Mechanism Of Regulation Of Smad3 Induced Autophagy In Ameliorating Sepsis-induced Lung Injury

BackgroundSepsis is a common severe trauma,burn and complications after operation within a short period of time can develop multiple organ dysfunction syndrome,is the main reason for the current trauma,burn and death in critically ill surgical patients.Sepsis related acute lung injury is not only the earliest,the highest incidence,and rapid progress,the mortality rate can be as high as 70% ~ 90%,there is no effective treatment now.We found in the previous study,Smad3 can reduce the sensitivity of the LPS to remote organ.At the same time,it is found that the TGF-beta /Smad signaling pathway is closely related to autophagy,and autophagy is a widespread phenomenon in eukaryotic cells,which has the effects of phagocytosis of pathogens,mediation of inflammatory response and apoptosis.Therefore,we suggest that Smad3 is closely related to the protective mechanism of autophagy induced by sepsis.In addition,the expression of Smad3 and activation can also be regulated by post transcriptional regulation,the main factors of this class is composed of non-encodingRNA including microRNA mediated,but as human body secrete an important class of exosomes,which contains mainly non encoding RNA(miroRNA,LncRNA).It has been found that the cell can regulate the function of signaling molecules through the secretion of exosomes to distant tissues or cells.But in the micro environment of sepsis,exosome release lung tissue cell is contained in the effect of Smad3 microRNAs expression and activation,and further affect the prognosis of patients with sepsis,there is no report before.In view of the protective effect of Smad3 in sepsis,it is one of the strategies to screen the clinical drugs which activating Smad3 for the treatment of sepsis.In the absence of Smad3,a study on the prevention and treatment of sepsis was carried out by using the LPS-TLR4 signaling pathway,a key signaling pathway in sepsis pathogenesis.In summary,we use Smad3 to investigate the Smad3 gene deficient mice in relationship with autophagy in septic lung injury at the same time,through the use of serum samples from the patients with sepsis and find targeted exosomes miRNAs regulation of Smad3,and finally through the screening of drugs to explore integrated pathway inhibitors and new strategies for the prevention and treatment of sepsis.Part 1: The role of Smad3 in regulating autophagy in lung injury induced by sepsisObjective:Through the establishment of Smad3(-/-)mice and septic lung injury model,observe the expression changes after Smad3 knockdown of autophagy related genes.Methods:In vivo by airway instillation of LPS to build acute lung injury model,BEAS-2B cell lines in vitro to construct stable Smad3 interference by lentivirus packaging,then establish LPS stimulation model in vitro.Thenbased on electron microscopy,HE staining,immunohistochemistry,ELISA,qPCR,Western blot and other molecular biological methods to observe the expression of inflammatory and detection of autophagy related genes.Results: 1.Smad3 gene knockout mice with sepsis after lung injury aggravated,lung wet weight ratio increased(*p<0.05);HE staining in KO group showed more obvious hemorrhage,alveolar collapse,neutrophil infiltration in more than WT group;serum and bronchoalveolar lavage were detected by ELISA,TNF-a and IL-6 production were significantly higher than that in WT control group(*p <0.05).2.Early injury(6h)in KO group under the electron microscopy the formation,we found that autophagosomes increased significantly.Besides,the expression of LC3 mRNA(*p<0.05)decreased.but the level of protein expression increased in 6h;.3.si-Smad3 BEAS-2B cell line was successfully constructed,and the expression of autophagy protein LC3 B in LPS-shSmad3 group was significantly higher than that of LPS-NC group in 6h,Conclusion:The septic lung injury in mice was aggravated after Smad3 knock out,and the autophagy increases in the early stage of injury.Smad3 may reduce lung susceptibility to LPS by regulating autophagy.Part 2:The miRNAs in exosome regulate Smad3 to reduce lung injury in sepsisObjective: To detect the Smad3 in the serum of animals and patients by using the high throughput sequencing of small RNA and gene chip technology to screen the miRNAs,and to verify the mechanism of regulating autophagy.Methods:The model of septic acute lung injury,Smad3 activation was detected by WB with a part of the lung tissue,the other part used for a high throughput sequencing,and extraction of serum and bronchoalveolar lavage exosomes for gene chip detection.Combined small RNA high-throughput sequencing with serum exosomes in different periods in patients with sepsis,we analysizedthe differences between groups and the cluster analysis method of bioinformatics screening Smad3 regulated downstream of miRNAs and targeting Smad3 upstream of miRNAs.Finally the target miRNAs were validated with in vitro BEAS-2B cells.Results:1.Phosphorylation smad3 expression was significantly increased after acute lung injury(6h).2.Lung tissue sequencing revealed that after Smad3 knockdown,miR-574-5p expression was reduced,while the serum and bronchoalveolar lavage fluid revealed that miR-1196-5p,miR-574-5p expression were increased in KO/WT.But mi R-1196-5p was not detected in lung tissue.3.miR-574-5p stimulated the expression of autophagy in BEAS-2B cell line stimulated by LPS,the inhibition of autophagy could be blocked by miR-574-5p inhibitor.4.By analysisingthe difference between the 3 groups after the sequencing of small RNA in serum of patients with sepsis exosomeswe found that compared with the control group,exosomes miRNA in 10 daysafter burned patients there were69 in significant difference(p<0.01);and in 20 days after burn group,there were 74 with significant ifference(p<0.01).In burns after 30 days group,there were 42 with significant difference(p<0.01).5.After time series analysis screened out the differential expression of miRNAs,and then we found there were 5 miRNAs related to the target gene of Smad3,LC3B: miR-9-5p,miR-3135 b,miR-432-5p,mi R-409-3p,miR-744-5p.The resultsl were confirmed by the in vitro sepsis model.Conclusion: 1.Smad3 deficiency induced autophagy in early stage of sepsis in mice,and the mechanism may be related to miR-574 induced autophagy.2.The protective effect of smad3 gene in patients with sepsis may be related to the decline of miRNAs in upstream target of smad3.Part 3:The mechanism of LPS-TLR4 signaling pathway inhibitor DMF in the prevention and treatment of sepsisObjective: To explore the molecular mechanism of DMF inhibiting the activation of LPS-TLR4 signaling and its effect on the prevention and treatment of sepsis.Methods:Through screening of LPS-TLR4 signaling pathway inhibitor DMF from clinical medicine database,and using LPS to stimulate RAW264.7 cells with DMF concentration gradient of different group,the activation of 4 key proteins by immunoblot detection in the NFk B signaling pathway.Finally,we established the model of sepsis by LPS i.p.and the effect of the animal survival rate was observed.Results:1.RAW cells were treated with DMF intervention group after LPS stimulation,the concentration of NO decreased,the expression of iNOS was inhibited;DMF also inhibited the LPS-TLR4 pathway downstream of IKK,Ik B expression,NFkB phosphorylation,IRAK protein expression was not affected.2.Survival time effect of gradient experiments found that survival rate is only the preoperative administration of 36 h days and 48 h to reach 50%,(*p<0.05),the survival rate of gradient experiments confirmed that 30mg/Kg can improve the dose effect of sepsis mice survival rate was 50%,the 15mg/Kg can enhance the 25%(*p<0.05).3.In vivo experiments showed that DMF can effectively inhibit the inflammatory factor TNF-a,IL-6,the expression of IL-10(*P<0.05),and HE staining showed that lung,liver and kidney damage in DMF group were significantly reduced compared with the LPS injury group in sepsis.4.Under the combined action of DMF and UTI,the levels of proinflammatory cytokines IL-6 and IL-1?,the transcription and translation of IL-10 were significantly inhibited,and the combination therapy could furtherly improve the survival rate of septic mice by 90%-100%.Conclusion: the inhibitory effect of DMF targeting LPS-TLR4 signaling pathway to inhibit the phosphorylation of IKK,and can effectively prevent sepsis,combined with ulinastatin can further reduce mortality of sepsis to 0.