组织蛋白酶B通过瞬时受体电位黏蛋白-1对核苷酸结合寡聚化结构域样受体蛋白3炎症小体活化的影响

doi:10.3881/j.issn.1000-503X.10847

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 Juanjuan^1,^2,⁵,ZHANG Qifang^2,³(),HUANG Zonghua⁴,ZENG Hongmei¹,BAI Hua^1,^5,⁶()

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

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

CLC Number:

R392

DUAN Juanjuan,ZHANG Qifang,HUANG Zonghua,ZENG Hongmei,BAI Hua. 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[J].Acta Academiae Medicinae Sinica, 2019, 41(2): 208-215.

Figures/Tables 4

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References 20

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分组Group	n	半胱天冬酶-1 Caspase-1(pmol/L)	白细胞介素-1β Interleukin-1β(pg/ml)
PBS	5	35.7±14.5	216.5±48.5
H₂O₂	5	86.3±26.2^a	292.9±68.2
H₂O₂+GdCl₃	5	92.5±46.6^a	445.2±97.3^b
H₂O₂+GdCl₃+ CA-074Me	5	39.4±16.7^c	221.8±50.8^c

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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

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