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
Xiaoxuming Decoction regulates NLRP3 ubiquitination level by down⁃regulating USP9X in treatment of acute lung injury
- 上海中医药杂志 2022年56卷第3期 页码：48-55
- 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：
项忆瑾，女，硕士，主治医师，主要从事中西医结合治疗急性脑血管疾病研究工作
蔡定芳，教授，主任医师，博士研究生导师； E-mail：cai.dingfang@zs-hospital.sh.cn
- 基金信息：国家自然科学基金项目(82004123);上海市科委基金项目(20YF1407000)
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
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