Xiaoxuming Decoction regulates NLRP3 ubiquitination level by down⁃regulating USP9X in treatment of acute lung injury

Yijin XIANG; Dingfang CAI; Ping WANG; Jiawei YU; Min CAI; Xiangting LI

doi:10.16305/j.1007-1334.2022.2105105

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Xiaoxuming Decoction regulates NLRP3 ubiquitination level by down⁃regulating USP9X in treatment of acute lung injury

更新时间：2022-07-10

- Xiaoxuming Decoction regulates NLRP3 ubiquitination level by down⁃regulating USP9X in treatment of acute lung injury
- Shanghai Journal of Traditional Chinese Medicine Vol. 56, Issue 3, Pages: 48-55(2022)
- 作者机构：
  
  1.复旦大学附属中山医院中西医结合研究院（上海 200032）
  2.复旦大学附属中山医院康复科（上海 200032）
  3.复旦大学附属中山医院肾病科（上海 200032）
- 作者简介：
- 基金信息：
- DOI：10.16305/j.1007-1334.2022.2105105
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项忆瑾,蔡定芳,王平等.小续命汤通过下调USP9X调控NLRP3泛素化水平干预急性肺损伤的机制研究[J].上海中医药杂志,2022,56(03):48-55.

XIANG Yijin,CAI Dingfang,WANG Ping,et al.Xiaoxuming Decoction regulates NLRP3 ubiquitination level by down⁃regulating USP9X in treatment of acute lung injury[J].Shanghai Journal of Traditional Chinese Medicine,2022,56(03):48-55.
项忆瑾,蔡定芳,王平等.小续命汤通过下调USP9X调控NLRP3泛素化水平干预急性肺损伤的机制研究[J].上海中医药杂志,2022,56(03):48-55. DOI： 10.16305/j.1007-1334.2022.2105105.

XIANG Yijin,CAI Dingfang,WANG Ping,et al.Xiaoxuming Decoction regulates NLRP3 ubiquitination level by down⁃regulating USP9X in treatment of acute lung injury[J].Shanghai Journal of Traditional Chinese Medicine,2022,56(03):48-55. DOI： 10.16305/j.1007-1334.2022.2105105.

摘要

目的,2,探究小续命汤通过下调泛素特异性蛋白酶9X（USP9X）干预脂多糖（LPS）诱导的急性肺损伤（ALI）炎症反应机制。,方法,2,制备含药血清，使用10%小续命汤含药血清预处理Ⅱ型肺泡上皮细胞。①小续命汤对LPS诱导的Ⅱ型肺泡上皮细胞炎症及功能的影响：实验分为空白组、LPS诱导组、LPS+小续命汤组。ELISA检测炎症因子肿瘤坏死因子-α（TNF-α）、白介素-1β（IL-1β）、白介素-8（IL-8）水平。qRT-PCR和Western blot检测紧密连接蛋白Occludin、ZO-1、Claudin的表达。②小续命汤通过USP9X发挥改善ALI细胞模型的炎症及功能：实验分为空白组、LPS诱导组、LPS+小续命汤组。 PCR和Western blot检测USP9X的表达。③USP9X与核苷酸结合寡聚化结构域样受体蛋白3（NLRP3）蛋白相互作用：人肺泡上皮细胞内USP9X过表达后，Co-IP联合质谱检测USP9X与NLRP家族互作，通过Western blot验证确定NLRP3与USP9X的关系。④小续命汤缓解LPS导致的人肺泡上皮细胞焦亡：实验分为空白组、LPS诱导组、LPS+小续命汤组。Western blot检测NLRP3、IL-1β、Pro-IL-1β、Caspase-1、Pro-Caspase-1，PCR检测NLRP3、IL-1β、Caspase-1，CCK-8检测细胞焦亡。,结果,2,①与模型组比较，小续命汤预处理显著减少LPS诱导的Ⅱ型肺泡上皮细胞炎症因子TNF-α、IL-1β、IL-8的蛋白表达水平（,P,<,0.05），显著增加紧密连接蛋白Occludin、ZO-1、Claudin的蛋白和mRNA表达水平。②与模型组比较，小续命汤含药血清预处理显著降低USP9X的转录及蛋白水平的表达（,P,<,0.05）。③Co-IP联合质谱检测USP9X与NLRP家族互作，通过Western blot验证确定NLRP3与USP9X存在互作。④与模型组比较，小续命汤预处理显著减少LPS诱导的Ⅱ型肺泡上皮细胞NLRP3、IL-1β、Pro-IL-1β、Caspase-1、Pro-Caspase-1的蛋白和转录水平，缓解细胞焦亡的发生（,P,<,0.05）。,结论,2,小续命汤能有效改善急性肺损伤，该作用可能与下调USP9X调控NLRP3泛素化水平的表达相关。

Abstract

Objective,2,To investigate the mechanism of Xiaoxuming Decoction on acute lung injury （ALI） induced by lipopolysaccharide （LPS） by down-regulating USP9X， and to explore possible action mechanism.,Methods,2,The medicated serum was prepared， and the type Ⅱ alveolar epithelial cells were pretreated with 10% Xiaoxuming Decoction medicated serum. ① Effect of Xiaoxuming Decoction on the inflammation and function of alveolar type Ⅱ epithelial cell induced by LPS： The experiment was divided into the blank group， the LPS induction group and the LPS+Xiaoxuming Decoction group. Inflammatory factors including TNF-α， IL-1β and IL-8 were detected by ELISA. QRT-PCR and Western blot were used to detect the expression of tight junction proteins including Occludin， ZO-1 and Claudin. ② Xiaoxuming Decoction improved the inflammation and function of ALI cell model through USP9X： The experiment was divided into the blank group， the LPS induction group and the LPS+Xiaoxuming Decoction group. The expression of USP9X was detected by PCR and Western blot. ③ The interaction between USP9X and NLRP3 protein： After USP9X overexpressed in human alveolar epithelial cells， Co-IP combined with mass spectrometry was used to detect the interaction between USP9X and NLRP family， and Western blot was used to confirm the relationship between NLRP3 and USP9X. ④Xiaoxuming Decoction alleviating the pyrolysis of human alveolar epithelial cells caused by LPS： The experiment was divided into the blank group， the LPS induction group and the LPS+Xiaoxuming Decoction group. NLRP3， IL-1 β， Pro-IL-1 β， Caspase-1 and Pro-Caspase-1 were detected by Western blot. NLRP3， IL-1β Caspase-1 and CCK-8 were detected by PCR.,Results,2,① Compared with the model group， Xiaoxuming Decoction significantly decreased the protein expression levels of inflammatory cytokines including TNF-α， IL-1β and IL-8 in type Ⅱ alveolar epithelial cells induced by LPS（,P,<,0.05）， and significantly increased the mRNA and protein expression levels of tight junction proteins including Occludin， ZO-1 and Claudin. ② Compared with the model group， the serum pretreatment of Xiaoxuming Decoction significantly reduced the transcription and protein expression of USP9X （,P,<,0.05）. ③ There was an interaction between NLRP3 and USP9X family detected by Co-IP combined with mass spectrometry and confirmed by Western blot. ④ Compared with the model group， the Xiaoxuming Decoction significantly reduced the protein and transcription levels of NLRP3， IL-1β， Pro-IL-1β， Caspase-1 and Pro-Caspase-1 in type Ⅱ alveolar epithelial cells induced by LPS， and alleviated the occurrence of pyrolysis （,P,<,0.05）.,Conclusion,2,Xiaoxuming Decoction can effectively improve acute lung injury， which may be related to the down-regulation of USP9X to regulate the expression of NLRP3 ubiquitination.

关键词

小续命汤急性肺损伤脂多糖泛素特异性蛋白酶9X核苷酸结合寡聚化结构域样受体蛋白3中药研究

Keywords

Xiaoxuming Decoctionacute lung injuryLPSUSP9XNLRP3traditional Chinese herbal medicine research

references

中华医学会重症病学分会. 急性肺损伤/急性呼吸窘迫综合征诊断与治疗指南（2006）［J］.中华急诊医学杂志，2007， 16（4）： 3-4.

LAN R， XIANG J， ZHANG Y， et al. PI3K/AKT pathway contributes to neurovascular unit protection of Xiao-Xu-Ming Decoction against focal cerebral ischemia and reperfusion injury in rats［J/OL］. Evid Based Complement Alternat Med， 2013［2021-04-10］. https：//www.ncbi.nlm.nih.gov/pmc/articles/PMC3678438/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3678438/.

CAO H， FENG Y， NING Y， et al. Edaravone protects rats and human pulmonary alveolar epithelial cells against hyperoxia injury： heme oxygenase-1 and PI3K/AKT pathway may be involved［J］. Exp Lung Res， 2015， 41（7）： 404-414.

YANG B， XIE C H. Ubiquitin specific peptidase 9X and tumor［J］. J Int Oncol， 2011， 38（12）： 900-902.

WANG Q， TANG Y， XU Y， et al. The X-linked deubiquitinase USP9X is an integral component of centrosome［J］. J Biol Chem， 2017， 292（31）： 12874-12884.

XIANG Y， ZHANG S， LU J， et al. USP9X promotes LPS-induced pulmonary epithelial barrier breakdown and hyperpermeability by activating an NF-κBp65 feedback loop［J］. AM J Physiol Cell Physiol， 2019， 317（3）： C534-C543.

茅月存，王爱菊，鲍丹莲. 肺部感染与急性脑卒中患者预后的相关性分析［J］. 浙江医学，2008， 30（7）： 761-762.

陆红，吴庆，刘媚娜，等. 脑卒中患者深部真菌感染的菌群分布及耐药性［J］. 中华医院感染学杂志，2009， 19（6）： 689-691.

吴芳玲，黄冬雅. 急性脑卒中并发肺部感染分析［J］. 中国实用内科杂志，2005， 25（7）： 619-620.

王威，陈路燕，刘建平. 脑卒中后肺部感染的危险因素与预后［J］. 中华医院感染学杂志，2007， 17（7）： 805-806.

KATAZAN I L， CEBUL R D， HUSAK S H， et al. The effect of pneumonia on mortality among patients hospitalized for acute stroke［J］. Neurology， 2003， 60（4）： 620-625.

邓伟，李新建. 小续命汤加减治疗类风湿关节炎39例疗效观察［J］. 长春中医药大学学报，2006， 22（3）： 11-12.

张金梅，马俊华，谯凤英. 小续命汤治疗中重度持续性变应性鼻炎临床疗效观察［J］. 四川中医，2014， 32（4）： 132-133.

严寒. 小续命汤加减配合艾灸治疗面神经麻痹35例总结［J］. 湖南中医杂志，2014， 30（8）： 70-71.

HERRERO R， SANCHEZ G， LORENTE J A. New insights into the mechanisms of pulmonary edema in acute lung injury［J］. Ann Transl Med， 2018， 6（2）： 32.

PETERANDERL C， SZNAJDER J I， HEROLD S， et al. Inflammatory responses regulating alveolar ion transport during pulmonary infections［J］. Front Immunol， 2017 （8）： 446.

SHAO L， MENG D， YANG F， et al. Irisin-mediated protective effect on LPS-induced acute lung injury via suppressing inflammation and apoptosis of alveolar epithelial cells［J］. Biochem Biophy Res Commun， 2017， 487（2）： 194-200.

FANG M， ZHONG W H， SONG W L， et al. Ulinastatin ameliorates pulmonary capillary endothelial permeability induced by sepsis through protection of tight junctions via inhibition of TNF-α and related pathways［J］. Front Pharmacol， 2018（9）： 823.

LIN X， BARRAVECCHIA M， KOTHARI P， et al. β1-Na+， K+-ATPase gene therapy upregulates tight junctions to rescue lipopolysaccharide-induced acute lung injury［J］. Gene therapy， 2016， 23（6）： 489-499.

THÉARD D， LABARRADE F， PARTISANI M， et al. USP9X-mediated deubiquitination of EFA6 regulates de novo tight junction assembly［J］. EMBO J， 2010， 29（9）： 1499-1509.

SHEN S M， ZHANG C， GE M K， et al. Author Correction： PTENα and PTENβ promote carcinogenesis through WDR5 and H3K4 trimethylation［J］. Nat Cell Biol， 2020， 22（1）： 135.

WANG P， XIA J， ZHANG L， et al. SNX17 recruits USP9X to antagonize MIB1-mediated ubiquitination and degradation of PCM1 during serum-starvation-induced ciliogenesis［J］. Cells， 2019， 8（11）：1335.

JOHNSON B V， KUMAR R， OISHI S， et al. Partial loss of USP9X function leads to a male neurodevelopmental and behavioral disorder converging on transforming growth factor β Signaling［J］. Biol Psychiatry， 2019， 87（5）： 100-112.

AQAQE N， YASSIN M， YASSIN A A， et al. An ERG enhancer-based reporter identifies leukemia cells with elevated leukemogenic potential driven by ERG-USP9X feed-forward regulation［J］. Cancer Res， 2019， 79（15）： 3862-3876.

YANG J， ZHAO Y， ZHANG P， et al. Hemorrhagic shock primes for lung vascular endothelial cell pyroptosis： role in pulmonary inflammation following LPS［J］. Cell death Dis， 2016， 7（9）： e2363.

HE X， QIAN Y， LI Z， et al. TLR4-Upregulated IL-1β and IL-1RI promote alveolar macrophage pyroptosis and lung inflammation through an autocrine mechanism［J］. Sci Rep， 2016（6）： 31663.

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