Acta Academiae Medicinae Sinica

Acta Academiae Medicinae Sinica

Acta Academiae Medicinae Sinica ›› 2019, Vol. 41 ›› Issue (2): 208-215.doi: 10.3881/j.issn.1000-503X.10847

• Original Articles • Previous Articles     Next Articles

Cathepsin B Affects the Activation of Nucleotide-binding Domain and Leucine-rich-repeat-containing Family and Pyrin Domain-containing 3 Inflammasome via Transient Receptor Potential Mucolipin-1

DUAN Juanjuan1,2,5,ZHANG Qifang2,3(),HUANG Zonghua4,ZENG Hongmei1,BAI Hua1,5,6()   

  1. 1 Department of Neurology,the Third Affiliated Hospital in Guizhou Medical University,Tuyun,Guizhou 558000,China
    2 Key Laboratory of Medical Molecular Biology,Guizhou Medical University,Guiyang 550004,China
    3 Key Lab of Local and Ethnic Diseases,Ministry of Education,Guizhou Medical University,Guiyang 550004,China
    4 Department of Clinical Laboratory,the Third Affiliated Hospital in Guizhou Medical University,Tuyun,Guizhou 558000,China
    5 Department of Neurology,Affiliated Hospital in Guizhou Medical University,Guiyang 550004,China
    6 Medical Experimental Center,the Third Affiliated Hospital in Guizhou Medical University,Tuyun,Guizhou 558000,China
  • Received:2018-09-14 Online:2019-04-28 Published:2019-05-07
  • Contact: Qifang ZHANG,Hua BAI E-mail:abcde9981@21cn.com;842031616@qq.com
  • Supported by:
    Project from Department of Science and Technology in Guizhou Province(QKH2016-1131);Fund from Bureau of Traditional Chinese Medicine in Guizhou Province(QZYY-2018-044);the National Natural Sciences Foundation of China(81560482)

Abstract:

Objective To explore the effects of cathepsin B(CTSB)on the activation of nucleotide-binding domain and leucine-rich-repeat-containing family and pyrin domain-containing 3(NLRP3)inflammasome via transient receptor potential mucolipin-1(TRPML1)in cell oxidative stress model and specific gene silencing cell model.Methods BV2 cells cultured in vivo were treated separately or simultaneously with hydrogen peroxide(H2O2),calcium-sensitive receptor agonist gadolinium trichloride(GdCl3),and CTSB inhibitor CA-074Me,and interleukin-1(IL-1)beta and caspase-1 protein were detected by enzyme-linked immunosorbent assay.The growth activity of BV2 cells in each group was measured by MTT.BV2 cells were treated with different concentrations of H2O2.Cystatin C mRNA and TRPML1 mRNA in BV2 cells were detected by real-time quantitative polymerase chain reaction and the proteins of TRPML1,CTSB,cathepsin D(CTSD),cathepsin L(CTSL)and cathepsin V(CTSV)were detected by Western blot.Specific small interfering RNA was designed for TRPML1 gene target sequence.TRPML1 gene silencing cell lines(named Tr-si-Bv2 cells)were established in BV2 cells and treated with or without H2O2.TRPML1,CTSB and transcription factor EB(TFEB)proteins in Tr-si-Bv2 cells or control cells were detected by Western blot.Results After treatment with H2O2,the expression of caspase-1 protein and NLRP3 mRNA in BV2 cells was increased,and IL-1beta protein in BV2 cells was significantly increased after treatment with GdCl3(P=0.0036).After treatment with CA-074Me,the doses of NLRP3 mRNA(P=0.037),caspase-1(P=0.021),and IL-1β(P= 0.036)were significantly reduced.Cells in the H2O2 group and H2O2+GdCl3 group grew more slowly.The expressions of CTSB mRNA and TRPML1 mRNA,or CTSB and TRPML1 proteins in BV2 cells in the treatment group with 200 μmol/L of H2O2 concentration were similar.H2O2-induced CTSB protein expression was inhibited after silencing TRPML1 gene.The changes of other cathepsins were not affected for the different concentration of H2O2.In the BV2 cells treated with TRPML1 gene silencing,the expression of CTSB protein was significantly reduced and the difference was statistically significant(P=0.021)between the H2O2 +siRNA treatment group and the H2O2 treatment group.Conclusion CTSB regulates the activation of NLRP3 inflammasome in the oxidative stress model of microglia cells,probably mediated by calcium channel protein TRPML1.

Key words: hydrogen peroxide, cathepsin B, inflammasome, gene silencing, transient receptor potential mucolipin-1

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