| Background and Purpose:Acute pancreatitis(AP) is a clinical acute abdominal emergency,which is mainly caused by the reversible inflammatory process caused by the excessive activation of exocrine pancreatic digestive enzymes.The incidence of AP is about 4.9 to 40 cases per 100,000 people,and the mortality rate is 1%.Most AP patients experience a mild disease process,but 10%-20%of patients develop a more severe AP(Severe acute pancreatitis,SAP),causing multiple organ failure and even death.Acute lung injury(ALI)is the most common cause of death in patients with severe AP(SAP),accounting for approximately 10%-25%of SAP cases and approximately 60%of SAP-related deaths.So far,there are many difficulties in the clinical treatment of S AP-related ALI.Therefore,the research and development of relevant specific drugs has become a current research hotspot.The screening of drugs with clear therapeutic effects,fewer adverse reactions and moderate treatment costs,and research on related drugs and their mechanism of action has become an important part of the current scientific research.High mobility group box 1(HMGB1) is a highly conserved DNA-binding nuclear protein,which plays an important role in the pathogenesis of inflammatory diseases.HMGB1 has a close relationship in the pathogenesis of SAP.The secreted HMGB1 released from necrotic acinar cells has been shown to aggravate the pancreatic inflammation process.First of all,HMGB1 has cytokine-like properties,which can induce a cascade of local and systemic inflammation,which ultimately leads to multiple organ dysfunction.Second,HGMB1 can bind to a variety of cell surface receptors,including Toll-like receptor(TLR)2,TLR4 or TLR9 and advanced glycation end products(RAGE),which activates the nuclear factor-κB(NF-kB)signaling pathway.Increase the gene expression of pro-inflammatory cytokines and chemokines,thereby further aggravating the inflammatory response.Finally,HMGB1 has also been shown to act as a chemokine,attracting neutrophils to infiltrate the site of inflammation,and prevent neutrophil apoptosis and aggravate tissue damage.Therefore,HMGB1 is a potential therapeutic target of SAP-ALI,and inhibiting the expression of HMGB 1 is of great significance for the treatment of SAP-ALI.Traditional Chinese medicine has been used to treat AP,and the adjuvant treatment of AP with traditional Chinese medicine has a positive effect on multiple organ failure,mortality and systemic infections.Flavonoids are a kind of small molecule natural plant components that exist in plants,and are a kind of secondary metabolites produced by plants in the long-term natural evolution and selection process.Flavonoids are diverse in nature,have complex structures,and have various physiological,pharmacological and biochemical effects.Calycosin(Cal)is a kind of plant isoflavone,which has been shown to have various biological effects,including powerful anti-inflammatory properties,as well as anti-tumor,neuroprotection,anti-Parkinson’s disease,anti-Alzheimer’s disease,Anti-diabetic and pro-angiogenic activity.However,the potential use of Cal in the treatment of SAP-related ALI is unclear.Therefore,the purpose of this study is to investigate the effects and related mechanisms of Cal administration on L-arginine(L-arginine,L-arg)-induced SAP-ALI in vivo model and LPS-induced ALI in vitro.The treatment provides new methods,while providing a new paradigm for the application of Chinese medicine.Method:1.Mice were randomly divided into four groups(n=6 in each group):control(saline),L-arg group(saline+4 g/kg),L-arg+low-dose Cal group(L,25 mg/kg)And L-arg+high-dose Cal group(H,50 mg/kg).H&E histological staining to detect the pathological damage of the pancreatic tissue and lung tissue of each group of experimental mice,and the pathology of the pancreatic tissue of each group of mice through necrosis,vacuolization and inflammatory cell infiltration of pancreatic injury Pathologically score the damage.The pathological damage of the lung tissue of each group of mice was scored by the thickness of the alveolar wall of the lung tissue,the infiltration of inflammatory cells and the dry-wet weight ratio of the lung tissue,and statistical analysis was performed;2.Detect the amylase,HMGB1,chemokine CXCL-1 and inflammatory factors(TNF-α,IL-6 and IL-1β)in the serum of each group of experimental mice by enzyme-linked immunosorbent assay(ELISA);3.Detect the expression levels of HMGB1,CXCL-1,TNF-α,IL-6 and IL-1βmRNA in the lung tissues of each group of mice by RT-PCR,and perform statistical analysis;4.Analyze the expression of Ly6G in the lung tissue of each group of mice by immunohistochemical staining,and conduct semi-quantitative analysis and comparison.5.Detect the expression level of HMGB1 and p-NF-κB in the lung tissue of each group of mice by immunofluorescence staining method,detect the protein expression of HMGB1 and NF-KB pathway by western blot and perform statistical analysis;6.Different concentrations of Cal were incubated with A549 cells for 24 hours,and CCK-8 was used to detect cell viability;A549 cells(alveolar epithelial cells)were stimulated with LPS to simulate an in vitro ALI inflammation model,and A549 cells with different concentrations of Cal were pretreated 1 hour in advance,followed by LPS incubation Observe whether Cal at different concentrations can reverse the decline in A549 cell viability caused by LPS for 24 hours,and perform statistical analysis.7.Select the optimal Cal concentration for follow-up experiments,and detect the levels of HMGB1 and p-NF-κB in A549 cells in the Con group(control group),Cal drug control group,LPS group,and LPS+Cal group by immunofluorescence staining.The expression level,western blot was used to detect the protein expression of HMGB1 and NF-κB pathway and perform statistical analysis.8.The interaction between Cal and HMGB1 is predicted by molecular docking.Results:1.Cal reduces pathological damage of pancreas and lung tissues in mice.Compared with the control group,the pancreas and lung tissue of the mice in the L-arg group was significantly damaged,and the pathological scores were significantly increased;Compared with the L-arg group,pancreatic and lung tissue damage was significantly reduced in the Cal group,and the pathological scores were significantly reduced.The high-dose Cal treatment was more effective.2.Cal reduces serum amylase and inflammatory factor levels.Compared with the Con group,the serum levels of amylase,HMGB1,CXCL-1,TNF-α,IL-6 and IL-1β were significantly increased in the L-arg group;While Cal treatment group reversed the demage induced by L-arg especially with a higher dose Cal.3.Cal reduces mRNA expression of inflammatory factors in lung tissue.Compared with the Con group,the mRNA expression levels of HMGB1,CXCL-1,TNF-α,IL-6-and IL-1β in the lung tissues of the L-arg group mice were significantly increased;Compared with the L-arg group,the expression levels of HMGB 1,CXCL-1,TNF-α,IL-6 and IL-1β mRNA in the lung tissue of the Cal treatment group were significantly reduced especially with a higher dose Cal.4.Cal reduces neutrophil infiltration in lung tissue.Immunohistochemical staining analysis showed that compared with the Con group,the expression of Ly6G in the lung tissue of the L-arg group was significantly reduced,and the difference was statistically significant;Compared with the L-arg group,the lung tissue of the Cal treatment group was significantly reduced,and the high-dose reduction was more obvious;5.Cal reduces the expression of HMGB1 and p-NF-κBp65 in lung tissue.Immunofluorescence staining analysis and western blot showed that compared with the Con group,the expression of HMGB1 and p-NF-κBp65 in the lung tissue of the L-arg group was greatly reduced;compared with the L-arg group,the expression of HMGB1 and p-NF-κBp65 in the lung tissue of the Cal treatment group The expression of p-NF-κBp65 was significantly reduced,and the high-dose reduction was more obvious.6.The protective effect of Cal on ALI model in vitro.Different concentrations of Cal had no significant effect on the viability of A549 cells;Cal and treatment reversed the decline in cell viability induced by LPS;Immunofluorescence staining analysis and western blot showed that compared with the Con group,the Cal drug control group A549 cells.No significant difference was seen in the expression of HMGB1 and p-NF-κBp65;The expression of HMGB1 and p-NF-κBp65 in A549 cells of the LPS group increased;The expression of HMGB1 and p-NF-κBp65 in A549 cells of the LPS+Cal treatment group was higher than that of the LPS group obviously decrease.7.Molecular docking analysis provides evidence for the direct interaction between Cal and HGMB1.Conclusion:1.In the in vivo SAP model,Cal treatment reduced the pathological damage of pancreatic tissue and lung tissue,reduced the expression level of serum inflammatory factors in mouse SAP,and down-regulated the expression of inflammatory factor mRNA in lung tissue;Cal treatment reduced lung tissue The infiltration of neutrophils(Ly6G)in the tissues inhibits the expression of HMGB1 and p-NF-κBp65 in lung tissues,thereby improving the pathological development of SAP-ALI.2.In an in vitro ALI inflammation model,Cal treatment reversed the upregulation of HMGB1 and p-NF-κBp65 expression in A549 alveolar epithelial cells induced by LPS;3.The simulated molecular docking shows that Cal can directly interact with HMGB1,providing a theoretical basis for Cal inhibiting the expression of HMGB1 and protecting SAP-ALI;suggesting that Cal has great potential for the treatment of SAP-ALI. |
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