Influence Of Secreted And Transmembrane 1b From Alveolar Epithelial Cell On Sepsis With Acute Respiratory Distress Syndrome And Its Mechanisms

Sepsis is the main cause of death in ICU patients which the incidence remains high refers to the systemic inflammatory response syndrome(SIRS)caused by severe infections,in which host-specific immune responses to infection is disordered,moreover can lead to septic shock,acute respiratory distress syndrome(ARDS),and then trigger multiple organ dysfunction syndrome(MODS).Simultaneously,the pathophysiology of acute respiratory distress syndrome in sepsis is very complex,including inflammatory activation,inflammatory cell infiltration,activation of the coagulation system,and damage to the pulmonary epithelium and so on.Among then,the lung epithelium is an important structure involved in the pathogenesis and rehabilitation mechanisms of acute respiratory distress syndrome.During the inflammatory response,a variety of adhesion molecules,chemokines and cytokines continue to release such as tumor necrosis factor,IL-6,IL-8,IL-1IL-1βand interferon are involved in the pathogenesis of ARDS.Pulmonary epithelial cells are able to secrete a variety of cytokines to regulate the innate immunity of the lung,there are currently no secreted proteins that express from alveolar epithelial cells which can regulate innate immunity in the lung.Thus,it is worth well for a study on the gene expression of lung epithelial cells in acute respiratory distress syndrome and the related secretory proteins in sepsis,secreted protein expressed by lung epithelial cells is expected to become a new molecular target for the clinical treatment of sepsis.The sectm1 b gene is a novel human coding gene whose gene is express the sectm1 b protain,which is a transmembrane and secreted protein 1b and belongs to the immunoglobulin superfamily.At present,the mechanism of sectm1 b gene regulation and its effect are not clear,and the research on sectm1 b is very few.There are some studies suggest that sectm1 b can inhibit TCR-mediated T cell activation and play a pro-inflammatory function.As we all know that pro-inflammatory and anti-inflammatory imbalance is the main cause of ARDS.Dexmedetomidine,as a commonly used sedative drug in ICU,has been gradually noticed by many people because of its anti-inflammatory activity in recent years.However,there is no research on the effect of dexmedetomidine on the expression of sectm1 b and the anti-inflammatory effect of ARDS induced by sepsis.In addition,sectm1 b secreted by alveolar epithelial cells can promote neutrophil expression and release of proinflammatory cytokines,and play a regulatory role in the recruitment of neutrophils to the site of pulmonary infection.However,the role of sectm1 b on lung epithelial function in sepsis with ARDS and its possible mechanism have not been reported so far.Our study investigated the effect of sectm1 b from alveolar epithelial cell on sepsis with acute respiratory distress syndrome and its mechanisms through in vitro and in vivo experiments.First of all,we screened and validated the high expression of sectm1 b in the lung epithelial cells of ARDS with septic mice.Secondly,we discussed the proinflammatory effect and its mechanism of sectm1 b expressed from lung epithelial cells on ARDS with sepsis.Finally,we also study the anti-inflammatory effect and mechanism of dexmedetomidine and explored which influence on the expression of sectm1 b form the lung epithelial cells on ARDS with sepsis mice.Part 1 Screening and verification of sectm1 b highly expressed in lung epithelial cells of ARDS with septic miceObjectiveGene chip screening and analysis of sectm1 b highly expressed in lung epithelial cells of ARDS with septic mice,sectm1 b was highly expressed in lung epithelial cells of ARDS with septic mice by in vivo and in vitro experiments.Methods1.C57 mice were used to construct model of ARDS with septic mice by CLP operation,left lung tissues were separated after CLP 0h,6h,12 h and 24 h.The differences of gene expression in lung epithelial cells between CLP and normal control mice at different time points were detected by gene expression profile chip.Using gene expression heat map analysis,genecard software analysis and access to literature focused on analysis of cytokines and chemokines and other secreted protein expression.2.C57 mice were randomly divided into four groups:0h after CLP,6h after CLP,12 h after CLP,24 h after CLP group.a.Using the left lung tissue in each group was stained with HE and pathological score.Extracting the right lung m RNA to detect the m RNA expression of sectm1 b by RT-PCR.b.taking the bronchoalveolar lavage fluid(BALF)of each group to measure the levels of IL-6,IL-1βand TNF-α by ELISA.The neutrophil count was determined by flow cytometry.c.Taking for blood through the eyeball in each group,to take the serum by centrifugation,to measure the levels of IL-6,IL-1β and TNF-α by ELISA.3.LPS stimulation of the lung epithelial cell MLE12,extracting the m RNA in LPS-induced at 0h,6h,12 h,24h,and detecting the expression of sectm1 b m RNA by RT-PCR and the level of IL-6,IL-1β,TNF-α in supernatant by ELISA.Results1.The lung tissue gene expression differences in ARDS with septic mice were significant,the m RNA of sectm1 b expression increased significantly after CLP 12 h and decreased shortly after CLP 24h(P<0.05).2.a.HE staining showed that the pathological damage of lung tissue of CLP mice was significantly aggravated,and the damage was stronger at 12 h after CLP than that 24 h after CLP,the pathological score was higher(P <0.05).The expression of sectm1 b m RNA was significantly higher(12h after CLP than normal about 30 times)(P<0.05).b.ELISA showed that the levels of IL-6,IL-1β and TNF-α in bronchoalveolar lavage fluid and serum were significantly higher than those in normal controls(P <0.05).Neutrophil count increased significantly after CLP(P <0.05).3.Compared with normal group,the expression of sectm1 b mRNA was increased(P <0.05)in MLE12 after induced by LPS,and the levels of IL-6,IL-1β and TNF-α in supernatant were increased(P <0.05).ConclusionThe lung epithelial cells highly expressed sectm1 b on ARDS induced by sepsis.The experimental model were established successfully for Sepsis-induced ARDS in vivo and in vitro experimental.For the follow-up experiment provides a reliable experimental basis.Part 2 Proinflammatory effects of sectm1 b expressed from lung epithelial cells on ARDS induced by sepsis and its mechanismObjectiveTo investigate the effect of sectm1 b expressed from lung epithelial cells on ARDS with LPS-induced and its mechanism.MethodsThe effect of silencing sectm1 b on LPS-induced MLE12 of ARDS with sepsis was investigated by silencing the expression of sectm1 b gene by siRNA transfection technique and its mechanism.The experiment is divided into two group: NC siRNA group and sectm1 b siRNA group.After transfecting 48 h,MLE12 was stimulatied by LPS,to extract the total m RNA,protein,supernatant after LPS stimulation 。The m RNA of sectm1 b,IL-6,IL-1βand TNF-αwere detected by RT-PCR.The levels of IL-6,IL-1βand TNF-αin the supernatant were detected by ELISA.Western-blot was used to detect the expression of molecules in inflammation related signaling pathways(NF-κB / MAPK).Results1.Using siRNA transfection of MLE12,after LPS induction 12 h,RT-PCR showed that the m RNA expression of sectm1 b in sectm1 b siRNA group was significantly lower than that of NC-siRNA group,the difference was statistically significant(P<0.05).2.Using siRNA transfection of MLE12,after LPS induction 6h,12 h,24h,the m RNA expression of IL-6,IL-1βand TNF-α in sectm1 b siRNA group was lower than that in NC siRNA group(P<0.05).The ELISA showed that the levels of inflammatory cytokines IL-6,IL-1βand TNF-α in the sectm1 b siRNA group were lower than those in NC siRNA group(P<0.05).3.Using siRNA transfection of MLE12,after LPS induction 12 h,the phosphorylation of P65 / P38 / ERK1 / 2 in sectm1 b siRNA group was decreased compared with NC siRNA group(P <0.05).ConclusionSectm1b from alveolar epithelial cell may has a pro-inflammatory effect on ARDS of sepsis,which effection may be related to the activation of NF-κβ / MAPK signaling pathway.It is suggested that sectm1 b may play an important role in sepsis-induecd ARDS.Part 3 The influence of dexmedetomidine on the expression of sectm1 b from alveolar epithelial cells on septic mice and its protective effectsObjectiveTo investigate the effect of dexmedetomidine(Dex)on inflammatory reaction of alveolar epithelial cells in septic mice and its influence on the expression of sectm1 b.MethodsC57 mice were randomly divided into three groups:cecal ligation and puncture(CLP)group,CLP with Dex administration group and Sham group.And these mice were treated with dexmedetomidine(50ug/Kg)while CLP group mice were treated with the equal volume of sterile physiological saline by intraperitoneal injection at 15 minute before CLP and then detected at different time point after CLP.Blood and bronchoalveolarlavage fluid(BALF)in each group were collected at various time point,and detection of interleukin-6(IL-6),interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in serum and bronchoalveolar lavage fluid by enzyme-linked immunosorbent assay(ELISA).Also,the survival rate of mice within 24 hours was recorded.In vitro studies of mouse alveolar epithelial cells MLE12 were divided into three groups: control group(without any treatment),LPS group(LPS,1ug/ml),LPS(LPS,1ug/ml)+ Dex(0.2ug/ml)group.IL-6,IL-1β and TNF-α in supernatant were measured by ELISA.And the extracellular signal-regulated kinase ERK1/2 and JNK phosphorylation were measured by western blot.Results1.the production of IL-6,IL-1β and TNF-αboth in serum and BALF of CLP-induced septic mice in dexmedetomidine group less than CLP group(P <0.05)and the survival rate of mice increased within 24h(P <0.05).2.Compared with the LPS group,the expression of IL-6,IL-1β and TNF-α in the supernatants of the dexmedetomidine group was decreased(P <0.05)and it is may be relatied to the activation of ERK1 / 2 and JNK was inhibited(P <0.05).3.Compared with the LPS group,the m RNA expression of sectm1 b was decreased in the dexmedetomidine group(P <0.05).ConclusionDex can affect the expression of sectm1 b in the lung epithelial cells of septic mice,reduce the production of inflammatory cytokines in the serum and BALF of septic mice and increase the survival rate in septic mice,thus playing a protective role in the inflammatory response of alveolar epithelial cells in septic mice,which may be related to the inhibition of dexmedetomidine on activation of ERK1/2 and JNK signal pathways.