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

目的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Westernblot检测USP9X的表达。(3)USP9X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(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)。(3)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。

Objective 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 The medicated serum was prepared,and the typeⅡalveolar epithelial cells were pretreated with 10%Xiaoxuming Decoction medicated serum.(1)Effect of Xiaoxuming Decoction on the inflammation and function of alveolar typeⅡepithelial 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-1βand 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.(4)Xiaoxuming 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-1β,Pro-IL-1β,Caspase-1 and Pro-Caspase-1 were detected by Western blot.NLRP3,IL-1βCaspase-1 and CCK-8 were detected by PCR.Results(1)Compared with the model group,Xiaoxuming Decoction significantly decreased the protein expression levels of inflammatory cytokines including TNF-α,IL-1βand IL-8 in typeⅡalveolar 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 typeⅡalveolar epithelial cells induced by LPS,and alleviated the occurrence of pyrolysis(P<0.05).Conclusion 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.