| Acute pancreatitis(AP)is a common abdominal disorder clinically.And 15-20 % of AP patients may develop into severe acute pancreatitis(SAP)eventually as the disease worsens.High mortality of SAP is a major problem in clinic because of its rapid progression and a variety of complications.Either the pathogenesis or treatment of SAP is still controversial until now.In the early stage of SAP,lungs are the first extra-pancreatic organs to be damaged,which could result in acute lung injury(ALI),an important complication of SAP.Without treatment timely,ALI is likely to develop into acute respiratory distress syndrome(ARDS),which is a major cause of death in the early stage of SAP.Pre-B cell colony-enhancing factor(PBEF),a peptide hormone,is closely related to the occurrence of lung injury.PBEF regulates cellular proliferation,differentiation and apoptosis of B cells.It has been found that PBEF inhibited polymorphonuclear leukocytes(PMN)apoptosis in some study,which could lead to an increased release of inflammatory cytokines and further aggravation of inflammatory response.A study of acute pancreatitis found that the expression of PBEF in patients’ blood was positively correlated with the progression of disease.In many years of clinical studies,Qingyi Decoction can be effective treatment of acute pancreatitis.It is mainly composed of rhubarb and mirabilite.Rhubarb is atraditional Chinese herbal medicine,in which the main active ingredient,emodin has been proved to have a variety of pharmacological functions,applied in the treatment of diseases.In recent years,the therapeutic effect of emodin in the development of SAP has also attracts more and more scientists.It has been found that emodin could reduce the inflammatory response and improve the organ damage induced by SAP.There is a lot of evidence that emodin plays an important role in preventing the further development of SAP.Although the mechanism of emodin in SAP has a variety of interpretation,the role of emodin in SAP still exist many controversy.Therefore,the present study will mainly try to explore whether Qingyi Decoction and its mainly active ingredient,emodin,have a treatment effect on SAP via influencing the release of PBEF and apoptosis of PMN or not.So we explored it here.It is of important significance for the exploration of new therapeutic targets.In this study,we established a rat model of SAP-ALI and all rats were divided into control group,SAP group and drug treatment group,including Qingyi Decoction,FK866 and DEX.We detected serum amylase activity of rats and observed histopathological features,apoptosis of lung and pancreas tissues by HE-staining and TUNEL-staining.Wet/dry weight(W/D)ratio was used to assess the degree of tissue edema.Then the relative expression of PBEF in lung and pancreas was tested by immunohistochemical staining.Meanwhile,PBEF in PMN separated from peripheral blood was tested by Real-time PCR and Western blot,which will make it clear whether Qingyi Decoction could inhibit expression of PBEF.Apoptosis of PMN was detected by flow cytometry analysis and expression of apoptosis-related protein was tested by Western blot to know the influence of Qingyi Decoction on apoptosis of PMN.For further validation the hypothesis,we separated PMN from normal rats’ peripheral blood and simulated inflammation using LPS in vitro.The apoptosis of PMN and expression of protein related apoptosis were also detected after drug treatment just as before.Combined experiments in vivo and in vitro,the influence of Qingyi Decoction and emodin on expression of PBEF and PMN apoptosis was analyzed and we hoped to illustrate a mechanism of emodin in SAP.It not only provided theoretical basis on futuretreatment of SAP,but also had important significance for inflammatory response in other diseases.PART 1: The effect of Qingyi Decoction on SAP-ALI in rats Objective: To investigate the effect of Qingyi Decoction on tissue injury,edema and apoptosis induced by SAP in lung and pancreas.Methods: The SAP model was induced by retrograde injection of sodium taurocholate solution into biliopancreatic duct.Serum amylase activity was analyzed by Iodine-starch spectrophotometer.Pathologic changes of lung and pancreas were observed by HE staining.TUNEL staining was used to detect the apoptosis of lung and pancreatic tissue.Wet/dry weight(W/D)ratios represented the degree of tissue edema.Results: Amylase expression activity in SAP rats increased but Qingyi Decoction could inhibit this effect.HE staining showed that Qingyi Decoction could alleviate the degree of bleeding,inflammatory cell infiltration in lung and pancreas induced by SAP.Edema was also relieved.The results of TUNEL staining showed that treatment with Qingyi Decoction decreased apoptotic cells in the lung and pancreatic tissuses compared with the SAP group.Conclusion: 1 Qingyi Decoction can reduce the enhanced activity of serum amylase in the SAP rats.2 Qingyi Decoction significantly ameliorated histopathological changes,tissue edema induced by SAP-associated ALI and decreased apoptotic cells in the lung and pancreatic tissues.PART 2: The mechanism of Qingyi Decoction on SAP in rats Objective: To explore the effect of Qingyi Decoction on expressin of PBEF and apoptosis of PMN;To illustrate the molecular mechanisms of Qingyi Decoction in the treatment of SAP.Methods: The expression of PBEF in lung and pancreas tissue is detected by immunohistochemical staining.The mRNA levels of PBEF in PMN,isolated from peripheral blood,were analyzed by Real-time PCR.Then Western blot were performed with anti-PBEF antibody.The apoptosis of PMN was examined with flow cytometry using Annexin V/PI staining.In addition,another Western blot was performed to detectthe expression of proteins related apoptosis,including Fas,FasL,Bax,cleaved caspase-3,Bcl-xL.Results: Immunohistochemical results showed that the expression of PBEF in lung and pancreas was increased in the rats of SAP group compared with that of the control group.However,after treatment with Qingyi Decoction,it decreased.The results of Real-time PCR and Western blot were consistent with that of immunohistochemical,which indicated Qingyi Decoction could reduce PBEF expression.SAP rats showed lower apoptosis rate of PMNs than the control rats,but Qingyi Decoction significantly increased it.Bax,cleaved caspase-3,Fas and FasL expression levels in the SAP group were significantly decreased compared with the control group,whereas Bcl-xL expression was increased in the SAP group.After the SAP rats were treated with Qingyi Decoction,the expression levels of Bax,cleaved caspase-3,Fas and FasL were notably increased and Bcl-x L expression was decreased compared with the un-treated SAP rats.Conclusion: 1 Qingyi Decoction can inhibit the expression levels of PBEF in SAP rats.2 Qingyi Decoction can promote the apoptosis of PMN and affect the expression of apoptosis related proteins in SAP rats.3 Qingyi Decoction may promote the apoptosis of PMN by inhibiting the expression of PBEF,and play a therapeutic role in the treatment of SAP.PART 3: The effect of emodin on the apoptosis of PMN in vitro Objective: To further validate the effect of emodin on the apoptosis of PMN in vitro and explore the mechanism of it.Methods: PMN,isolated from peripheral blood in normal rats,was treated with LPS to simulate the inflammatory environment in vitro.Then corresponding drug was given as before.The apoptosis of PMN was examined with flow cytometry using Annexin V-FITC/PI staining and the expression of Fas,FasL,Bax,cleaved caspase-3,Bcl-x L was detected by Western blot.Results: Apoptosis detection showed that LPS could significantly inhibit the apoptosis of PMN and emodin could antagonize the effect.The results of Western blot were consistent with that in vivo.Emodin could lead to up-regulation of Fas,FasL,Bax,cleaved Caspase-3 and down-regulation of Bcl-x L.Conclusion: 1 Emodin can promote the apoptosis of PMN.2 Emodin may induce apoptosis of PMN both through mitochondria-dependent pathway and death receptor pathway.3 Emodin has the potential to treat other inflammatory diseases by inducing apoptosis of PMN. |
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