The Role And Mechanism Research Of TLR9/MyD88/JNK Signaling Pathway In Paraquat Induced Acute Lung Injury

Introduction:Paraquat poisoning is one of the most dangerous toxic diseases in clinical practice,with a lethality of up to 30-70%.It is clinically painful with no specific antidote.Acute lung injury is the main manifestation of moderate to severe paraquat poisoning in the early stage.Its rapid progress and multiple complications can rapidly progress to respiratory failure and multiple organ dysfunction leading to death.As a herbicide,paraquat with bipyridine structure blocks the intracellular electron transport chain,generates a large amount of reactive oxygen species(ROS)in situ,causing intense lipid peroxidation and directly destroying the integrity of the cell membrane The role of plant killing.Paraquat poisoning into the body after the accumulation of polyamine transport system in the lungs,causing acute lung injury,but its specific mechanism is not yet fully understood.Toll-like receptors(Toll-like receptors,TLR)as part of the innate immune system,play an important role in identifying pathogens and activating the immune system to start immune killer.However,recent studies have found that pathogen-associated molecular patterns(PAMPs),such as lipopolysaccharide(LPS)on gram-negative bacterial membranes,unmethylated common to bacteria and viruses,DNA(CpG DNA)or endogenous unmethylated DNA such as mitochondria DNA(mt DNA)is recognized by TLR on the surface of pulmonary capillary endothelial cells.Thereby activating the TLR-mediated signaling pathway leading to a massive release of inflammatory mediators through Myeloid differentiation factor 88(MyD88),which aggravates the inflammation in the lungs and aggravates the damage to pulmonary capillary endothelial cells,Leading to increased pulmonary microvascular permeability,leading to pulmonary edema and the formation of a clear membrane,causing acute lung injury.TLR9,an important member of the Toll-like receptor family,is an essential receptor for the recognition of CpG,an immunostimulatory sequence in viral and bacterial DNA.In the early stage of paraquat poisoning,TLR9 should not have any significant viral or bacterial DNA attack.However,abnormal activation of TLR9 occurs in the early stage of paraquat poisoning.Therefore,it is very important to study the mechanism of TLR9 signaling pathway inacute lung injury caused by paraquat poisoning,which has important clinical guidance for the targeted treatment of paraquat poisoning acute lung injury significance.Its downstream c-Jun N-terminal kinase(JNK)also has an abnormal activation in paraquat poisoning,so this experiment with acute paraquat-induced acute lung injury as the starting point,from the organ level,protein level and nucleic acid level related detection to explore the mechanism.Through specific inhibitor intervention and gene silencing methods,the role of TLR9 / MyD88 / JNK signaling pathway in the development of paraquat poisoning acute lung injury maybe clarified,which provided the basis for clinical targeted therapy.Methods:First,the animal model making and specimen collection1.8-week-old wild-type SPF mice(C57BL / 6J)and MyD88 knockout mice(C57BL / 6J background)were purchased from the Model Animal Institute of Nanjing University,male or female,breeding,breeding in Shengjing Hospital,China Medical University Center Laboratory,feeding temperature control 20 ~ 25 ?,humidity control 40% to 70%,circadian rhythm control 12 h,free to eat water.Toxic mice were intraperitoneally injected with 30 mg / kg paraquat solution(10mg / mL dissolved in saline)and control group were given equal volume of normal saline.2.specimen collection and preservation48 h after paraquat intraperitoneal injection,10% chloral hydrate 300 mg / kg intraperitoneal anesthesia.To be fully anesthetized,the mouse supine on the mouse fixed platform,routine disinfection,open the chest,the use of 1mL syringe into the right ventricle,slowly bleeding,to avoid hemolysis.The blood into 1.5mL centrifuge tube,3000 r / min,4 ?,centrifuged 10 min,the supernatant sub-1.5mL EP tube,each tube release about 0.2mL,set-80 ? refrigerator.After the blood is collected from the heart,the left atrial appendage is cut off,the lung tissue is washed with saline.Completely remove the lung tissue on both sides,observe the lung tissue color,texture,with or without bleeding and other pathological changes.After washing with saline,left lung and lungs were placed in 4% paraformaldehyde and fixed overnight at 4 ° C.The next day,30% sucrose was dehydrated at 4?.After complete dehydration,the cells were embedded in OCT and stored in a refrigerator at-80?.Right lung liquid nitrogenfrozen at-80 ? refrigerator to prepare for testing.Second,cell model constructionA549 cells were inoculated into 96-well plates.There are control group and paraquat poisoning groups(50,100,200,400,600,800 and 1000 ?mol / L),Incubation incubated 24 h screening out the experimental treatment concentration.At the end of each well by adding MTT solution 20?L set at 37? for 4h.Discard the supernatant,add DMSO150?L to each well and shake for 10 min to fully dissolve the blue crystal.The microplate reader detects the absorbance value(A value)of each well at a wavelength of 560 nm.Cell survival rate(%)=(average A value in experimental group / average A value in control group)× 100%.With the survival rate of the nearest50% of the treatment concentration as the screening of the experimental treatment concentration.In accordance with the intervention of different sub-negative control group,exposure group,silent exposure group,silent control group.A gene silencing intervention was added to the corresponding siRNA lentivirus particles(Santa Cruz,USA)for 72 hours.The challenge group was challenged with paraquat at the experimental concentration previously screened,and the control group was given normal saline at 24 h after initiation of challenge,the culture was terminated for further testing.1.ELISA method serum TNF-?,IL-1? content determination.2.Lung wet / dry weight ratio and lung tissue damage score assessment3.Realtime RT-PCR method was used to detect the expression of MyD88,TNF-?and IL-1? mRNA in lung tissue4.Immunofluorescence detection of MyD88 and p-p65 expression in lung tissue5.Western blot was used to detect the expression of TLR4,TLR9,MyD88 and p-p65 in lung tissue Third,TLR9 downstream genes MyD88 and JNK paraquat poisoning acute lung injury1.MyD88 effect using gene knockout mice and wild-type mice for study,each group by ELISA serum TNF-?,IL-1? content,lung wet / dry weight ratio and lung tissue damage score assessment,The expression of MyD88,TNF-? and IL-1? mRNA in lung tissue were detected by Real-time PCR.The expression of MyD88 and p-p65 in lung tissue was detected by immunofluorescence.The expressions of TLR4,TLR9,MyD88 and p-Mouse acquisition and lung wet weight / dry weight ratio assay were used to evaluate the role of MyD88 in paraquat poisoning acute lung injury.2.The effect of JNK was studied by in vitro and in vivo studies using specific inhibitors.HE staining and lung injury scores of rat lung tissue were used in each group.ROS production in lung tissue was detected by DHE staining and Annexin V-PI double staining.The levels of TNF-? and IL-6 were detected by ELISA,the mRNA levels of TNF-? and IL-6 were detected by RT-qPCR.The transcriptional factor DNA of AP-1and NF-?B p-p65 were detected by EMSA Activity of TLR9-specific antagonist A549 cells after paraquat poisoning Western blotting detection of cytoplasmic JNK phosphorylation levels in order to assess the role of JNK in paraquat poisoning acute lung injury.Fourth,the application of TLR9-specific inhibitors and gene silencing comparative study1.In vivo,TLR9-specific inhibitor ODN2088 was used to detect the levels of TNF-? and IL-1? in serum,lung injury scores,the expression of TLR9 and p-p65 by immunofluorescence,and the expressions of TLR9,MyD88,P-IRAK4,p-JNK,and p-p65 to verify TLR9-mediated paraquat poisoning of acute lung injury from in vivo levels.2.In vitro,A549 cells were treated with TLR9 gene silencing,and apoptosis was detected by Annexin V-PI double staining.Real-time RT-PCR was used to detect TLR9,TNF-? and IL--1?,and the expression of TLR9 and p-p65 was detected by immunofluorescence method to study the molecular mechanism of TLR9-mediated acute paraquat lung injury in vitro.Results:First,Acute lung injury and apoptosis1.Paraquat poisoning lung tissue light microscopy showed alveolar structural damage,alveolar hemorrhage,pulmonary interstitial edema,a large number of inflammatory cell infiltration,wet / dry weight ratio increased.2.Paraquat poisoning A549 apoptosis rate was significantly increased.Second,MyD88 and JNK involved in mediating paraquat poisoning acute lung injury1.MyD88 gene knockdown can reduce lung wet / dry weight ratio and lung injuryscore,lower serum TNF-?,IL-1? protein levels and mRNA expression,upstream TLR4,TLR9 no change but downstream p-p65 activation Nuclear translocation significantly reduced.2.JNK-specific inhibitor can reduce lung wet / dry weight ratio and lung injury score,less ROS generation of lung tissue,inhibit apoptosis,and down-regulate the DNA binding activity of AP-1 transcription factor to reduce TNF-? And IL-6transcriptional expression level,TLR9 specific inhibitors can reduce the level of JNK phosphorylation and reduce the degree of acute lung injury poisoning.Third,TLR9 initiates activation of MyD88 / JNK in paraquat-induced acute lung injury1.In vivo experiments showed that ODN2088,a specific inhibitor of TLR9,could decrease the level of TNF-? and IL-1? in lung tissue and decrease the levels of MyD88,p-IRAK4,p-JNK and p-p65.2.In vitro experiments confirmed that TLR9 gene silencing can significantly reduce the rate of apoptosis,and downregulate the levels of TNF-?,IL-1?mRNA and cell supernatant TNF-?,IL-1?,and p-p65 nuclear translocation in TLR9 gene After the silence also significantly reduced.Conclusion:1.Paraquat poisoning can cause activation of the TLR9 / MyD88 / JNK pathway leading to acute lung injury and apoptosis.2.Intervention with TLR9 / MyD88 / JNK pathway can improve acute lung injury and apoptosis induced by paraquat poisoning.