Insulin-like growth factor binding protein related protein 1 knockdown attenuates hepatic ?brosis via the regulation of MMPs/TIMPs in mice

Background: Previous research suggested that insulin-like growth factor binding protein related protein 1(IGFBPrP1), as a novel mediator, contributes to hepatic fibrogenesis. Matrix metalloproteinases(MMP) and tissue inhibitors of metalloproteinases(TIMP) play an essential role in hepatic fibrogenesis by regulating homeostasis and remodeling of the extracellular matrix(ECM). However, the interaction between IGFBPrP1 and MMP/TIMP is not clear. The present study was to knockdown IGFBPrP1 to investigate the correlation between IGFBPrP1 and MMP/TIMP in hepatic fibrosis. Methods: Hepatic fibrosis was induced by thioacetamide(TAA) in mice. Knockdown of IGFBPrP1 expression by ultrasound-targeted microbubble destruction-mediated CMB-shRNA-IGFBPrP1 delivery, or inhibition of the Hedgehog(Hh) pathway by cyclopamine treatment, was performed in TAA-induced liver fibrosis mice. Hepatic fibrosis was determined by hematoxylin and eosin and Sirius red staining. Hepatic expression of IGFBPrP1, α-smooth muscle actin( α-SMA), transforming growth factor β 1(TGF β1), collagen I, MMPs/TIMPs, Sonic Hedgehog(Shh), and glioblastoma family transcription factors(Gli1) were investigated by immunohistochemical staining and Western blotting analysis. Results: We found that hepatic expression of IGFBPrP1, TGF β1, α-SMA, and collagen I were increased longitudinally in mice with TAA-induced hepatic fibrosis, concomitant with MMP2/TIMP2 and MMP9/TIMP1 imbalance and Hh pathway activation. Knockdown of IGFBPrP1 expression, or inhibition of the Hh pathway, reduced the hepatic expression of IGFBPrP1, TGF β1, α-SMA, and collagen I and re-established MMP2/TIMP2 and MMP9/TIMP1 balance. Conclusions: Our findings suggest that IGFBPrP1 knockdown attenuates liver fibrosis by re-establishing MMP2/TIMP2 and MMP9/TIMP1 balance, concomitant with the inhibition of hepatic stellate cell activation, down-regulation of TGF β1 expression, and degradation of the ECM. Furthermore, the Hh pathway mediates IGFBPrP1 knockdown-induced attenuation of hepatic fibrosis through the regulation of MMPs/TIMPs balance.

鳖甲煎丸对肝癌大鼠黏着斑激酶/雷帕霉素靶蛋白通路的影响

目的:探讨鳖甲煎丸对肝癌模型大鼠肝组织病理特征及FAK/mTORC1通路的影响。方法:HepG2(1×10^(7))细胞注射于大鼠右腋皮下,建立肝癌模型,分为模型组、5-氟尿嘧啶组、鳖甲煎丸低剂量组、鳖甲煎丸高剂量组,另设正常组。各药物组给予相应药物灌胃,持续给药4周,正常组、模型组给予等体积的0.9%氯化钠溶液。实验结束后,测定肝癌组织重量、肝癌组织体积、肝癌组织抑瘤率、肝癌FAK、mTORC1水平、肝癌VEGFR-2、VEGF、IL-4、IL-8、TNF-α蛋白水平。结果:与正常组比较,模型组肝癌组织重量、肝癌组织体积、肝癌组织抑瘤率、VEGFR-2、VEGF、IL-4、IL-8、TNF-α蛋白、肝癌FAK、mTORC1 mRNA和蛋白表达升高(均P<0.05)。与模型组比较,5-氟尿嘧啶组、鳖甲煎丸低剂量组、鳖甲煎丸高剂量组肝癌组织重量、肝癌组织体积、肝癌组织抑瘤率、VEGFR-2、VEGF、IL-4、IL-8、TNF-α蛋白、肝癌FAK、mTORC1 mRNA和蛋白表达降低(均P<0.05)。鳖甲煎丸高剂量组肝癌组织重量、肝癌组织体积、肝癌组织抑瘤率、VEGFR-2、VEGF、IL-4、IL-8、TNF-α蛋白、肝癌FAK、mTORC1 mRNA和蛋白表达低于鳖甲煎丸低剂量组(均P<0.05)。结论:鳖甲煎丸对大鼠肝癌具有明显抑制作用,能减轻大鼠肝癌所致的病理损伤,其机制可能与鳖甲煎丸抑制肝癌FAK、mTORC1表达,诱导肝癌细胞凋亡,降低炎症反应有关。

Gehua Jiejiu Dizhi decoction(葛花解酒涤脂汤)ameliorates alcoholic fatty liver in mice by regulating lipid and bile acid metabolism and with exertion of antioxidant stress based on 4DLabel-free quantitative proteomic study

OBJECTIVE:To analyze the effect and molecular mechanism of Gehua Jiejiu Dizhi decoction(葛花解酒涤脂汤,GJDD)on alcoholic fatty live disease(AFLD)by using proteomic methods.METHODS:The male C57BL/6J mouse were randomly divided into four groups:control group,model group,GJDD group and resveratrol group.After the AFLD model was successfully prepared by intragastric administration of alcohol once on the basis of the Lieber-DeCarli classical method,the GJDD group and resveratrol group were intragastrically administered with GJDD(4900 mg/kg)and resveratrol(400 mg/kg)respectively,once a day for 9 d.The fat deposition of liver tissue was observed and evaluated by oil red O(ORO)staining.4DLabel-free quantitative proteome method was used to determine and quantify the protein expression in liver tissue of each experimental group.The differentially expressed proteins were screened according to protein expression differential multiples,and then analyzed by Gene ontology classification and Kyoto Encyclopedia of Genes and Genomes pathway enrichment.Finally,expression validation of the differentially co-expressed proteins from control group,model group and GJDD group were verified by targeted proteomics quantification techniques.RESULTS:In semiquantitative analyses of ORO,all kinds of steatosis(ToS,MaS,and MiS)were evaluated higher in AFLD mice compared to those in GJDD or resveratroltreated mice.4DLabel-free proteomics analysis results showed that a total of 4513 proteins were identified,of which 3763 proteins were quantified and 946 differentially expressed proteins were screened.Compared with the control group,145 proteins were up-regulated and 148 proteins were down-regulated in the liver tissue of model group.In addition,compared with the model group,92 proteins were up-regulated and 135 proteins were downregulated in the liver tissue of the GJDD group.15 differentially co-expressed proteins were found between every two groups(model group vs control group,GJDD group vs model group and GJDD group vs control group),which were involved in many biological processes.Among them,11 differentially co-expressed key proteins(Aox3,H1-5,Fabp5,Ces3a,Nudt7,Serpinb1a,Fkbp11,Rpl22l1,Keg1,Acss2 and Slco1a1)were further identified by targeted proteomic quantitative technology and their expression patterns were consistent with the results of 4D label-free proteomic analysis.CONCLUSIONS:Our study provided proteomics-based evidence that GJDD alleviated AFLD by modulating liver protein expression,likely through the modulation of lipid metabolism,bile acid metabolism and with exertion of antioxidant stress.

Cell-free synthesis of stable isotope-labeled internal standards for targeted quantitative proteomics

High-sensitivity mass spectrometry approaches using selected reaction monitoring(SRM)or multiple reaction monitoring(MRM)methods are powerful tools for targeted quantitative proteomics-based investigation of dynamics in specific biological systems.Both high-sensitivity detection of lowabundance proteins and their quantification using this technique employ stable isotope-labeled peptide internal standards.Currently,there are various ways for preparing standards,including chemical peptide synthesis,cellular protein expression,and cell-free protein or peptide synthesis.Cell-free protein synthesis(CFPS)or in vitro translation(IVT)systems in particular provide high-throughput and low-cost preparation methods,and various cell types and reconstituted forms are now commercially available.Herein,we review the use of such systems for precise and reliable protein quantification.

一种新型免疫层析技术在临床乳腺癌Her-2蛋白定量检测中的应用

建立一种靶点蛋白质快速定量检测方法。在原有侧向流动免疫层析技术的基础上,通过优化层析材料和纳米微球的均一性、改进检测区的检测方法,经逐点扫描技术,建立标准浓度曲线,以达到对临床靶点蛋白质的定量检测。以乳腺癌组织中的Her2表达为例,通过对已知浓度样品的检测,验证本技术方法的准确度大于96%。另外,以蛋白质免疫印迹作为组织中特定蛋白质检测金标准,分析临床肿瘤组织中Her2蛋白的含量,其准确率也达到95.5%,而免疫组织化学方法检测准确率仅为69.58%。新型免疫层析法检测结果与靶向治疗患者的愈后密切相关(P<0.01)。改进后的新型免疫层析方法能够准确地对临床靶点蛋白质进行定量检测,而且结合侧向流动技术的简单、快速和易用性,这种新型检测方法可以广泛应用于临床组织标本、血液标本和体液标本中靶点蛋白质的临场定量检测,在一定程度上可以替代免疫组化技术。

基于质谱的蛋白质绝对定量研究策略和建议

科学家已经研究获得了大量的蛋白质/多肽潜在生物标志物,这些生物标志物需要经过验证和确证才能进一步转化到临床应用。针对蛋白质/多肽的绝对定量研究在标志物验证和确证过程中起到关键作用。传统的蛋白质定量方法,如酶联免疫吸附试验(ELISA)技术存在蛋白质抗体难以获得、不同抗体批次之间存在差异、基于抗体的检测存在交叉反应等问题。近年来,随着质谱仪器性能的提升,质谱法已经广泛应用于蛋白质大分子以及代谢物小分子定量研究领域,基于质谱法的蛋白质/多肽定量方法也得到发展,其中靶向蛋白质组学技术,如SRM/MRM、PRM和MRM 3技术结合标准品可对目标蛋白质/多肽进行精确多重定量。因此,基于靶向质谱法的蛋白质/肽段绝对定量的策略应运而生。根据标准品不同,质谱绝对定量方法包括基于合成肽段、基于合成完整蛋白以及基于标记试剂。本文详细介绍了基于质谱法的蛋白质/多肽绝对定量策略的研究进展,对比各种策略的优势和限制,总结其在生物医学研究领域的应用。同时,还总结了目前蛋白质绝对定量面临的挑战,并根据已有研究报道对提高定量的精确度,促进蛋白质/多肽定量技术向临床转化提出建议,包括对标准品肽段的选择、处理、储存,标准曲线的建立,以及对整个流程严格的质控方法。

2型糖尿病患者肾实质硬度与尿蛋白成分的相关性研究

目的探讨2型糖尿病(T2DM)患者肾实质硬度与尿蛋白成分的相关性。方法选取2020年4-12月于该院住院的T2DM患者117例,应用声触诊组织定量技术(VTQ)测量患者的肾实质剪切波速度(SWV),同时收集患者一般临床资料如病程、年龄、血压;常规超声参数如左肾大小、肾实质厚度;肾功能指标如血肌酐、血清尿素氮及24 h尿蛋白定量;尿蛋白成分如α1-微球蛋白(α1-MG)、β2-微球蛋白(β2-MG)、免疫球蛋白、转铁蛋白和微量白蛋白,并计算各种尿蛋白占尿蛋白总量的百分比。应用SPSS22.0统计软件对数据进行统计分析。结果肾实质SMV与糖尿病病程呈正相关(r=0.304,P<0.05)。肾实质SWV与血肌酐、血清尿素氮及24 h尿蛋白定量呈负相关(r=-0.356、-0.281、-0.202,P<0.05)。肾实质SWV与α1-MG、β2-MG百分比呈负相关(r=-0.611、-0.578,P<0.05)。结论T2DM患者肾实质SWV与尿液中小分子蛋白百分比密切相关,二者联合应用或将在糖尿病肾病诊断和疗效评估中发挥重要作用。

基于PI3K/Akt/mTOR信号通路调控自噬探讨当归四逆汤对痛风性关节炎大鼠的影响

目的:基于磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(m TOR)信号通路,探讨当归四逆汤对痛风性关节炎(GA)大鼠自噬水平的影响。方法:60只雄性SD大鼠随机分为正常组、模型组、秋水仙碱组(0.3 mg·kg^(-1))和当归四逆汤低、中、高剂量组(6.54、13.08、26.16 g·kg^(-1)),每组10只,并分别给予相应药物灌胃,正常组、模型组给予等体积生理盐水灌胃,连续7 d。于第5天给药1 h后向各组(正常组除外)大鼠右侧踝关节注射尿酸钠混悬液(50 g·L^(-1))建立GA模型,正常组注射等体积的无菌生理盐水。观察大鼠踝关节肿胀情况;测定血清中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、IL-1β水平;观察踝关节病理形态变化;蛋白免疫印迹法(Western blot)检测滑膜PI3K、磷酸化(p)-PI3K、Akt、p-Akt、m TOR、p-m TOR、微管相关蛋白1轻链3Ⅱ/Ⅰ(LC3Ⅱ/Ⅰ)、自噬效应蛋白(Beclin-1)、泛素结合蛋白(p62)表达水平;实时荧光定量聚合酶链式反应(Real-time PCR)检测PI3K、Akt、m TOR、LC3、Beclin-1、p62 m RNA表达水平。结果:与正常组比较,模型组大鼠关节肿胀指数显著升高(P<0.01),血清TNF-α、IL-6、IL-1β水平升高(P<0.01),踝关节滑膜组织可见明显炎性细胞浸润和纤维组织增生,滑膜组织PI3K、p-PI3K、Akt、p-Akt、m TOR、p-m TOR、p62蛋白表达显著升高(P<0.01),PI3K、Akt、m TOR、p62 m RNA表达显著升高(P<0.01),LC3Ⅱ/Ⅰ、Beclin-1蛋白和LC3、Beclin-1 m RNA表达降低(P<0.01)。与模型组比较,当归四逆汤中、高剂量组大鼠关节肿胀明显减轻(P<0.05);血清中TNF-α、IL-6、IL-1β表达量显著明显降低(P<0.05);踝关节滑膜组织未见明显炎性细胞浸润,纤维组织增生减轻;滑膜组织PI3K、p-PI3K、Akt、p-Akt、m TOR、p-m TOR、p62蛋白表达量均明显降低(P<0.05),PI3K、Akt、m TOR、p62 m RNA表达量亦明显降低(P<0.05),而LC3Ⅱ/Ⅰ、Beclin-1蛋白和LC3、Beclin-1 m RNA表达量均明显上升(P<0.05)。结论:当归四逆汤可以抑制PI3K/Akt/m TOR信号通路,提高大鼠滑膜组织自噬水平,改善痛风性关节炎,并且以高剂量效果最佳。

氨基磁珠偶联法解析地榆皂苷Ⅱ作用蛋白

目的采用磁珠偶联法解析地榆皂苷Ⅱ作用蛋白。方法地榆皂苷Ⅱ具有羧基端,将其活化后,羧基端与氨基磁珠的氨基端偶联。将0、2、10 mmol·L-1地榆皂苷Ⅱ与磁珠形成配合物后,与等量TF-1细胞蛋白裂解产物共孵育,使地榆皂苷Ⅱ结合潜在作用靶蛋白;利用磁力架捕获上述磁珠,解离地榆皂苷Ⅱ的结合蛋白;经过SDS-PAGE电泳分离,采用高分辨质谱鉴定PAGE胶上的蛋白,呈现与地榆皂苷Ⅱ相结合的蛋白;通过基因本体论和京都基因与基因组百科全书数据库分析差异蛋白;通过特异性肽段数量及基于强度的绝对蛋白质定量(IBAQ)值的高低,从中筛选可信度较高的地榆皂苷Ⅱ作用蛋白。结果鉴定空白磁珠及化合物偶联磁珠结合蛋白共计226种。扣除空白磁珠结合蛋白本底,鉴定出66种地榆皂苷Ⅱ特异性作用蛋白,通过特异性肽段与IBAQ值综合分析可知:与地榆皂苷Ⅱ作用相关的蛋白主要为DNA依赖性蛋白激酶催化亚基、翻译激活剂GCN1及酰基甘油激酶等。结论利用氨基磁珠偶联法解析地榆皂苷Ⅱ的潜在作用蛋白,可为进一步阐释地榆皂苷Ⅱ及地榆的作用机制提供依据。