Exploring The Role Of Trim27 In Asthma Via NLRP3 Inflammasome

Background and Purpose:As a member of the Trim protein family,Trim27 is involved in the occurrence and development of various diseases by influencing intracellular signal transduction,transcriptional regulation,cell differentiation,immune and inflammatory responses.However,the role of Trim27 in asthma is currently unclear and needs further investigation.Therefore,the role of Trim27 in asthma was further studied at both in vivo and in vitro levels in this study,and the molecular mechanism of Trim27 affecting the onset of asthma was expounded,in order to provide a new perspective and basis for asthma treatment targets.Methods:This topic is divided into three parts.In the first part,the expression of Trim27 in asthma was studied using RT-qPCR,Western blot and IHC by constructing in vivo and in vitro asthma models.In the second part,a mouse model of acute asthma was constructed using ovalbumin(OVA).Trim27-sh RNA lentvirus(2.2 × 106 TU)was injected into the tracheal of mice 48 hours before OVA stimulation to knockdown Trim27 gene.In this part,24 C57BL/J mice were randomly divided into 4 groups,which were labeled as blank control group(control group),model group(OVA),MOCK+OVA group(MOCK/OVA)and Trim27-sh RNA+model group(Trim27-sh RNA/OVA).After the modeling was completed,airway hyperresponsiveness of mice was measured by whole-body labeling system,and blood of mice in each group was collected for serum Ig E detection.Alveolar lavage fluid(BALF)was collected for classification and counting of inflammatory cells,and cytokine levels were measured by enzym-linked immunosorbent assay(ELISA).Mouse lung tissues were isolated,the left lung tissues were fixed and embedded for histopathological detection(hematoxylin-eosin staining,periodic acid-schiff staining and immunohistochemical detection),and the right lung tissues were ground for protein extraction.SOD,MDA,GSH and CAT detection kits were used to evaluate the oxidative stress level.The expression of related proteins was detected by RT-qPCR and Western blot.In the third part,MLE-12 cells stimulated by lipopolysaccharide(LPS)combined with adenosine triphosphate(ATP)were used to construct an in vitro model of asthma.Meanwhile,the knockout,overexpression and E3 ubiquitin ligase mutant cell models of Trim27 were constructed.RT-qPCR and ELISA kit were used to detect the levels of inflammatory factors.Oxidative stress levels were detected by SOD,MDA,GSH and reactive oxygen species detection kits.Western blot was used to detect the expression of related proteins.Results:Part Ⅰ: The expression of Trim27 protein was significantly increased in the lung tissue of asthmatic mice,and the expression of Trim27 was closely related to the secretion of IL-1β,suggesting that Trim27 may be a regulatory factor in the pathogenesis of asthma.Part Ⅱ: In a mouse model of acute asthma induced by OVA,knockdown of Trim27 significantly reduced airway hyperresponsiveness,the total number of inflammatory cells and eosinophils in the airway,and downregulated the levels of inflammatory cytokines(such as IL-4,IL-5,IL-13,IL-1β and IL-18)in BALF,as well as reduced the level of oxidative stress in mice.At the same time,the pathological injury and goblet cell hyperplasia of mice lung tissue were alleviated.Moreover,the expression levels of p65 protein and NLRP3 inflammasome-related proteins(NLRP3,caspase-1 and IL-1β)in lung tissue of asthmatic mice were significantly decreased by Trim27 knockdown.The results suggest that Trim27 may influence the development of asthma by affecting the activation of NLRP3 inflammasome.Part Ⅲ: Trim27 affects inflammation,oxidative stress,NF-κB signaling pathway,and activation of NLRP3 inflammatorome in a cell model stimulated by LPS combined with ATP.Under LPS/ATP stimulation,the levels of IL-6 and TNF-α,oxidative stress and the expressions of p65(nuclear)protein and NLRP3inflammasome-related proteins(NLRP3,caspase-1 and IL-1β)in Trim27 knockout cells were significantly decreased compared with normal cells.After overexpression of Trim27,the levels of intracellular inflammatory cytokines and oxidative stress were significantly increased,and the NLRP3 inflammasome was activated.However,the E3 ubiquitin ligase mutant cell model of Trim27 could not activate the NLRP3 inflammasome or increase the levels of inflammatory response and oxidative stress.Conclusion:In conclusion,through in vitro and in vivo studies,our results showed that Trim27 can affect inflammation,oxidative stress,NF-κB signaling pathway and the activation of NLRP3 inflammasome,and then participate in the progression of asthma,which may be related to the E3 ubiquitin ligase activity of Trim27,suggesting that Trim27 may be a target for asthma treatment.