| Part 1.Correlation between peripheral blood free mt DNA(mitochondrial DNA)level and clinical severity of acute pancreatitisObjective: To investigate the relationship between the content of free mitochondrial DNA in peripheral blood and the severity and related clinical characteristics of acute pancreatitis(AP).Methods: Blood samples were collected from 10 healthy subjects as the control group,20 patients with mild acute pancreatitis(MAP),20 patients with moderate-severe acute pancreatitis(MSAP),and 10 patients with severe acute pancreatitis(SAP)on the first day of hospitalization,and plasma was extracted.The copy number of plasma free mitochondrial DNA was detected by digital PCR.The blood routine and related biochemical indicators were recorded.The copy number of plasma free mitochondrial DNA was compared between acute pancreatitis patients and healthy controls,severe acute pancreatitis patients and mild acute pancreatitis patients.The correlation of plasma free mitochondrial DNA copy number with common clinical indicators and clinical scores was analyzed.Results: 1.By digital PCR,we found that the copy numbers of plasma free mitochondrial DNA in healthy control group,mild acute pancreatitis group,moderate severe acute pancreatitis group and severe acute pancreatitis group were: Based on mitochondrial copies of MT-ND1,the mean was 1374.4(±541.2)copies/ml in the healthy control group and 10694.8(±10504.4)copies/ml in the mild acute pancreatitis group.The mean in the moderately severe pancreatitis group was 26471.5(±27990.4)copies/ml and351076.3(±314709.2)copies/ml.The mean of MT-7S was 1374.4(±541.2)copies/ml in the healthy control group and 10694.8(±10504.4)copies/ml in the mild acute pancreatitis group.The mean in the moderately severe pancreatitis group was 26471.5(±27990.4)copies/ml and 351076.3(±314709.2)copies/ml.2.We also measured the expression of genomic intrinsic markers TRMT10 C in each sample,and calculated the ratio of mitochondrial gene markers to genomic intrinsic markers to eliminate experimental errors.Then,we combined clinical indicators for correlation analysis.The ratio of MT-ND1 to TRMT10 C was positively correlated with peripheral blood white blood cell count,neutrophil count,total cholesterol and procalcitonin(P<0.001).The ratio of MT-ND1 to TRMT10 C was also positively correlated with the concentration of C-reactive protein in peripheral blood(P<0.05).The ratio of MT-7S to TRMT10 C was strongly positively correlated with peripheral blood leukocyte count,neutrophil count,total cholesterol,calcitonin concentration and C-reactive protein(P<0.001).The ratio of MT-ND1 to TRMT10 C was also positively correlated with the concentration of blood lipase(P<0.05).Conclusions: Based on the above results,we found that the concentration of free mitochondrial DNA in peripheral blood increased significantly in patients with acute pancreatitis,especially in patients with severe pancreatitis.In addition,we found that the concentration of peripheral blood free mitochondrial DNA was significantly associated with two inflammatory markers,C-reactive protein and procalcitonin,in patients with acute pancreatitis.Part 2.Single cell sequencing reveals cell heterogeneity and STING signaling pathway activation during acute pancreatitisObjective: To explore the number and proportion changes of cell subsets and functional differences of cell subsets during the occurrence and development of acute pancreatitis,as well as the activation of STING signaling pathway in different stages of acute pancreatitis.Methods: This part of the study was based on the reanalysis of single cell sequencing data from a paper published in SCIENCE on the transformation of pancreatic inflammatory carcinoma.The data set was based on single cell sequencing of pancreatic parenchymal cells from mouse pancreatic tissue after 1,7,and 28 days of caerulein modeling,and single cell sequencing of pancreatic tissue from untreated mice as a control group.The original single cell sequencing data was downloaded from the public database,and the original data was compared and expressed quantitatively through cellranger based on Linux system.The processed matrix data were analyzed and processed downstream by seurat package in R language,including quality control and data filtering,data standardization,data integration and batch effect removal,data dimension reduction,unsupervised clustering,determination of subpopulation specific genes and subpopulation annotation.Then the annotated acinar cells were extracted and subdivided separately,and the differential genes and change pathways of each subgroup of acinar cells were determined.Results: The gene expression of pancreatic parenchymal cells showed high heterogeneity during acute pancreatitis.The signature genes and activated signaling pathways of pancreatic acinar cells also changed significantly during pancreatitis.STING signaling pathway is continuously activated in pancreatic acinar cells during acute pancreatitis.Conclusions: In acute pancreatitis,parenchymal cells of the pancreas,dominated by acinar cells,exhibit greater pro-inflammatory cellular heterogeneity.STING signaling pathway was continuously activated in acinar cells of acute pancreatitis.Part 3.To explore the mechanism of mt DNA-STING signaling pathway in acinar injury of pancreas in acute pancreatitis based on animal and cell modelsObjective: Through the animal models of severe and mild pancreatitis and the cell models induced by cholecystokinin in vitro,the mitochondrial DNA in pancreatic acinar cells under the condition of acute pancreatitis is leaked to the cytoplasm,and can be sensed by CGAS enzyme,a key receptor of STING signaling pathway,and its influence on STING signaling pathway are investigated.Thus,the activation of STING signaling pathway in pancreatic acinar cells was further confirmed in molecular biology experiments,and the promoting mechanism of STING signaling pathway on the release of inflammatory cytokines and the intensification of acute pancreatitis was discussed.Methods: A mouse model of severe acute pancreatitis was successfully constructed by retrograde bile duct injection of sodium taurocholate,and a mouse model of mild acute pancreatitis was successfully constructed by intraperitoneal injection of caerulein.The existing methods of mouse pancreatic acinar cell dissociation and primary culture were improved,and a large number of acinar cells were obtained from mouse pancreatic dissociation.Acinar cell models of acute pancreatitis were constructed by adding cholecystokinin to primary acinar cells and acinar cell lines of pancreas.The leakage of mitochondrial DNA from mitochondria was detected by electron microscopy and mitochondrial DNA staining in pancreatic acinar cell lines stimulated by cholecystokinin.Sh RNA and overexpressed plasmids were used to perform knockdown and overexpression function experiments on the upstream and downstream mitochondrial DNA-STING signaling pathway at the level of pancreatic acinar cells.The transcriptional levels of downstream inflammatory cytokines were verified by fluorescence quantitative PCR.Results: STING signaling pathway was activated during the occurrence and development of pancreatitis in mice,and mt DNA leakage caused by mitochondrial dysfunction of pancreatic acinar cells could be recognized by CGAS upstream of STING signaling pathway and activate STING signaling pathway.Increased activation of STING signaling pathway leads to secretion of proinflammatory cytokines by pancreatic acinar cells.Conclusion: In pancreatic acinar cells in the condition of pancreatitis,mt DNA leakage to the cytoplasm caused by mitochondrial disorders can lead to the activation of STING signaling pathway in the cytoplasm,as well as the activation of downstream inflammatory pathways and the release of pro-inflammatory cytokines.The release of pro-inflammatory factors continues to stimulate the pancreatic acinar cells,resulting in more severe mitochondrial dysfunction,thus causing a vicious cycle.Summary:In the occurrence and development of acute pancreatitis,mitochondrial DNA of pancreatic acinar cells can leak into cytoplasm and extracellular matrix from damaged mitochondria,accompanied by mitochondrial disorders caused by calcium overload,endoplasmic reticulum stress and autophagy overload.On the one hand,STING signaling pathway is recognized by CGAS,which activates STING signaling pathway and downstream NFKB inflammatory pathway and releases a large number of inflammatory factors,thus forming a vicious cycle and continuously promoting the progression of pancreatitis.On the one hand,mitochondrial DNA released into the extracellular can enter the blood and improve the level of free mitochondrial DNA in peripheral blood.Therefore,mitochondrial DNA can also be used as a good indicator to judge the severity of acute pancreatitis.This study is the first to comprehensively explore the relationship between STING signaling pathway of pancreatic acinar cells,mitochondrial DNA leakage from defective mitochondria and downstream inflammatory cytokines release during acute pancreatitis.On the one hand,the important role of mitochondrial DNA and STING signaling pathway in the progression of acute pancreatitis was confirmed.On the other hand,it reveals a new therapeutic target for acute pancreatitis. |
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