| Background:Acute pancreatitis is an inflammatory disease caused by abnormal activation of trypsin and subsequent digestion of pancreatic tissue,which was manifested as acute epigastric pain,nausea and vomiting with a sudden onset,rapid progress,serious complications,and high mortality.At present,the treatment of acute pancreatitis is mainly focused on jejunitas,pain relief,fluid resuscitation,nutritional support and surgical treatment to improve symptoms.However,there is no specific drug available yet.Moreover,acute pancreatitis could be classified into the four categories of “abdominal pain”,“hypochontic pain”,and “Yi Dan” in Chinese medicine according to its characteristics.The basic pathogenesis of it is “Yangming Rejie”and “Fuqi Butong”.Qingyi Granule is an in-hospital preparation of the First Affiliated Hospital of Dalian Medical University,which is made based on the classic prescription Dachengqi Decoction.It’s good at purging heat,soothing the liver and regulating qi,and have remarkable clinical effects on acute pancreatitis.However,the effective ingredients and molecular mechanisms of Qingyi Granule in the treatment of acute pancreatitis have not been elucidated.Aim:1.To map the chemical profile of Qingyi Granule by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLCQTOFMS).2.The core components and potential mechanisms of Qingyi Granule in the treatment of acute pancreatitis was screened and evaluated based on network pharmacology combined with molecular docking technology.3.The protective effect of Qingyi Granule on acute pancreatitis was evaluated through rat models,and the predicted results of network pharmacology and molecular docking screening was validated by molecular biology.Materials and methods:1.The sample of Qingyi Granule was prepared by boiling method.The chemical profile of Qingyi Granule was mapped by UPLC-QTOFMS.Identification and elucidation of chemical composition in Qingyi Granule was obtained based on the accurate information of quasi-molecular and fragment ions provided by high-resolution mass spectrum and comparison of retention time and chromatographic behavior with the reference substances.2.The active components of Qingyi Granule identified by UHPLC-QTOFMS were screened by Molispiration Smiles database.The targets of active components were predicted from The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform and Swiss Target Prediction database.Related targets of acute pancreatitis were obtained from OMIM,Gene Cards,Dis Ge NET and GAD databases.The Cytoscape software was used to obtain candidate therapeutic targets of Qingyi Granule for acute pancreatitis.The core therapeutic targets of Qingyi Granule on acute pancreatitis were further screened by protein-protein interaction(PPI)analysis and topological analysis.The KEGG pathway enrichment analysis was used to predict the action mechanism of core targets.Molecular docking technique was employed to evaluate the binding between active components and core targets.3.The rat model of acute pancreatitis was established by retrograde injection of sodium taurocholate into the biliopancreatic duct.40 SD rats were randomly divided into four groups(n=10): control group(only slightly turning the small intestine),acute pancreatitis group(model was established by retrograde injection of 3.5% sodium taurocholate into the biliopancreatic duct),Qingyi Granule group(treated with Qingyi Granule after model construction)and dexamethasone group(dexamethasone treatment after model construction).After the establishment of the model,the success of the model was verified based on the pathological changes of pancreatic tissue and the index of serum amylase in rats.Expression of protein that predicted by network pharmacology and molecular docking were extracted from pancreatic tissue and evaluated by Western blottingResults:1.110 chemical constituents of Qingyi Granule were identified by UPLC-QTOFMS,including 28 flavonoids,16 organic acids,15 alkaloids,15 monoterpenes,11 iridoid terpenoids,8 triterpenes,5 phenylethanol glycosides,5 anthraquinones and 7 others.2.Through the method of network pharmacology,47 active components of Qingyi Granule(including 423 drug targets)and 6593 acute pancreatitis-related targets were obtained.Based on PPI,topological analysis and KEGG enrichment analysis,11 core components,47 core therapeutic targets and 15 related signal pathways were screened.The results of molecular docking showed that ERK1/2,c-Fos and p65 had stronger binding to the core components of Qingyi Granule.3.The results of pancreatic histopathology showed that there were obvious pathological changes such as edema,vacuolation,hemorrhage and necrosis in the acute pancreatitis group compared with the control group(p < 0.01).Qingyi Granule treatment could significantly alleviate the above pathological changes,the difference was statistically significant(p < 0.01).The results of biochemical indexes showed that the level of serum amylase in the acute pancreatitis group was significantly higher than that in the control group,treatment with Qingyi Granule and dexamethasone decreased the serum amylase level significantly(p < 0.01).The results of Western blotting assay showed that the expressions of p-ERK1/2 and c-fos were significantly decreased and the expression of p-p65 was significantly increased in acute pancreatitis group compared with the control group.However,the expression of p-ERK1/2 and c-fos in Qingyi Granule group was up-regulated,and the expression of p-p65 was down-regulated(p < 0.01).Conclusions:1.The chemical constituents of Qingyi Granule could be identified by UPLC-QTOF-MS,which revealed its material basis.2.The method of network pharmacology could explain the active components and integral mechanism of Qingyi Granule in the treatment of acute pancreatitis from the point of view of "multi component-multi target-multi pathway".3.Qingyi Granule could reduce the pathological injury of pancreas caused by acute pancreatitis and reduce the high level of serum amylase caused by acute pancreatitis.The protective mechanism may be related to apoptosis and inflammation mediated by pERK1/2,c-Fos and p-p65. |
