodyweight was significantly increased in QCS treatment.At the same time,DAI was significantly decreased in QCS treatment.Colon length and weight and colonic histology were significantly improved in QCS treatment.Furthermore,the expression of IL-1β,IL-6,TNF-α,MPO,SOD,COX2,and iN OS were significantly decreased in QCS treatment.Finally,the expression of NF-κB signaling pathway-related proteins NF-κB,iκBα,p-iκBα,and the expression of NLRP3 inflammasome related proteins caspase-1,NLRP3 and ASC were significantly decreased in QCS treatment.CONCLUSION:Traditional Chinese drug QCS could treat UC by inhibiting the NF-κB signaling pathway and NLRP3 inflammasome formation in mice.

过表达ATAD3A诱导大鼠正常肝干细胞发生上皮-间质转化

目的探讨过表达ATP酶家族AAA结构域蛋白(ATPase family AAA domain protein,ATAD3A)后对大鼠肝脏干细胞上皮-间质转化(epithelial-to-mesenchymal transition,EMT)、迁移和侵袭能力的影响。方法体外培养大鼠肝干细胞系(WBF-344),分别转染含过表达ATAD3A基因的慢病毒(ATAD3A过表达组)和不含过表达ATAD3A基因的载体(空载对照组),以不做处理的细胞作为正常对照组。显微镜下观察细胞形态变化,细胞划痕和Transwell实验检测各组细胞的迁移和侵袭能力,RT-qPCR法和Western blot法检测EMT标志物和转录因子的基因和蛋白表达水平,体外裸鼠皮下成瘤实验验证肝干细胞恶性转化程度。结果与2个对照组比较,ATAD3A过表达组:①细胞由卵圆形向长条形和梭形的间质细胞形态特征转变;②细胞迁移和侵袭能力得到明显增强(P<0.05);③E-cadherin的基因和蛋白表达明显降低,而N-cadherin的基因和蛋白表达明显升高(P<0.05);④转录因子ZEB1和ZEB2的表达也显著增加(P<0.05)。但体外成瘤实验未能观察到肿瘤块的形成。结论过表达ATAD3A后能诱导大鼠肝干细胞EMT的发生。

Sini powder ameliorates the inflammatory response in rats with stress-induced non-alcoholic fatty liver disease by inhibiting the nuclear factor kappa-B/pyrin domain-containing protein 3 pathway

OBJECTIVE: To evaluate the effectiveness of Sini powder for the treatment of non-alcoholic fatty liver disease(NAFLD) in rats and the molecular mechanisms involved.METHODS: A rat model of stress-induced NAFLD was established by a combination of long-term tethering and feeding of a high-fat, high-calorie diet. These rats were then intragastrically administered with either simvastatin, Sini powder, or vehicle for 1 week. The body mass and field test scores for each group were recorded weekly. Serum aspartate aminotransferase and alanine aminotransferase activities, and triglyceride, total cholesterol,and free fatty acid concentrations were measured.Liver tissue histopathology was examined on hematoxylin and eosin-stained paraffin sections and oil red O-stained frozen sections. The hepatic m RNA expression of nuclear factor kappa-B(NF-κB),NOD-, LRR-, and pyrin domain-containing protein 3(NLRP3), apoptosis-associated speck-like protein containing CARD(ASC), and caspase-1 were measured by reverse transcription-polymerase chain reaction(RT-PCR). The hepatic protein concentrations of NF-κB and NLRP3, ASC, caspase-1, interleukin-1β(IL-1β), interleukin-6(IL-6), and the serum concentrations of IL-1β and IL-6 were measured by enzyme-linked immunosorbent assay.RESULTS: Compared with the Blank group, rats in the Compound model group showed significant pathologic manifestations of NAFLD, and the expression of NF-κB, NLRP3, ASC, caspase-1, IL-1βand IL-6 were significantly higher(all P < 0.01). Both simvastatin and Sini powder significantly ameliorated the NAFLD pathology and the abnormal expression of NF-κB, NLRP3, ASC, caspase-1, IL-1β, and IL-6(all P < 0.01).CONCLUSION: Sini powder inhibits the inflamma-tory response in rats with NAFLD, which is mediated by NF-κB/NLRP3, IL-1β, and IL-6, reduces the effects of psychological stress, and improves lipid metabolism. Therefore, Sini powder may be effective for the treatment of stress-related NAFLD through multiple mechanisms.

Qingre Jianpi decoction(清热健脾汤)attenuates inflammatory responses by suppressing NOD-like receptor family pyrin domain-containing 3 inflammasome activation in dextran sulfate sodium-induced colitis mice

OBJECTIVE:To investigate the effects of Qingre Jianpi decoction(清热健脾汤,QRJPD)on dextran sulfate sodium(DSS)-induced colitis mice and explore its mechanism.METHODS:All mice were randomly divided into six groups.Weight changes,disease activity index values,and histological damage were detected.Inflammatory cytokines and immune cell infiltration were measured using enzyme-linked immunosorbent assays(ELISA)and immunohistochemistry(IHC)method.The key protein expression levels of the NOD-like receptor family pyrin domain-containing 3(NLRP3)inflammasome were detected by western blot analysis,IHC,and quantitative reverse transcription polymerase chain reaction.RESULTS:QRJPD played an anti-inflammatory role in the treatment of ulcerative colitis(UC),reduced the secretion of inflammatory cytokines,and inhibited the inflammatory infiltration of immune cells by suppressing DSS-induced activation of the NLRP3 inflammasome.CONCLUSION:QRJPD exerts protective effects by inhibiting DSS-induced NLRP3 inflammasome activation.

人源fidgetin like-1蛋白AAA结构域的重组表达及酶学性质分析

Fidgetin是2000年发现的一个AAA蛋白家族新成员,同源序列分析表明它与ka-tanin、spastin属于AAA家族的同一亚家族,因此可能也具有ATP依赖的微管切割活性,ATP的结合和水解应该发生于fidgetin蛋白中保守的AAA结构域.在大肠杆菌中重组表达的人源fidgetin like-1(FIGL-1)蛋白的AAA结构域片段(HsFIGL-1-AAA),纯化后的最终产率为每升5 mg蛋白.与AAA蛋白通常形成六聚体不同,HsFIGL-1-AAA在溶液中为单体,且其ATPase活性很低,仅为0.0063 s-1.与其他AAA蛋白的序列比对发现,Sensor 2 motif中保守的Arg残基在HsFIGL-1-AAA结构域中为Thr,但该位点的突变T609R并未明显改变该蛋白的ATPase活性.这些结果提示HsFIGL-1可能以一种特殊的方式发挥功能,这为进一步研究fidgetin及其同源蛋白的结构和功能奠定了基础.

AstragalosideⅣplays a role in reducing radiation-induced liver inflammation in mice by inhibiting thioredoxin-interacting protein/nod-like receptor protein 3 signaling pathway

OBJECTIVE:To investigate the efficacy of Astragaloside IV(AS-IV)on radiation-induced liver inflammation in mice.METHODS:The mice were divided into normal group,dimethyl sulfoxide solvent group,irradiation group(IR),irradiation+AS-IV(20 mg/kg)group(IR+AS-20)and irradiation+AS-IV(40 mg/kg)group(IR+AS-40).One month after intraperitoneal injection of AS-IV,the mice were irradiated with 8Gry Co60γ,the blood was collected for biochemical analysis,and the liver was collected for hematoxylin-eosin staining,immunofluorescence and electron microscopic observation,oxidative stress,and Western blot analysis.RESULTS:The AS-IV treatment significantly ameliorated the pathological morphology of liver and reduced the alanine aminotransferase and aspertate aminotransferase levels in serum induced by radiation;AS-IV treatment also significantly reduced the expression of inflammatory factors tumor necrosis factor alpha and interleukin 6 and antagonized malonaldehyde content and superoxide dismutase activity in liver caused by radiation;in addition,AS-IV treatment can significantly inhibited the positive expression of thioredoxin-interacting protein(TXNIP)and nod-like receptor protein 3(NLRP3)inflammasome in liver tissue after radiation;The expression of TXNIP,NLRP3 inflammasome,apoptosisassociated speck-like protein containing a CARD,cysteinyl aspartate-specific proteinase 1 and interleukin 1beta in the AS-IV prevention group decreased significantly compared to the radiation group.CONCLUSIONS:These findings suggested that Co60γradiation can cause structural and functional damage to the liver,which may be related to the NLRP3 mediated inflammatory pathway;AS-IV may play a protective role by inhibiting the TXNIP/NLRP3 inflammasome signaling pathway in the radiation-induced liver injury model.

A Novel Mutation in the Pyrin Domain of the NOD-like Receptor Family Pyrin Domain Containing Protein 3 in Muckle-Wells Syndrome

Background： Cryopyrin-associated periodic syndrome （CAPS） is a group of rare, heterogeneous autoinflammatory disease characterized by interleukin （IL）-1β-mediated systemic inflammation and clinical symptoms involving skin, joints, central nervous system, and eyes. It encompasses a spectrum of three clinically overlapping autoinflammatory syndromes including familial cold autoinflammatory syndrome, Muckle-Wells syndrome （MWS）, and neonatal-onset multisystem inflammatory disease. CAPS is associated with gain-of-function missense mutations in NOD-like receptor family pyrin domain-containing protein 3 （NLRP3）, the gene encoding NLRP3. Moreover, most mutations leading to MWS occurred in exon 3 ofNLRP3 gene. Here, we reported a novel mutation occurred in exon 1 ofNLRP3 gene in an MWS patient and attempted to explore the pathogenic mechanism. Methods： Genetic sequence analysis of NLRP3 was performed in an MWS patient who presented with periodic lever, arthralgia, and multiform skin lesions. NLRP3 was also analyzed in this patient＇s parents and 50 healthy individuals. Clinical examinations including X-ray examination, skin biopsy, bone marrow aspiration smear, and blood test of C-reactive protein （CRP）, erythrocyte sedimentation rate （ESR）, serum levels oflL-1β, immunoglobulin E （lgE）, antineutrophil cytoplasmic antibodies, antinuclear antibodies, and extractable nuclear antigen were also analyzed. The protein structure of mutant NLRP3 inflammasome was calculated by SWISS-MODEL software. Proteins of wild type and mutant components ofNLRP3 inflammasome were expressed and purified, and the interaction abilities between these proteins were tested by surface plasmon resonance （SPR） assay. Results： X-ray examination showed no abnormality in the patient＇s knees. Laboratory tests indicated an elevation of CRP （233.24 nag/L） and ESR （67 mm/h） when the patient had fever. Serum IL-1β increased to 24.37 pg/ml, and serum lgE was higher than 2500.00 IU/ml. Other blood tests were normal. Bone marrow aspiration smear was normal. A novel point mutation c.92A〉T in exon 1 of NLRP3 gene was identified, which caused a p.D31V mutation in pyrin domain （PYD） of NLRP3. SPR assay showed that this point mutation may strengthen the interaction between the PYD of NLRP3 and the PYD of the apoptosis-associated speck-like protein. The mutation c.92A〉T in exon 1 of the NLRP3 gene was not lbund in the patient＇s parents and 50 healthy individuals. Conclusions： The rnutation c.92A〉T in exon 1 of the NLRP3 gene is a novel mutation associated with MWS. The p.D31V mutation might promote the activation ofNLRP3 inflammasome and induce MWS in this patient.

转染三磷酸腺苷酶家族蛋白2 shRNA的人骨肉瘤细胞株U2-OS增殖、凋亡能力观察

目的观察转染三磷酸腺苷酶家族蛋白2(ATAD2)shRNA的骨肉瘤细胞株U2-OS增殖、凋亡的变化。方法以qRT-PCR法和Western blotting法分别检测人骨肉瘤细胞(U2-OS、MG-63、SaOS-2)和人正常成骨细胞(h FOB1.19)中ATAD2 mRNA、蛋白。在U2-OS细胞中转染ATAD2 shRNA慢病毒载体,采用qRT-PCR法和Western blotting法检测转染效果,CCK8方法检测细胞增殖变化,平板克隆形成实验检测细胞克隆形成数目,碘化丙啶(PI)单染法检测细胞周期,膜联蛋白V-FITC(Annexin V-FITC)/PI双染法检测细胞凋亡率,Western blotting法检测细胞剪切的含半胱氨酸的天冬氨酸蛋白水解酶3(C-Caspase-3)、细胞周期依赖性蛋白激酶4(CDK2)、剪切的含半胱氨酸的天冬氨酸蛋白水解酶9(C-Caspase-9)、细胞周期蛋白D1(CyclinD1)蛋白。结果骨肉瘤细胞中ATAD2 mRNA、蛋白表达水平高于正常成骨细胞(P均<0.05)。ATAD2 shRNA慢病毒载体转染后的骨肉瘤细胞中ATAD2表达水平下降(P<0.05)。转染ATAD2 shRNA后的骨肉瘤细胞增殖能力和克隆形成能力均降低,细胞G1期比例升高,细胞凋亡率升高,细胞中C-Caspase-3、C-Caspase-9蛋白表达升高,CDK2、CyclinD1蛋白表达降低(P均<0.05)。结论转染ATAD2 shRNA可抑制U2-OS细胞增殖,并诱导细胞凋亡。

三磷酸腺苷酶家族蛋白2在不同病变胃组织中的表达及其意义

目的探讨三磷酸腺苷酶家族蛋白2(ATAD2)在不同病变胃组织中的表达及意义。方法收集12例新鲜胃腺癌及相应癌旁相对正常组织标本,利用实时荧光定量PCR方法(RT-qPCR)检测两种组织中ATAD2 mRNA的表达;同时应用免疫组化法检测ATAD2在121例胃腺癌及相应癌旁相对正常组织、121例非瘤变胃组织、29例高低级别上皮内瘤变组织中的表达情况。结果 RT-qPCR方法检测结果表明,与相应癌旁相对正常组织相比,新鲜胃腺癌组织中ATAD2 mRNA表达明显上调,差异具有统计学意义(t=4.664,P<0.01);ATAD2在非瘤变胃组织中均不表达,在高低级别上皮内瘤变中均为低表达,在胃腺癌组织中有23例(23/121)呈高表达。在胃腺癌组织中ATAD2的表达明显高于相应癌旁相对正常组织(z=-12.588,P<0.01);在低级别上皮内瘤变中的表达高于非瘤变胃组织(z=-7.984,P<0.01);在胃腺癌、高级别上皮内瘤变组织中的表达均高于低级别上皮内瘤变组织(z=-2.531、-2.285,P<0.05);在胃腺癌与高级别上皮内瘤变、慢性萎缩性胃炎与慢性浅表性胃炎中的表达差异均不具有统计学意义(P>0.05)。ATAD2在胃腺癌组织中的表达与肿瘤大小、分化程度相关(χ~2=5.506、8.412,P<0.05),而与病人年龄、性别、淋巴结转移、浸润深度、TNM分期无关(P>0.05)。结论 ATAD2参与了胃腺癌的发生发展过程;ATAD2表达检测对临床判定某些胃黏膜病变性质有重要的参考价值。

肝癌中线粒体膜蛋白ATAD3A表达与临床病理特征及预后的关系

目的:检测肝癌组织中线粒体膜蛋白ATAD3A表达情况,探究其与肝癌临床病理特征及预后的关系。方法:选取2016年7月至2018年3月行手术治疗的85例肝癌患者癌组织及其对应癌旁组织(距癌灶边缘3~5 cm)标本进行研究。采用免疫组织化学染色法和qRT-PCR检测肝癌组织及癌旁组织中ATAD3A蛋白及mRNA表达情况。采用SPSS 25.0统计软件进行数据分析,计量资料以(xˉ±s)表示,计数资料用[n(%)]表示,ATAD3A表达与临床病理特征的相关性采用χ^(2)或t检验,Kaplan-Meier生存曲线分析预后,COX回归分析预后生存危险因素。P<0.05认为差异有统计学意义。结果:肝癌组织中ATAD3A蛋白阳性表达率为28.2%(24/85),mRNA相对表达水平为(2.6±0.5),明显低于癌旁正常组织的76.5%(65/85)和(4.8±0.9),差异有统计学意义(χ^(2)=39.641,t=19.701,P<0.001)。ATAD3A蛋白及mRNA表达与肝癌肿瘤直径、分化程度、TNM分期及脉管癌栓等病理特征关系密切,差异有统计学意义(χ^(2)=39.641,P<0.05)。ATAD3A阳性组患者术后5年累积总生存率为58.3%,阴性组患者术后5年累积总生存率为41.0%,差异有统计学意义(Log-Rank χ^(2)=4.317,P=0.038)。肿瘤直径、分化程度、TNM分期、伴脉管癌栓及ATAD3A阴性表达是影响肝癌患者预后不良的独立危险因素(P<0.05)。结论:肝癌组织中ATAD3A显著阴性/低表达,与肿瘤直径、分化程度、TNM分期、脉管癌栓等病理特征关系密切,可能参与病情进展;ATAD3A阴性表达是影响肝癌患者预后不良的独立危险因素,可将其作为判断预后的分子标志物。

卵巢癌腹水细胞块中ATAD-2的表达及其临床意义

目的研究卵巢癌腹水细胞块中三磷酸腺苷酶家族蛋白2抗体(ATAD-2)的表达及其临床意义。方法收集2021年8月-2022年8月在赣州市人民医院治疗的卵巢癌患者40例,将其作为观察组,收集同期卵巢良性病变患者40例作为对照组,采集两组患者卵巢腹水细胞块的石蜡包埋标本,检测其ATAD-2水平,并研究其与卵巢癌患者肿瘤标志物[人附睾上皮分泌蛋白4(HE4)、糖类抗原125(CA125)]、临床病理特征及预后的关系。结果观察组患者ATAD-2蛋白表达较对照组高(P<0.05);ATAD-2高表达患者HE4、CA125水平均较ATAD-2低表达患者高(P<0.05);卵巢癌浆液型、低分化及Ⅲ-Ⅳ期患者ATAD-2蛋白表达较卵巢癌黏液型、高分化及Ⅰ-Ⅱ期患者高(P<0.05);当ATAD-2呈高表达时,其Youden指数为0.428,曲线下面积(AUC)为0.714,预测卵巢癌预后敏感度为100.00%,特异度为42.86%;当HE4的截断值为324.94pmol/L时,其Youden指数为0.524,AUC为0.742,预测卵巢癌预后敏感度为66.67%,特异度为85.71%;当CA125的截断值为693.89U/mL时,其Youden指数为0.556,AUC为0.774,预测卵巢癌预后敏感度为55.56%,特异度为100.00%。结论ATAD-2蛋白在卵巢癌腹水细胞块中呈高表达状态,且ATAD-2呈高表达时,其HE4、CA125水平提高,与卵巢癌病情进展及预后有关。

Ginsenoside Rb1 Reduces D-GalN/LPS-induced Acute Liver Injury by Regulating TLR4/NF-κB Signaling and NLRP3 Inflammasome

Background and Aims:The effect of ginsenoside Rb1 on D-galactosamine(D-GalN)/lipopolysaccharide(LPS)-induced acute liver injury(ALI)is unknown.The aim of this study was to evaluate the effect of ginsenoside Rb1 on ALI and its underlying mechanisms.Methods:Mice were pretreated with ginsenoside Rb1 by intraperitoneal injection for 3 days before D-GalN/LPS treatment,to induce ALI.The survival rate was monitored every hour for 24 h,and serum biochemical parameters,hepatic index and histopathological analysis were evaluated to measure the degree of liver injury.ELISA was used to detect oxidative stress and inflammatory cytokines in hepatic tissue and serum.Immunohistochemistry staining,RT-PCR and western blotting were performed to evaluate the expression of toll-like receptor 4(TLR4),nuclear factorkappa B(NF-κB),and NLR family,pyrin domain-containing 3 protein(NLRP3)in liver tissue and Kupffer cells(KCs).Results:Ginsenoside Rb1 improved survival with D-GalN/LPS-induced ALI by up to 80%,significantly ameliorated the increased alanine and aspartate transaminase,restored the hepatic pathological changes and reduced the levels of oxidative stress and inflammatory cytokines altered by D-GalN/LPS.Compared to the control group,the KCs were increased in the D-GalN/LPS groups but did not increase significantly with Rb1 pretreatment.D-GalN/LPS could upregulate while Rb1 pretreatment could downregulate the expression of interleukin(IL)-1β,IL-18,NLRP3,apoptosis associated specklike protein containing CARD(ASC)and caspase-1 in isolated KCs.Furthermore,ginsenoside Rb1 inhibited activation of the TLR4/NF-κB signaling pathway and NLRP3 inflammasome induced by D-GalN/LPS administration.Conclusions:Ginsenoside Rb1 protects mice against D-GalN/LPS-induced ALI by attenuating oxidative stress and the inflammatory response through the TLR4/NF-κB signaling pathway and NLRP3 inflammasome activation.

沉默ATAD3A对黑素瘤A375细胞增殖、侵袭和迁移的影响及机制研究

目的探讨沉默三磷酸腺苷酶家族蛋白3A(ATAD3A)对黑素瘤A375细胞增殖、侵袭和迁移能力的影响。方法2019年8-12月在重庆市渝北区人民医院收集经病理确诊的3例黑素瘤患者的黑素瘤组织及癌旁组织,应用Western印迹检测ATAD3A在上述组织中的表达。分别采用沉默ATAD3A慢病毒和空载慢病毒感染黑素瘤A375细胞系,构建沉默ATAD3A(shATAD3A)实验组和空载对照(shCtrl)组,经实时荧光定量聚合酶链反应(qRT-PCR)和Western印迹验证干扰效率。采用CCK-8实验、克隆形成实验比较两组细胞的增殖能力和克隆形成能力,采用Transwell细胞侵袭实验、划痕实验比较两组细胞的侵袭、迁移能力,采用Western印迹检测两组细胞自我更新相关蛋白(NANOG、SOX2、OCT4)和侵袭、迁移相关蛋白[基质金属蛋白酶2(MMP2)、波形蛋白、SLUG]的表达水平。两组间各指标的比较采用独立样本t检验。结果Western印迹显示,相对于癌旁组织,ATAD3A在3例黑素瘤组织中高表达(t=10.825,P<0.001)。qRT-PCR和Western印迹显示,shATAD3A组A375细胞ATAD3A mRNA为0.230±0.073,shCtrl组为1.000±0.244,两组比较,t=9.461,P<0.001,蛋白表达水平分别为0.279±0.267和0.867±0.115,t=8.595,P=0.002,表明成功构建沉默ATAD3A的黑素瘤A375细胞系。克隆形成实验和CCK-8实验显示,shATAD3A组克隆形成率低于shCtrl组(22.667%±2.510%比43.667%±5.030%,t=6.464,P=0.003),且细胞增殖活性自第2天开始直至第4天均显著低于shCtrl组。划痕实验及Transwell细胞侵袭实验显示,与shCtrl组相比,shATAD3A组划痕愈合减慢,划痕愈合率自第12 h开始(32.920%±4.642%比49.302%±1.448%,t=5.835,P=0.004)至24 h明显降低,通过Transwell小室下室的侵袭细胞数减少(68.330±13.050比234.330±19.139,t=12.411,P<0.001)。Western印迹显示,shATAD3A组细胞NANOG、SOX2、OCT4、MMP2、波形蛋白、SLUG的表达水平均显著下降(P<0.05或0.001)。结论ATAD3A在黑素瘤组织中高表达,沉默ATAD3A能抑制黑素瘤A375细胞的增殖、侵袭和迁移能力。

Effect of electroacupuncture on inflammatory signal expression in local tissues of rats with chronic pelvic pain syndrome based on purinergic 2X7 receptor/NOD-like receptor pyrin domaincontaining 3 signal pathway

OBJECTIVES: To study the expression of inflammatory signal in local prostate tissue of chronic pelvic pain syndrome(CPPS) rats by electroacupuncture(EA) of Guanyuan(CV4), Zhongji(CV3), Huiyang(BL35) and Sanyinjiao(SP6), and to explore the possible mechanism of anti-inflammatory and analgesic effects of EA. METHODS : A total of 36 Sprague-Dawley male rats were randomly divided into three groups: control, model and EA(n=12 rats/group). The CPPS model was made by injection of CFA into ventral lobes of the prostate(0.1 m L). Electric acupuncture apparatus was applied to stimulate Guanyuan(CV4), Zhongji(CV3), bilateral Huiyang(BL35) and Sanyinjiao(SP6) acupoints in EA group. The general condition of rats was observed and the prostate index(PI) was calculated. The thermal pain threshold was collected after each therapeutic course. Histopathological changes of the prostate tissue were examined by hematoxylin-eosin staining method. The expression levels of tumor necrosis factor α(TNF-α), interleukin-1β(IL-1β) and prostaglandin E2(PGE2) in prostatic homogenates were measured by enzyme linked immunosorbent assay(ELISA). Moreover, the expression levels of purinergic 2X7 receptor(P2X7R), NOD-like receptor pyrin domain-containing 3(NLRP3), caspase-1 and interleukin-18(IL-18) m RNA were quantified by quantitative real-time polymerase chain reaction. RESULTS: Compared with control group, the PI of rats increased, and the thermal pain threshold decreased significantly in model group. The morphological structure of prostate tissues of rats in model group was severely damaged with a large number of inflammatory cells infiltration. Additionally, the levels of TNF-α, IL-1β and PGE2 were higher, and the expressions of P2X7R, NLRP3, caspase-1 and IL-18 m RNA were higher than those in control group. After EA treatment, the PI was significantly decreased, the thermal pain threshold was significantly increased, and the tissue damage was significantly improved. The expressions of inflammatory cytokines were lower in EA group, and expression of P2X7R/NLRP3 pathway was down-regulated. CONCLUSION: The effect of EA at Guanyuan(CV4), Zhongji(CV3), Huiyang(BL35) and Sanyinjiao(SP6) can improve inflammation and pain symptoms of CPPS rats induced by Complete Freund’s adjuvant(CFA). This suggests that EA at Guanyuan(CV4), Zhongji(CV3), Huiyang(BL35) and Sanyinjiao(SP6) can produce antiinflammatory analgesia effect by preventing the activation of P2X7R/NLRP3 signal pathway, inhibit the release of inflammatory cytokines in CPPS rats, which may provide a putative novel target for the treatment of CPPS.

Reduning plus ribavirin display synergistic activity against severe pneumonia induced by H1N1 influenza A virus in mice

OBJECTIVE:To investigate synergistic effect of Reduning(RDN)injection plus ribavirin against severe pneumonia induced by H1 N1 influenza A virus in mice.METHODS:We established a mouse model of severe pneumonia induced by influenza A virus by infecting Balb/c mice with CA07 virus.We randomly assigned the infected mice into four groups,and treated them with normal saline(NS group),RDN(injection,86.6 mg/kg),ribavirin(injection,66.6 mg/kg)or double Ribavirin plus RDN group,the same dosage as used in the single treatments)for 5 d.Lung index and lung pathology were recorded or calculated in terms of the curative effective.Cytokines,NOD-like receptor family pyrin domain containing 3(NLRP3)inflammasome related protein including caspase-associated recruitment domain(CARD)domain Apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC),caspase-1 and NOD-like receptor family,pyrin domain containing 3(NLRP3),and reactive oxygen species were simultaneously investigated.RESULTS:RDN plus ribavirin treatment,not RDN or ribavirin alone,provided a significant survival benefit to the influenza A virus-infected mice.The combination treatment protected the mice against severe influenza infection by attenuating the severe lung injury.The combined treatment also reduced the viral titers in mouse lungs and lung index,downregulated their immunocytokine levels,including IL-1βand IL-18,and down regulated the NLRP3,especially the transcription and translation of caspase-1.Meanwhile NS group had significantly higher reactive oxygen species(ROS)expression which could was dramatically reduced by the treatment of RDN plus ribavirin.CONCLUSION:Our study showed that RDN combined with ribavirin could protect the mice,and reduce the lung immunopathologic damage caused by severe influenza pneumonia.The mechanism could be that it reduced ROS produce and inhibited NLRP3 inflammasome activation so that mainly lower the downstream inflammatory cytokines IL-1βand IL-18.

雄激素受体与三磷酸腺苷酶家族蛋白2在肝细胞癌中的表达及二氢睾酮对其影响

目的分析肝细胞癌（HCC）中雄激素受体（AR）、三磷酸腺苷酶家族蛋白2（ATAD2）的表达与患者临床指标的关系，探讨二氢睾酮（DHT）、AR与ATAD2对肝癌细胞增殖的影响。方法收集2012年2月至12月中国医科大学附属第一医院75例HCC患者标本及临床资料。LM3和Huh7细胞随机分为对照组、DHT组、DHT＋CDX（比卡鲁胺）组、CDX组；LM3和Huh7细胞均分为Ri-ATAD2组（加入干扰片段）和Ri-C组（加入对照载体序列）。免疫组化检测AR、ATAD2表达，并分析其与临床指标及患者生存的关系。Real-time PCR、Western印迹检测AR、ATAD2表达；CCK-8检测各组细胞增殖。结果HCC患者按AR和ATAD2表达高低分组。与31例AR低表达比较，44例高表达患者肿瘤≤5 cm比例升高，TNM分期Ⅰ＋Ⅱ比例降低；与35例ATAD2低表达比较，40例高表达患者转移和肿瘤分化Ⅲ＋Ⅳ级比例升高，差异有统计学意义（P〈0.05）。ATAD2高表达患者术后总生存率低于其他患者，差异有统计学意义（P〈0.05）。多因素Cox回归分析显示ATAD2表达（HR＝1.935，95%CI：1.066～3.515）、转移（HR＝2.212，95%CI：1.059～4.619）为患者术后预后不良独立预测指标。与正常人肝细胞LO2比较，LM3及Huh7组AR、ATAD2蛋白和mRNA相对表达升高，差异有统计学意义（P〈0.05）。加入DHT后，与对照组比较，肝癌细胞在48、72小时后增殖率明显升高，差异有统计学意义（P〈0.05）。加入CDX后，DHT诱导的LM3及Huh7增殖受到抑制。DHT可诱导ATAD2的蛋白表达，而CDX可抑制ATAD2的表达。肝癌细胞加入DHT 48、72小时后，与Ri-C组比较，Ri-ATAD2组增殖下降，差异有统计学意义（P〈0.05）。结论DHT及AR可通过诱导ATAD2的表达进一步促进肝癌细胞的增殖。ATAD2可能是DHT及AR促进肝癌细胞增殖的潜在机制之一。