| Background:Ischemia-reperfusion(IR)injury refers to the physiological,biochemical and pathological changes of the body caused by the re-restoration of blood flow by tissues in an ischemic state,which can affect the function of ischemic tissues,induce inflammatory reactions and damage remote organs.The lung is one of the most vulnerable remote organs during IR.Many diseases,such as pulmonary embolism,thrombosis,cardiopulmonary bypass surgery and lung transplantation,can cause lung injury caused by IR.Acute lung injury(ALI)induced by LIR is a common problem for clinicians in the 21 st century.ALI is a very common type of critical illness in clinical practice,which is caused by a variety of intrapulmonary and extrapulmonary factors such as severe trauma,shock,acidosis or severe infection,with a mortality rate as high as 40 to 31%,characterized by inflammation and increased vascular permeability,clinically manifested as progressive hypoxemia and respiratory distress,and progresses to a severe stage known as acute respiratory distress syndrome.Insufficiency and structural immaturity of pulmonary surfactant increase lung sensitivity to IR injury,while inflammation and oxidative stress contribute to the pathological process of ALI induced by LIR.Although ALI has been extensively explored for decades,no major progress has been made in related research,and patients with ALI still face long-term physical injury.At present,the understanding of ALI is deepening,and most scholars believe that inflammatory storm is a key factor in the occurrence of ALI.Rapamycin(RAPA)is an immunosuppressive agent belonging to macrolide compounds,which inhibits immunity by inhibiting the expression of interleukin(IL)-2 thereby hindering the activation of T and B cells.RAPA is approved by the Food and Drug Administration as an immunosuppressive agent for renal transplant anti-rejection therapy and lymphangioleiomyoma.RAPA has been largely studied to inhibit cancer progression,and also has a significant effect in diabetes,neurodegenerative diseases,as well as hematological diseases.In conclusion,RAPA can reduce inflammation and has the potential to treat ALI.LncRNAs are type of RNA transcripts over 200 nucleotides in length that are involved in many physiological and pathological processes.It has been found that lncRNAs may regulate the process of cell pyroptosis in the development of diseases by directly or indirectly regulating proteins associated with pyroptosis.The study of lncRNA in tissues injury induced by LIR has been widely reported,and existing studies have shown that it is involved in the development of the disease.In retinal IR injury,increased expression of lncRNA H19 significantly promoted the imbalance of NLRP3/6 inflammasome,leading to microglial pyroptosis,cytokine overproduction and neuronal death,and these injuries were effectively inhibited by lncRNA H19 silencing,indicating that lncRNA H19 is a key factor in IR-induced inflammation.However,studies of lncRNAs in lung injury induced by LIR have not been reported,but they are widely involved in the disease process of ALI.Therefore,we used ALI models induced by LIR in rats and high-throughput sequencing technology as supports,found that down regulation of LOC102553434 expression via RAPA to inhibit pyroptosis and thus attenuate ALI induced by LIR,and gradually revealed the treatment mechanism of RAPA in ALI and provided potential therapeutic targets and new drug candidates for ALI.Part I RAPA protects ALI induced by LIR via inhibiting pyroptosisObjective:To investigate the therapeutic effect and mechanism of RAPA on ALI model induced by LIR.Methods:In this study,a LIR-induced ALI rat model(IR group)was constructed by ligating the roots of both lower limbs and using the sham group as the control,and the experimental group was treated with RAPA(IR+RAPA group).The pathological changes of rat lung tissues were determined by HE staining;the changes of MDA content and SOD activities in rat were detected by kits;the expressions of NLRP3 protein and caspase-1 protein were detected by immunohistochemistry and western blot;and the changes of IL-1? and IL-18 were detected by ELISA.Results:1.Compared with the IR group,the IR+RAPA group had significantly reduced pulmonary edema and congestion,reduced inflammatory cell infiltration,and significantly preserved lung tissue with atypia.2.Compared with the IR group,IR+RAPA group significantly revised the changes of SOD activity,MDA level,NLRP3 and caspase-1 protein levels,and 1L-1?and 1L-18 contents caused by IR treatment.Part II Transcriptome sequencing analysis of lung tissue induced by LIR in ratsObjective:Differentially expressed lncRNAs and mRNAs involved in the process of RAPA treatment of ALI and associated with pyroptosis and injury were screened by high-throughput sequencing.Methods:In this study,ALI in rats was induced by LIR(IR group),and RAPAwas used to pretreat LIR rats(IR+RAPA group).Then,the expression profiles of lung tissues of rats in the two groups were characterized by high-throughput sequencing to obtain the differential expression characteristics of mRNA and lncRNA in lung tissues between IR and IR+RAPA group.GO functional enrichment analysis and KEGG pathway enrichment analysis explored the enrichment of differentially expressed mRNA and lncRNA;Miranda and RNAhybird software predicted the relationship between differentially expressed mRNA and lncRNA and miRNA,and constructed the ce RNA regulatory relationship.Results:1.A total of 220 differentially expressed mRNAs were identified between the IR group and the IR+RAPA group.Compared with the IR group,there were 149 downregulated mRNAs and 71 up-regulated mRNAs in the IR+RAPA group.In addition,63 differentially expressed lncRNAs were identified.Compared with the IR group,27 lncRNAs were down-regulated and 36 lncRNAs were up-regulated in the IR+RAPA group.2.The differentially expressed mRNA and lncRNA between IR and IR+RAPA groups were mainly involved in the cellular response of IL-1,the positive regulation of IL-6 biosynthesis process,neutrophil chemotaxis,the interaction of cytokinecytokine receptors,and cell adhesion molecules and other biological processes and signaling pathways.3.A total of 1945 lncRNA-miRNA-mRNA regulatory relationships were obtained based on differentially expressed mRNA and lncRNA,indicating that there are differentially expressed lncRNAs affected the mRNA expression by sponging adsorption miRNAs to impact the occurrence and treatment of ALI.4.The expression of lncRNA LOC102553434 and MMP9 in IR group were significantly higher than those in Sham and IR + RAPA groups,and MMP9 was the downstream target gene of LOC102553434/rno-miR-674-5p/MMP9 axis.Therefore,RAPA treatment decreased the expression of LOC102553434 in ALI lung tissue,which may improve acute lung injury by targeting rno-miR-674-5p/MMP9 axis.Part III Functional study of LOC102553434 in alveolar epithelial cell injuryObjective:To investigate the mechanism of RAPA inhibiting pyroptosis through ce RNA in alleviating lung epithelial cell injury.Methods:In this study,alveolar epithelial cell lines of rat were selected in vitro experiments,and the expression changes of inflammatory factors(1L-1?,1L-18)and LOC102553434,rno-miR-674-5p and MMP9 in Blank,LPS and LPS+RAPA groups were detected by fluorescence quantitative PCR;the expression of LOC102553434 was silenced by si RNA,and the cell proliferation ability was detected by CCK-8;and the expression of NRPL3,caspase-1 and MMP9 was detected by WB in Blank,LPS,LPS+RAPA+si-NC and LPS+RAPA+si RNA groups.Results:1.Compared with the Blank and LPS+RAPA groups,the expression of 1L-1?,1L-18,LOC102553434 and MMP9 in alveolar epithelial cells was significantly increased in the LPS group,while the expression of rno-miR-674-5p was significa-ntly decreased.2.After silencing LOC102553434,the proliferation of alveolar epithelial cells in LPS+si RNA group was significantly enhanced compared with the LPS+si RNA-NC and LPS groups.3.Compared with LPS group or LPS+RAPA+si-NC group,the expression of NRPL3,caspase-1 and MMP9 protein in alveolar epithelial cells of LPS+RAPA+si RNA group was significantly decreased.Conclusions:1.RAPA protects ALI induced by LIR via inhibiting pyroptosis.2.Differentially expressed lncRNAs affect the expression of mRNAs through sponged miRNAs to involve in the pathological process of ALI in rats,while rapamycin may effectively treat ALI by regulating the expression of lncRNAs.3.LOC102553434 may be involved in alveolar epithelial cell injury develop-ment by acting on the rno-miR-674-5p/MMP9 axis and is expected to be a potential preventive and therapeutic target for ALI.4.RAPA improve ALI by down-regulating the LOC102553434 expression and then down-regulating the MMP9 expression as well as inhibiting pyroptosis,which may be an effective candidate for ALI treatment. |
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