| Background:Acute pancreatitis(AP)is a common acute abdominal condition with an increasing incidence worldwide.Severe acute pancreatitis(SAP)is a severe form of AP with the potential to cause life-threatening complications.SAP is mainly treated with symptomatic therapy and preventive medicines.Nevertheless,hospitalization and the requirement for advanced medical technology make the management of such patients expensive and time-consuming.Therefore,there is a need to develop novel SAP treatments.Dexmedetomidine(Dex)is a highly effective α2-adrenergic receptor.It is extensively applied in the intensive care unit and analgesic during surgery as a sedative.Studies have shown that it effectively reduces the need for perioperative anesthetics.Dex has been shown to have anti-inflammatory effects.It can attenuate oxidative stress,and mitigate systemic inflammatory response.Given its high safety,it is considered as a promising multiorgan protective agent.Toll-like receptor 4(TLR4)is a member of the Toll-like receptors(TLRs)family which is highly expressed in alveolar cells,and stellate cells.The binding of ligands such as lipopolysaccharide to TLR4 can activate the downstream NF-κB signaling pathways and induce the expression of TNF-α and other pro-inflammatory cytokines.Under certain conditions,the binding of TLRs to ligands may trigger necroptosis.Necroptosis is a new form of programmed cell death that differs from apoptosis and plays a role in the development of several diseases.Necroptosis in pancreatic can initiate AP.Targeting necroptosis is an effective option for SAP treatment.Lung injury is the leading cause of death in the early stages of SAP.Approximately one-third of SAP patients develop acute pancreatitis associated-lung injury(APALI)or even ARDS.In this study,the inhibitory effect of Dex on SAP and lung injury was evaluated through in vitro and in vivo experiments,and its possible mechanism of action was investigated.Patients with acute SAP require surgical intervention and are admitted to the intensive care unit for non-invasive ventilation.Dex is currently used by intensive care clinicians and anaesthesiologists as an adjunctive sedative.Therefore,the clinical availability of Dex makes it easier to implement in SAP treatment than developing new drugs.In this study,the anti-inflammatory and organ-protective benefits of Dex were initially investigated in SAP rats.Second,transcriptomics and molecular biology techniques were employed to determine the effect of Dex on necroptosis during AP and the molecular pathways underlying its action.Moreover,the effect of Dex in the treatment of SAP was elucidated,providing an experimental basis for the future clinical application of Dex in the treatment of SAP.Method:Part Ⅰ: A total of 45 rats were randomly divided into sham-operated(Sham),SAP,and Dex groups.In the SAP group,5% sodium taurocholate was injected retrogradely into the biliopancreatic duct.Rats in the Dex group were intraperitoneally administered with Dex(10μg/kg).Serum amylase activity and inflammatory factor levels were measured using commercially available kits.The IL-6,TNF-α,and IL-1β m RNA expression level were quantified by qRT-PCR.HE staining was performed to assess the degree of pancreatic tissue injury in each rat.Immunohistochemistry was conducted to detect the expression of MPO,CD68,and 4-HNE.The GEO2 R tool was used to identify DEGs associated with necrotizing apoptosis in acute pancreatitis.Differential protein expression of RIPK1,RIPK3,and MLKL in pancreatic tissues of rats in each group was determined by Western blot and immunohistochemistry.The subcellular organelle structure of pancreatic tissue was examined using a transmission electron microscope.TUNEL staining was carried out to determine apoptosis of pancreatic tissue.The STC was used to establish a model of Inflammatory in pancreatic alveolar cells AR42 J.Cell morphological changes were observed using a light microscope.The expression of inflammatory was detected by ELISA.The apoptosis and ROS levels were measured using the flow cytometry and DCFH-DA fluorescence method.Part Ⅱ: The regulatory effect of Dex on the gene expression profile of SAP rat pancreas tissue was investigated through RNA sequencing.Differentially expressed genes(DEGs)were identified via functional enrichment analysis and construction of a PPI network.qRT-PCR was performed to measure the m RNA expression levels of critical DEGs in rat pancreatic tissues.qRT-PCR and Western blot analysis were conducted to quantify expression of TLR4/NF-κB signaling pathway-related RNA and proteins.Part Ⅲ: HE staining of lung tissues was performed,and the pathohistological score standard was assigned in line with the traditional scoring criteria.Inflammatory cytokines,including interleukin IL-6 and TNF-α of BALF were detected based on ELISA.The lung wet/dry ratio was calculated from weights of lung tissues.The expression of TNF-α and IL-1β m RNA in lung tissues was quantified by qRT-PCR.The trans-differentiation of type II alveolar epithelial cells into type I alveolar epithelial cells was observed through immunofluorescence double staining.The specific protein markers of type Ⅱ and type Ⅰ alveolar epithelial cells and the expression levels of proteins related to apoptosis were detected by Western blot analysis.Results:Part Ⅰ: The survival rate of rats in Dex group was significantly increased following 24 hours of modeling.Compared with the SAP group,the level of serum amylase and inflammatory factors in the Dex group was decreased.The pancreatic IL-6,TNF-α,and IL-1β m RNA levels were detected by qRT-PCR,and similar results as the serum ELISA tests were obtained.The pathological damage to the pancreatic tissue was examined by the HE staining assay.Many inflammatory cells infiltrated the pancreas of SAP rats,and a large number of necrosis of acinar cells was observed.In contrast,Dex treatment significantly improved pancreatic tissue’s bleeding,necrosis,and inflammatory cell infiltration.The pancreatic pathological damage scores in the Dex group were significantly decreased.Analysis of IHC results for MPO and CD68 in pancreatic tissue revealed that Dex significantly reduced the number of positive inflammatory cells.The IHC results of 4-HNE suggested that administration of Dex decreased oxidative stress in rats with SAP.Analysis of key necroptosis-related proteins in pancreatic tissue showed that Dex downregulated RIPK3,RIPK1,and p-MLKL expression levels in SAP rats.The IHC results were consistent with this finding.The electron microscopic results showed that in the SAP group of rats,the mitochondrial surface appeared wrinkled,neutrophils with nuclear fission images were visible,and Dex treatment improved these ultrastructural changes.Tunnel assay results demonstrated that Dex reduced the rate of apoptosis.In vitro,cellular assays revealed that Dex treatment inhibited STC-induced inflammation and apoptosis levels in AR42 J cells.Moreover,the ROS levels in the Dex group were reduced after treatment for 24 hours with the two doses,suggesting that Dex decreased ROS levels in AR42 J cells.Part Ⅱ: The RNA-sequencing showed that 1037 genes were up-regulated,and 598 genes were down-regulated in the SAP group.In the Dex group,309 genes were up-regulated whereas 1204 genes were down-regulated.The DEGs among the three groups were subjected to intersection analysis.It was found that Dex regulated SAP-associated 473 DEGs.The GO analysis showed that Dex-regulated SAP-associated DEGs were mainly enriched in biological and cellular functions such as neutrophil chemotaxis,apoptosis,chemokine activation,among others.KEGG enrichment analysis revealed that the DEGs were significantly enriched in neutrophil extracellular trap(NET)formation,TLR,and NF-κB signaling pathway.Subsequently,the STRING database and Cytospace software were used to construct a PPI network of473 DGEs.In addition,gene clustering analysis was performed for the DEGs using the MOCDE plugin and three gene modules with K-score greater than 3 were performed,with scores of 9.185,7.4,and 4.96.GSEA analysis showed that DEGs that were targeted by Dex were mainly enriched in multiple pathways,including NET formation,TLR,NF-κB,JAK-STAT,and PI3K-Akt signaling pathway,and MAPK signaling pathway.To further confirm these results,we screened for the most critical overlapping DEGs between the Dex and SAP groups and validated their expression in pancreatic tissues using qRT-PCR.The results obtained were consistent with RNA sequencing findings.In the SAP group,the expression of AQP9,ATF3,CXCL1,CXCR2,S100A8,S100A9,and TREM1 was increased,whereas Dex treatment decreased their expression.In contrast to the SAP group,CLDN5 expression was increased in the Dex group.qRT-PCR and western blot analysis showed that the relative expression of TLR4 and p-NF-κB was down-regulated in pancreatic tissues of rats in the Dex group.Part Ⅲ: Pathological findings of rat lung tissue indicated that Dex treatment reduced lung injury,and ELISA results revealed that Dex suppressed lung inflammation by based on the analysis of alveolar lavage fluid and inflammation-related m RNA in lung tissue.The levels of SFTPC and AQP5 in lung tissues of rats in the SAP group were significantly lower compared with of the sham-operated group,whereas Dex treatment upregulated the levels of both proteins.These results suggest that Dex can promote the proliferation of alveolar epithelial cells in APALI.The expression of pro-apoptotic protein Bax was down-regulated and that of anti-apoptotic protein Bcl-2 was up-regulated after Dex treatment,implying that Dex can inhibit apoptosis of lung tissue in APALI.Immunofluorescence double staining of labeled alveolar type II epithelial cells and type I alveolar epithelial cells,showed that the number of both types of alveolar epithelial cells was higher in the Dex group than in the SAP group,and some type II alveolar epithelial cells were converted to type I.Conclusion:1.Dex attenuated SAP-induced inflammatory response and pancreatic injury in vitro and in vivo.2.Dex influenced SAP-induced 473 DEGs,including AQP9,ATF3,CXCL1,CXCR2,CLDN5,S100A8,S100A9,and TREM1,as determined by RNA sequencing.The relative expression of TLR4 and p-NF-κB was down-regulated in pancreatic tissues of rats in the Dex group.3.Dex inhibited APALI in rats by inhibiting apoptosis and promoting the repairment of alveolar epithelial cells. |
