The Role Of CD4~+CD25~+ Regulatory T Cells In Peripheral Blood And Intestinal Endotoxin During Liver Injuries In Rats With Severe Acute Pancreatitis And Its Impact Of Forsythia Suspensa

Severe acute pancreatitis(SAP) is a serious life-threatening systemic disease, by whichsystemic inflammation response syndrome (SIRS) and multiple organ dysfunction syndrome(MODS) easily induced. The liver is the first station of the pancreas’blood backflow, which isone of the main organ SAP involved .Except uncontrolled inflammatory response, immunedysfunction may also lead to the SAP hepatic injury in one of the main reasons, while liverinjury may also have an important impact on the progress of SAP’s condition. In the early of SAPliver injury the damage of mechanical barrier and immune gut barrier function results theintestinal bacteria and bacterial endotoxin (ET) shifting, then arouse congenital inflammatoryreaction and immune function disorder is one of the important mechanism of SIRS or evenMODS. CD4~+CD25~+regulatory T cells (Treg) is discovered in recent years has a unique negativerole in immune regulation of T cell subsets, the constitutively high expression of the IL-2receptor alpha chain (CD25), is considered to play a crucial role in the suppression ofautoimmune disease progression, control of potentially harmful inflammatory responses andmaintain immune homeostasis. Transcription factors Foxp3 (Fork head/winged helixtranscription factor) is one of the symbols of the Treg characteristic. In the infected state, theprimary function of Treg is to adjust the balance between the T cell which control pathogenseffectively and the overexpression of T cells response which lead to severe inflammation andtissue destruction Recent studies indicate that traditional Chinese medicine forsythia (Forsythiasuspensa (Thunb.) vah1) have the effect of anti endotoxin and immune regulating, but itsmechanism is not very clear, immune balance effects of SAP liver injury has not been reported.This study proposed to analysis ET and Treg changes in the peripheral blood in the early stage ofSAP liver injury and the relationship between them through rats in vivo, and explore the effect offorsythia to ET and Treg, so as to lay the foundation to clarify the role of ET and Treg in immuneimbalance of early SAP liver injury, and provide a certain experiment basis and theoretical basisto the effective treatment of SAP liver damage.ChapterⅠThe role of CD4~+CD25~+regulatory T cells in peripheral blood and intestinalendotoxin during liver injuries in rats with severe acute pancreatitisObjective To observe the CD4~+CD25~+regulatory T cells(Treg) in peripheral blood and intestinal endotoxin(ET) in early liver injury of rat severe acute pancreatitis(SAP) at differenttime points, and explore its role and mechanism of liver injury in SAP.Methods Sixty Sprague-Dawley(SD)rats were randomly allocated into sham operationgroup(SO group)(n=30) and severe acute pancreatitis group(SAP group)(n=30), The rats in SOgroup just turning the pancreas after laparotomy, and then closed the abdomen, The rats in SAPgroup received the injection of Sodium Taurocholate(NaTc) into their far-end of bile-pancreaticduct(0.1ml/100g,0.1ml/min)Each group sacrificed at 3、6、12h under the condition of anesthesia,asepsis, and then get blood from abdominal aortic, keep the pancreas, liver specimens to bemeasured serum amylase(AMY) and alanine aminotransferase(ALT) determined byfull-automatic biochemistry instrument, Radioimmunoassay of plasma tumor necrosisfactor(TNF-α), limulus reagent method is used to determine ET content in plasma, the proportionof Treg among peripheral lymphocytes was determined by Flow Cytometry(FCM), HE stain isused to observe pathological changes in liver and pancreas, the protein of Foxp3 activity wasevaluated by immunohistochemistry staining, and the relationships between these indicatorswere analyzed using Pearson correlation analysis test. Measurement data the single factorvariance analysis, P<0.05 for the difference is statistically significant, statistic software isSPSS16.0.Results SAP rats 3h ET content in peripheral blood and Treg percentage began to increase(0.74±0.11,2.26±0.32), increased gradually with the modeling compared with the SO group(0.38±0.08,1.86±0.06) and at 12h to achieve peak(0.85±0.11,2.80±0.35;P<0.01); The expressionof Foxp3 protein on the livers synchronization with Treg. The pathological score of pancreas andliver at each time point were significantly higher than the SO group(P<0.01); AMY、ALT andTNF-αconcentration increased gradually(P<0.01). Pearson correlation analysis teat showed thatquantities of Treg were positively proportional to the levels of ET and liver pathology score afterliver injury of SAP(p<0.01).ChapterⅡThe effects of Forsythia suspense on the intestinal endotoxin andCD4~+CD25~+Treg during liver injuries in rats with severe acute pancreatitisObjective To observe the effects of different doses aqueous of Forsythia suspense(AFS)on thelevels of ET、Treg in peripheral blood of rats 12h after liver injuries in rats with severe acutepancreatitis, to explore the anti-endotoxin and immunity modulation function of AFS and itspossible mechanisms.Methods Sixty SD rats were randomly allocated into A group(SO group), B group(SAP group),C、D and E respectively forsythia low、medium and high dose group(AFS1,AFS2,AFS3 group),F group as the positive control of polymyxin B group(PloyB group)(n=10). The rats of AFS1 group、AFS2group and AFS3 group of them were given AFS 1.25g/kg、2.5g/kg、5g/kg once a dayby Po.F group (PloyB 3500U/ml 1.5ml/100g) once a day by Intraperitoneal injection, pathwayfor seven days before SAP, respectively. Each modeling method with the chapterⅠ,Each groupsacrificed at 12h under anesthesia,asepsis,abdominal aortic blood, keep the liver specimens usedto observe pathological changes by HE stain, alanine aminotransferase(ALT) determined byfull-automatic biochemistry instrument, limulus reagent method is used to determine ET contentin plasma, the proportion of Treg among peripheral lymphocytes on A、B、C、D and E wasdetermined by Flow Cytometry(FCM). Measurement data the single factor variance analysis,P<0.05 for the difference is statistically significant, statistic software SPSS16.0.Results SAP rats 12h ALT、ET content in peripheral blood level and Treg percentagesignificantly increased, the pathology score of liver increased significantly compared with SOgroup, AFS1、AFS2 and AFS3 group the index was significantly lower and show dose dependentrelationship, AFS3 group and PloyB group ET level no significant difference.Conclusions1 Application of 5% sodium taurocholate retrograde pancreatic duct injection preparation SAPliver injury model, the pathology detection of pancreatic edema, hemorrhage,necrosis and livercongestion,edema,inflammatory cell infiltration,model stability,good repeatability.2 Pancreas and liver damage occurs pathological changes and liver dysfunction, and with theprolonged time damage are aggravated. SAP can be complicated with liver pathological injuryand dysfunction.3 With the progress of SAP, plasma ET content and Treg percentage elevated.liver dysfunctionand pathological damage increased, indicating that ET and Treg play an important role in theSAP liver injury, the inhibition of Treg on the immune system may be one of the mechanisms.4 Traditional Chinese medicine forsythia preconditioning can decrease levels of ET、Treg inperipheral blood of SAP and reduce the liver pathological damage. Forsythia suspensa hascertain treatment function by anti endotoxin and immune regulation effects on SAP rat liverinjury.This experimental study the role of CD4~+CD25~+Treg and ET in SAP liver injury in rats, andfor the application of Forsythia suspensa in treatment for SAP complicated with liver injuryprevention to provide certain animal experimental and theoretical basis.