| Sepsis is a systemic inflammatory response caused by infection and frequently results in MODS of which there are clinical manifestations from fever,fast heart beat to dyspnea,hypotension,confusion,oliguria etc.Sepsis is leading cause of death in critically ill patients in the ICU,even after anti-infection and support treatment timely,its mortality rate is as high as 20% ~ 60%.The major cause of death in septic patients is MODS induced by sepsis,in which ARDS is the most common form.ARDS is a life-threatening clinical syndrome,with mortality rate 30% ~60%,clinical manifestations of which are refractory hypoxemia and non-cardiac respiratory failure;generally with chest radiograph of bilateral infiltrated pulmonary edema.Studies on the mechanism of ARDS have shown that over activation of alveolar macrophage and uncontrolled release of inflammatory cytokines cause ALI in sepsis.In these inflammatory cytokines,TNF-? and IL-1? are of the most biological activity at the early stage,which potentially start the cascade of inflammatory response and cause ALI.Autophagy acts as one basic metabolic mechanism in organism,which plays a key role in nutrient metabolism,tumorigenesis,immune inflammation and so on.Recently,the research has shown that autophagy can remove impaired mitochondria,and block proIL-1? to be cleaved into mature form via preventing the reactive oxygen species and mitochondrial DNA to activate Caspase-1 in cytoplasm.In addition,microRNA also have a powerful function in regulating various biological actions in organism.Ingredients of pathogenic microbe and inflammatory mediators at early stage,such as LPS,TNF-? and IL-1?,can stimulate mono-nuclear macrophage resulting in upregulation of microRNA-155.MicroRNA-155,in turn,combines with the message RNA of adaptor protein and inhibits its expression post-translationally;followed with quenching the amplication of inflammatory response.Given that autophagy and miR-155 can regulate inflammatory response through their own pathway independently,the link between autophagy and miR-155 in response to inflammation remains unclear.To determine the relationship between inflammation,autophagy and miR-155,and the mechanism where they interact with each other,could be helpful for prevention and treatment of ALI induced by sepsis,and for research of novel special medicine.Part ? Changes of inflammatory response,autophagy and miR-155expression in NR8383 cell after LPS challengeObject: The relationship of inflammatory response,autophagy and miR-155 was explored through the determination of their corresponding biomarkers in NR8383 cell after LPS challenge.Method: NR8383 cells were cultured with regular methodology.Cells and supernatant were collected after challenge with LPS for 0.5/3/6/12/24 hrs.TNF-? and IL-1? in supernatant were determined by ELISA,proteins LC3?/?,TAB2 and Caspase-1 by Western Blot and miR-155 expression by real-time qPCR.Result: TNF-? and IL-1? of normal group were 20.12±7.20 and 894.34± 77.98pg/ml respectively,and cytokines of PBS group were 19.63±6.51 and 1024.50± 113.47pg/ml,there was no difference between normal and PBS group(P>0.05).Correspondingly,TNF-? and IL-1? of groups with LPS challenge for 0.5/3/6/ 12/24 hrs were 230.37±19.28 and 3281.50±263.29pg/ml,360.35±33.28 and 3908.50± 228.47pg/ml,560.35±43.62 and 4601.84±301.27pg/ml,860.46±84.62 and 6201.83± 173.93pg/ml,601.21±54.73 and 4008.62±247.45pg/ml.All groups with LPS challenge were different from norm group(P<0.05);furthermore,there was difference between 12 hrs and 24hrs(P<0.05).Protein LC3?/? ratio was 0.11±0.04 in normal group,and 0.09±0.06 in PBS group;There was no difference between groups(P<0.05).Protein LC3?/? ratio in groups with LPS challenge for 0.5/3/6/12/24 hrs was 0.46±0.07,0.51±0.13,0.59±0.09,0.81±0.13,0.56±0.07.All LPS groups were different from PBS group in protein LC3?/? ratio(P<0.05);furthermore,there was difference between 12 hrs and 24hrs(P<0.05).Comparison to normal group,TAB2 in PBS group was 0.83±0.11 times;TAB2 in groups with LPS challenge for 0.5/3/6/12/24 hrs was correspondingly 1.2±0.17,0.93±0.12,1.2±0.04,1.04±0.17,1.27±0.09 times.Difference was not found between every two groups(P<0.05).Comparison to normal group,Caspase-1 in PBS group was 1.1±0.20 times(P>0.05);Caspase-1 in groups with LPS challenge for 0.5/3/6/12/24 hrs was correspondingly 4.82±0.32 times(P<0.05),8.23±0.41 times(P<0.05),11.29±0.54 times(P<0.05),12.63±0.39 times(P<0.05),9.87±0.49 times(P<0.05).Furthermore,there was difference between 12 hrs and 24hrs(P<0.05).Comparison to normal group,miR-155 expression in PBS group was 0.91±0.33 times(P>0.05),mi R-155 expression in groups with LPS challenge for 0.5/3/6/12 /24 hrs was correspondingly 1.21±0.19 times(P>0.05),1.41±0.20 times(P>0.05),1.74±0.18 times(P<0.05),3.11±0.12 times(P<0.05),7.82±0.30 times(P<0.05).Conclusion: Inflammatory response mounted to peak at 12 hrs after LPS challenge.Expression of miR-155 and autophagic response escalated with time and both displayed time-dependent manner.Additionally,the trend of elevated protein Caspase-1 geared to the trend of inflammatory reaction completely.Part ? The mechanism in which miR-155 impacts inflammatoryresponse,as well as autophagyObject: To explore the mechanism in which miR-155 impacts inflammatory response induced by LPS,as well as autophagyMethod: With upregulation of miR-155,and after challenge with LPS for 12 hrs,cells and supernatant were collected for determination of TNF-? and IL-1 by ELISA,LC3?/?by Western Blot,miR-155 by real-time qPCR.Secondarily,with inhibition of miR-155,and after challenge with LPS for 12 hrs,cells and supernatant were collected for determination of the same biomarkers above.Result: 1.Comparison to miR-155 expression in normal NR8383 cells,miR-155 expression in NR8383 cells transfected with miR-155 mimics was 236.73±46.49 times in 24 hrs after transfection(P<0.01).TNF-? and IL-1 in normal group were 18.83±5.65 and 583.83±47.29pg/ml respectively,both in LPS group were 774.84±59.23 and 5806.28±167.01pg/ml,and both in LPS+mimics group were 347.84±27.16 and 2087.27±83.20pg/ml.Difference was found between every two groups(P<0.05).Protein LC3?/? ratio in normal group was 0.13±0.04,ratio was 0.49±0.11 in LPS group,and ratio was 0.79±0.14 in LPS+mimics group.Difference was found between every two groups(P<0.05).Comparison to normal group,TAB2 in LPS group was 0.92±0.08 times(P>0.05),TAB2 in LPS+mimics group was 0.32±0.04 times(P<0.05).There was difference between LPS+mimics group and LPS group(P<0.05).Comparison to normal group,Caspase-1 in LPS group was 11.57±0.23 times and Caspase-1 in LPS+mimics group was 7.27±0.16 times.Difference was found between every two groups(P<0.05).2.Comparison to miR-155 expression in normal NR8383 cell,miR-155 expression in NR8383 cells transfected with miR-155 inhibitor was 1.6±0.34 times(P>0.05)in 24 hrs after transfection.TNF-? and IL-1 in normal group were 17.45±3.90 and 558.40±45.31pg/ml respectively,both in LPS group were 581.27±60.14 and 5506.37±114.47pg/ml,and both in LPS+inhibitor group were 811.38±45.68 and 7036.08±120.63pg/ml.Difference was found between every two groups(P<0.05).Protein LC3?/? ratio in normal group was 0.14±0.02,ratio was 0.65±0.11 in LPS group,and ratio was 0.43±0.06 in LPS+inhibitor group.Difference was found between every two groups(P<0.05).Comparison to normal group,TAB2 in LPS group was 1.23±0.16 times(P>0.05),TAB2 in LPS+inhibitor group was 2.11±0.30 times(P<0.05).There was difference between LPS+inhibitor group and LPS group(P<0.05).Comparison to normal group,Caspase-1 in LPS group was12.60±0.31 times and Caspase-1 in LPS+inhibitor group was 15.16±0.29 times.Difference was found between every two groups(P<0.05).Conclusion: Autophagic response was enhanced with upregulation of miR-155,and conversely autophagic response was dampend with inhibition of miR-155.MiR-155 oscillated with autophagy through targeting mRNA of protein TAB2,which was called “autophagy switch”.Autophagy negatively regulated Caspase-1 activity to modulate inflammatory reaction via preventing the maturation of IL-1 cleaved from proIL-1.Part ? Mi R-155 syringed into SD rat impacts inflammatoryresponse as well as autophagyObject: To study the lung protection and its mechanism by evaluation of inflammatory injury and count of autophagosomes under electro microscope in SD rats that were instilled with miR-155 loaded by in vivo-jetPEITM through airway.Method: Nine male SD rats randomly entered into normal,LPS,and LPS+mi R-155 group with three per group.Rats in LPS group were instilled with LPS(7.5mg/kg)through airway to produce rat ALI model and were executed after 6 hrs.Rats in LPS+miR-155 group were instilled with miR-155(40?g/body)loaded by in vivo-jetPEITM via airway,after 24 hrs were treated with a serial of manipulations as above.Rats in normal group were treated with all manipulations as same as in LPS group but LPS.After execution,blood samples were collected from abdominal artery for blood gas analysis.Pulmonary tissue from the same site of left lung was determined for W/D ratio and regular pathological section dyed by HE.Autophagosome under electro microscope were analysed by specialist.Supernatant samples from BALF were determined for TNF-? and IL-1 by ELISA,and total RNA samples were extracted from cells that were isolated from BALF for miR-155 expression determination by real-time qPCR.Result: Comparison to normal group,miR-155 expression was 5.87±0.34 times in LPS group(P<0.05)and 95.23±16.84 times in LPS+miR-155 group(P <0.01).TNF-? and IL-1 in normal group were 48.51±8.21 and 678.64±55.32pg/ml respectively,both in LPS group were 745.99±34.45 and 5482.10±119.57pg/ml,and both in LPS+ mi R-155 group were 364.68±23.00 and 2349.38±55.09pg/ml.Difference was found between every two groups(P<0.05).There were no pulmonary edema and bleeding stains in lung of rats in normal group;however,there were severe pulmonary edema and multiple sites of bleeding in lung of rats in LPS group.Appearance in lung of rats in LPS+miR-155 group was between the two.Oxygenation index in normal group was 420.63±34.96 mmHg,oxygenation index in LPS group rats was 271.42± 23.46 mmHg,and oxygenation index in LPS+mi R-155 group was 315.76±16.08 mmHg.Difference was found between every two groups(P<0.05).Neat alveolar structure but no swelling epithelium was found in pulmonary tissue section from rats in normal group.However,disordered alveolar structure and swelling lung epithelium were found in pulmonary tissue section from rats in LPS group.Conditions of section from rats in LPS+miR-155 group were between the two groups above.No autophagosome was found in electroscrope graph(EG)of alveolar macrophages collected from rats in normal group.Two autophagosomes were found in EG from LPS group.Seven autophagosomes were found in EG from LPS+miR 155 groupConclusion: MiR-155,which was syringed into rat through airway,alleviated ALI induced by LPS via upregulation of autophagy. |
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