| Acute pancreatitis?AP?is a common severe disease in clinic,and the affected population tends to be younger[1].In the United States,more than 200,000 patients are hospitalized each year according to the latest statistics[2,3].The incidence of AP has increased year by year,however,there is currently no treatment for its pathogenesis.Revision of the Atlanta classification and definitions by international consensus[4]classifies AP into mild acute pancreatitis?MAP?,moderately severe acute pancreatitis?MSAP?,and severe acute pancreatitis?SAP?by the degree of severity.MAP have good self-limiting and prognosis,and can be alleviated after symptomatic treatment.MSAP has more peri-pancreatic exudation and may be associated with Systemic inflammatory response syndrome?SIRS?with many complications.SAP is dangerous and the mortality rate is as high as 15%-30%[4].Therefore,this study focuses on the pathogenesis of SAP.SAP with Mutiple organ failure?MOF?is the key factor in determining the severity of AP,acute lung injure?ALI?or acute respiratory distress syndrome?ARDS?is most important.ALI is a major cause of early death in SAP patients,with a mortality rate of up to 60%in SAP-ALI[5].Domestic scholars have found that there is a window period from the onset of SAP to the emergence of ALI[6],and the window period provides a possibility for early intervention to reduce the occurrence of ALI.Endothelial cell permeability changes is the central part to SAP-ALI,however,there is currently no research on the pathogenesis of SAP-ALI.The target of early prevention and treatment target of SAP-ALI remains unclear.MicroRNAs are non-coding,small-fragment RNAs consisting of approximately 18-22nucleotides that regulate post-transcriptional translation levels of proteins by complete or incomplete complementation binding to the non-coding region?3'-UTR?of the mRNA.Studies have shown that microRNAs are involved in the development of many diseases[7-11].Previous studies have suggested that miR-216a can be used as an molecular marker for AP[12].Our study foucus on explore the role of miR-216a in the development of ALI.Exosome is a micro-vesicle secreted by cells,between 35-100 nm in diameter.In recent years,it has been found that exosome vesicles are rich in proteins,nucleic acids,etc.They can shuttle between the blood vessel wall and the extracellular matrix,and can also avoid the phagocytosis of macrophages because of its small size.Exosomes are efficient means of material transporter within the body[13,14].MicroRNA is extremely unstable,and we suspect that miR-216a is transported through the exosome package and participates in the development of SAP-ALI.Part?:Expression of exosomal miR-216a in patients with acute pancreatitis and acute lung injuryObjective:To investigate the expression of exosomal miR-216a in plasma of patients with different kind of acute pancreatitis.Methods:Plasma was collected from 78 patients with AP admitted to the pancreatic intensive care unit?PICU?,department of gastroenterology,Shanghai Changhai Hospital from December 1,2015 to June 30,2016,including 26 cases of AP with ALI,52 cases of AP without ALI,and 8 cases of normal volunteers.Firstly,the level of miR-216a in plasma samples was determined by PCR.Secondly,plasma exosomes were extracted,and then exosome RNA was extracted according to the protocol of the kit.Finally,the content of miR-216a in plasma exosomes was detected by PCR.Results:PCR results showed that the relative expression of plasma exosomal miR-216a in AP with ALI patients,AP without ALI patients,and normal volunteers was15.11±0.38,10.64±0.18,and 4.89±0.71,respectively.The plasma exosomal miR-216a level in patients with AP with ALI was significantly higher than that of patients without ALI and normal volunteers?P<0.01?.Conclusion:Exosomal miR-216a was highly expressed in plasma of AP with ALI patients.Whether miR-216a is involved in the development of ALI remains to be further studied.Part?:Study on pancreatic tissue specificity of miR-216a and SAP-ALI animal model constructionObjective:To further clarify the source of miR-216a and its relationship with SAP-ALI at the animal level.Methods:The SAP-ALI rat model was established by retrograde pancreatic duct injection of 5%sodium taurocholate.Rats were sacrificed at the following time points:12h,24h,36h,and 72h after SAP-ALI model establishment.Whether the model was constructed successful was determined by HE staining,ascites volume measurement,lung dry-to-wet ratio,plasma amylase level,pancreatic tissue and lung tissue pathology score.Tissues of different organs in normal rat were taken,tissue RNA was extracted,and the levels of miR-216a in different tissues were detected by Northern Blot.At the same time,the plasma exosomal microRNA of the control group and the model rats were extracted,and the level of miR-216a was detected by RT-PCR.Results:The ascites volume of the model rats was 4.77±0.37g?12h?,7.32±0.5g?24h?,7.57±0.35g?48h?and 8.22±0.37g?72h?,which were significantly higher than the control group?0.55±0.1,P<0.01?.The dry-to-wet ratio of the model group was 4.29±0.61?12h?,5.88±0.69?24h?,6.5±0.82?48h?and 6.83±0.75?72h?,which was significantly higher than that of the control group?2.73±0.44,P<0.01?.The plasma amylase levels of the model group were 2667.8±432.57 U/L?12h?,7535.53±592.6 U/L?24h?,3650.61±464.79 U/L?48h?and 3184.92±677.08 U/L?72h?,significantly higher than control group?1635.1±159.63 U/L,P<0.01?.The HE staining of the 72h model group pancreatic tissue could not distinguish the acinar structure,the nucleus was dissolved,the structure of the pulmonary lobule was seriously damaged,and a large amount of congestion and inflammatory cell infiltration were observed.The pathological scores of pancreatic tissue were 7.17±1.07?12h?,10.33±0.94?24h?,10.83±0.69?48h?and 11.5±0.96?72h?,which were significantly higher than the control group?0.33±0.47,P<0.01?.The histopathological scores of the model group were 4.17±0.69?12h?,5.17±0.69?24h?,5.33±0.47?48h?and 5.17±0.9?72h?,which were significantly higher than the control group?0.17±0.37,P<0.01?.Northern Blot results showed that miR-216a was detected in pancreatic tissue,and can not be detected in other organs:lung,large intestine,small intestine,spleen,heart,kidney,and brain.The RT-PCR results of rat plasma exosomal microRNA showed that the level of plasma exosomal miR-216a increased compared with the control group after the SAP-ALI model establishment:2.82±0.61?12h?,13.27±0.96?24h?,7.51±0.56?48h?,and 4.48±0.68?72h?.Conclusion:MiR-216a is a pancreas-specific microRNA.The SAP-ALI rat model was successfully constructed and miR-216a was significantly increased in the plasma exosomes of SAP-ALI rats.Part?:Mir-216a regulates endothelial cell permeability through the exosome/LAMC1/ZO-1 pathwayObjective:To investigate whether miR-216a is involved in the regulation of HUVEC endothelial cell permeability in the form of exosome vesicles and its specific molecular mechanism..Methods:MiR-216a mimics and NC were transfected into HUVEC cells,and trans-endothelium electrical resistant?TEER?was measured 24 hours later.Plasma exosomes of AP with ALI patients,AP without ALI patients,and normal volunteers were stained by DiI and added to serum-free medium to culture HUVEC cells.Exosome traces were observed by fluorescence microscopy.The TEER of HUVEC cells was measured.The protein of HUVEC cells transfected with miR-216a mimics was extracted.The expression of tight junction protein ZO-1 and Occludin was detected by RT-PCR,Western Blot and immunofluorescence.The target genes of miR-216a were predicted by TargetScan,PicTar and miRTarBase,and the downstream target genes of miR-216a with the greatest potential were selected.Luciferase,cell immunofluorescence and Western Blot were used for further verification.The permeability of HUVEC cells and the expression of ZO-1 were determined by down-regulating the expression of the selected target gene.Results:After transfected with miR-216a mimics,cell permeability of HUVEC cells was significantly increased?TEER:0.74±0.02 vs 1.02±0.06,P<0.01?.DiI staining exosomal tracer experiments showed that miR-216a can be encapsulated into HUVEC cells by exosomes,thereby increasing endothelial cell permeability.The results of RT-PCR,Western Blot and cellular immunofluorescence showed that the expression of ZO-1 in HUVEC cells was significantly lower than that in the control group after transfection of miR-216a mimics,and the expression level of Occludin remained unchanged.The three microRNA target gene prediction websites predicted LAMC1,HMGB1,JAK2,PTEN,RANBP10 and SMAD7 were the target gene of miR-216a.By referring to the literature,we locked the target gene in LAMC1.Luciferase experimental results showed that LAMC1 was indeed the target gene of miR-216a.Cell immunofluorescence assay and Western Blot assay further confirmed that LAMC1 was a downstream target gene of miR-216a.After HUVEC cells were transfected with si-LAMC1,the TEER value was significantly lower than that of NC group.Western Blot results showed that the expression level of ZO-1 protein in experimental group was significantly lower than that of NC group.Conclusion:MiR-216a enters endothelial cells through exosomes pathway targeting the LAMC-1 gene and then down-regulates the expression of HUVEC cell tight junction protein ZO-1,thereby increasing the permeability of endothelial cells.Part?:Study on the protective mechanism of anti-miR-216a against SAP related lung injury in ratsObjective:To further validated the role of miR-216a in SAP lung injury in vivo experiments.Methods:AAV-anti-miR-216a-GFPandAAV-miR-216a-mimics-GFPadeno-associated virus were constructed and verified,and the state of the rat was observed after tracheal instillation of adeno-associated virus into SD rats.The rats was sacrificed 3 weeks after the addition of the virus,and the lung tissues were collected.After the virus was successfully infected into the lung tissue of rats,5%sodium taurocholate was injected retrogradely through the pancreatic duct.After 12 hours,the lung tissue RNA was extracted and the expression level of miR-216a was detected by RT-PCR.Lung tissue sections were prepared from each group,HE staining and scoring were performed,and the expression of ZO-1 and LAMC1 protein was detected by immunohistochemistry.Results:The sequencing results indicated that the adeno-associated viruses AAV-anti-miR-216a-GFP and AAV-miR-216a-mimics-GFP were successfully constructed.Scattered green fluorescence was observed in lung tissue under the fluorescence microscope,indicating that the virus successfully infected the lung tissue of SD rats.The results of RT-PCR showed that the relative expression of miR-216a in the miR-216a mimics group?10.45±0.25?was significantly higher than that in the control group?2.30±0.13??P<0.01?,and the relative expression of miR-216a in the anti-miR-216a group?0.71±0.15?was significantly lower than that of the control group?P<0.01?.The pathological scores of lung tissue after modeling in control group,miR-216a mimics group,and anti-miR-216a group were 3.6±0.49,4.4±0.49,and 0.8±0.75,respectively.Immunohistochemical results showed that compared with the control group,the expression of ZO-1 and LAMC1 in lung tissue of miR-216a mimics group was significantly decreased.There were no significant differences in the expression of ZO-1 and LAMC1 protein in lung tissue between anti-miR-216a group and control group.Conclusion:The adeno-associated viruses AAV-anti-miR-216a-GFP and AAV-miR-216a-mimics-GFPweresuccessfullyconstructed.Anti-miR-216aalleviates SAP-associated lung injury. |
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