| Acute pancreatitis(AP)is one of the common causes of gastrointestinal disease hospitalization,with a high incidence worldwide,which brings a heavy burden to the healthcare system and patients.However,the pathological mechanism of SAP is not completely clear,among which abnormal activation of calcium ions leads to premature activation of trypsinogen is considered to be an important pathological mechanism of SAP.In the early stage of AP,due to various reasons,the concentration of calcium ions in the cytoplasm increases,and the organelle function is impaired,which induces the premature activation of trypsinogen and leads to pancreatic digestion.With the progression of the disease,AP patients were complicated with systemic inflammatory response syndrome(SIRS)and multiple organ failure(MOF),and the mortality rate was up to 30%-50% after the progression to severe acute pancreatitis(SAP).In recent years,a large number of studies have shown that there is a relationship between the pathogenesis of SAP and gene transcription regulation.Studies have shown that some non-coding RNAs can participate in the development of SAP by regulating downstream proteins.Therefore,it is of great clinical significance to explore effective measures to prevent or inhibit the progression of AP in the treatment of SAP.Long non-coding RNA(lncRNA),which is more than 200 nt in length,plays an important role in many disease processes.Currently,there are three mechanisms of lncRNA.Firstly,lncRNA can directly bind to protein and play a role.Secondly,lncRNA directly binds to transcription factors and regulates DNA transcription.Thirdly,the most classical mechanism of lncRNAs is to influence the level of downstream mRNA by binding miRNAs,namely,competing Endogenous RNAs(ceRNA).Studies have confirmed that a large number of lncRNAs can affect the occurrence and development of diseases through ceRNA.The mechanism of lncRNA in SAP is being explored.Studies have shown that lncRNA can directly participate in the disease process of SAP.However,only a few lncRNAs have been discovered that can affect the progression of SAP disease.In view of this,this project hopes to explore more lncRNAs that can play a role in the process of SAP disease through high-throughput sequencing,and to explore their downstream targets and their mechanism of action.Part Ⅰ : High-throughput sequencing results of pancreatic lncRNAs in SAP mice.Objective: Differentially expressed lncRNAs in pancreatic tissues were explored inSAP mouse model and their downstream targets were predictedMethods: Twelve SPF male C57BL/6 mice at 6-8 weeks were divided into two groups according to the random number table method: Sham operation group(n=6,Sham group,the pancreas was turned several times after laparotomy and the abdominal cavity was closed)and severe acute pancreatitis group(n=6,SAP group,after opening the abdominal cavity,the mouse was slowly injected with 5% sodium taurocholate(0.1m L /100g)retrograde through the pancreatic tube to construct the mouse SAP model.Blood and pancreatic tissue samples were collected 24 hours after surgery.HE staining was used to observe the pathological changes of pancreatic tissues in mice.Serum amylase lipase concentration was determined by ELISA kit.The differentially expressed lncRNAs in pancreatic tissues of SAP mice were explored by high-throughput sequencing,and the expression of lncRNAs in pancreatic tissues of SAP mice was detected by RT-qPCR.The six lncRNAs with the most obvious differential expression in the high-throughput sequencing results were sorted out and screened.The lncRNA-miRNA-mRNA network diagram was drawn by combining the sequencing results with the lncRNA-related mRNAs indicated in the database and the miRNAs verified in previous literatures.To explore possible downstream targets of lncRNA.Results: Compared with Sham group,serum amylase and serum lipase in SAP group were significantly increased.HE staining results showed that pancreatic tissue of mice in SAP group was obviously destroyed.Sequencing results showed that differentially expressed lncRNAs in SAP group were related to lipid metabolism,alcohol and protein metabolism.After a series of screening,five lncRNA with high expression in SAP group(AK035396,NR＿030738,TCONS＿00010866,UC007 HXV.1,UC029 SUg.1)and one lncRNA with low expression in SAP group(AK052913)were identified.Six differentially expressed lncRNAs were verified in mouse pancreatic tissues by expanding the sample size.CaMKⅡ,FUT8,IKK,PRKCE and RIP3 were highly correlated with 6 lncRNAs.Conclusion: The pathological results of pancreatic tissue and serum amylase and lipase in mice proved that the mouse model of SAP group was successfully constructed.Sequencing results showed that a large number of lncRNAs may be involved in the process of SAP disease,including lipid metabolism,protein metabolism and alcohol metabolism.qPCR results of six lncRNAs showed that the screened lncRNAs were widely expressed in pancreatic tissues of SAP mice.Six lncRNAs could be associated with CaMKⅡ,FUT8,IKK,PRKCE and RIP3 through SAP-related miRNAs.PartⅡ: Mechanism of lncRNA downstream target protein CaMKⅡin SAP Objective: To observe the expression of CaMKⅡ protein in pancreatic tissues ofSAP mice,and to explore the mechanism of its action in the process of SAP disease after the use of its specific inhibitor KN93 was combined.Methods: Thirty-six SPF C57 mice at 6-8 weeks were divided into 4 groups according to random number table method,including Sham group(Sham group),inhibitor group(KN93 group),severe acute pancreatitis group(SAP group),and severe acute pancreatitis + inhibitor group(SAP+KN93 group),with 9 mice per group.Beforemodeling,mice in the Sham group were fasted without water for 12 h.After laparotomy,we turned the pancreas of mice in the KN93 group before abdominal closure.Mice inthe SAP+KN93 group were injected KN93(1mg/kg)through tail vein,and 5% sodium taurocholic acid solution(1mg/kg)was retrograde injected through pancreatic ductafter laparotomy.Mice in SAP group was injected with sodium taurocholate solution and KN93(1mg/kg)by tail vein.The levels of lipase,amylase,IL-6,IL-10 and TNF-α inserum were detected by ELISA kits.The damage of pancreatic tissue was observed by HE staining.The expression of NF-κB in pancreatic tissue was observed byimmunohistochemical staining.The expression levels of CaMKⅡ,p-CAMK Ⅱ,NF-κB,p-NF-κB,ERK and p-ERK in pancreatic tissues were observed by western blot.ThemRNA expression levels of CaMKⅡ in pancreatic tissues were detected by RT-qPCR.Results:Compared with Sham group,serum amylase and lipase in SAP mice weresignificantly increased(P<0.05),levels of IL-6 and TNF-α in serum increased(P<0.05).The phosphorylation levels of CaMKⅡ,NF-κB and ERK in pancreatic tissues of mice inSAP group were increased,and the difference was statistically significant(P<0.05).After the activity of CaMKⅡwas inhibited by KN93,the pathological injury of pancreasof SAP mice was alleviated,and the serum inflammatory factors IL-6 and TNF-αwere decreased(P<0.05),the level of IL-10 increased(P<0.05).Meanwhile,thephosphorylation levels of NF-κB and ERK in pancreatic tissue decreased(P<0.05).Conclusion:The activity of CaMKⅡprotein in pancreatic tissues of mice in SAPgroup was significantly increased,but after specific inhibition of CaMKⅡprotein effectively inhibited the activity of CaMKⅡprotein in pancreatic tissues of mice in SAP,the degree of pancreatic injury of mice in SAP was significantly reduced,which may be caused by alleviating pancreatic injury and systemic inflammation through ERK/MAPK signaling pathway. |
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