An Experimental Study On The Effect Of Sinomenine On The Acute Rejection Reaction After Liver Transplantation And Evaluating The Hepatic Pathological Lesion By CDFI And IBS In Rats

Liver transplantation(LT) has become the major treatment for the end-stage chronic liver disease and severe acute failure.Development in LT has depended on researching in small and large animals, the orthotopic liver transplantation (OLT)has become the first choice in lots of experimental an- imal models for low price, feeding and management easier,simple technique and clear genetic background in rats.The OLT model is an ideal animal model for researching preserving transplanted organ,ischemia- reperfusion injury,hemodynamics,immunosuppressant,graft rejective reaction and imm- uneologic tolerance. After more than 30 years improving by the Chinese and foreign scholars, the two-cuff technique has become the most classic tech- nique. Because of absence other varieties(as Lewis or BN ) and low price, SD and Wistar rats are generally selected for transplantation in our coun- try.This SD-Wistar OLT model is considered a high rejection reaction.Although new immunodepressants constantly emerged, the rejection reaction was still inevitable after transplantation.Inducing immunotolerance of transplanted antigen might be an essential method to solve this problem. In addition, it was deficiency of drug treatment and treatment plans of immune tolenranc in clinical.Therefore, it was becoming the hot issue in clinical transplantation field to look for effective, low-toxicity and cheap immunodepressants.Recently some Chinese herbal medicines has been fou- nd to have the function of immunodepression.The medicinal plant sinom- enium acutum Rehd. Et Wils.has been used in china for more than 2000 years,for the treatment of various diseases.The main active constituent has been identified as the alkaloid sinomenine (7,8-didehydro-4-hydro-xy- 3,7-dimethoxy-17-methyl-9α13α,14α-morphinan-6-one).Sinomenine (SIN) was in clinical used in China and Japan due to analgetic and anti-inflannatory activities.In the pathology diagnostics field of liver transplantation, the color dopplor flow image(CDFI) has been the most used technique of imaging diagnosis.Because two-dimension is lack of objectivity, the diagnostic value of CDFI become weaken.The integrated backscatter(IBS) is a novel technique of tissue charac- terization, and related to pathological change.IBS is expected to be another important technology.In this study, we investigated the functional mechanisms of SIN against acute rejection reaction in the model of OLT, and observed the effect of SIN combining with sub-dose cyclosporine A(CsA), at the same time detecting the acoustic characteristics of liver by color Doppler flow image and IBS.In order to provide experimental evidence for clinical drug administration and application of ultrasonic technology.PartⅠEstablished and modified orthotopic liver transplantation in ratsObjective:To investigate modified technique and observed survival rate of orthotopic liver transplantation(OLT) in rats.Method:Classic operative technique was used in establishing of 70 OLT models in SD-Wistar rats, then established of 35 cases with modified operative technique.Compared the anhepatic phase, anastomosis time of suprahepatic cena cave and one-week survival rate.Observed the survival rate of classic opration group and the change of liver parenchyma.Result: The time of anhepatic phase and anastomosising the supra- hepatic inferior vene cavum (SVC) was 20±1.6min and 10.7±1.3min for using classic technique,26.7±4.1min and 15.1±1.7min for using modified technique.One-week survival rate after one week is 17.1% and 91.4% respecttively .There were 12 survivals in classic operation group,and the survival time was 11-22days(mean14.8±2.8days).After the death of those rats, liver pathology coincided toⅢof Williams system.Conclusion:The OLT model of SD-Wistar consistented with severe rejection reaction.It could improve the stability of OLT by ameliorating the way of retouching SVC and cuff technique.The key point of long-term surviving was favourable anastomoses of the SVC and PV, and the anastomoses quality was more important than anhepatic phase.PartⅡAn experimental study on the effect of sinomenine on the acute rejection reaction after liver transplantation in rats.Objective:To compare the effect of CsA,SIN and their combination on the rejection reaction after the orthotopic liver transplantation in rats.Method: we established the stable orthotopic liver transplantation model.The rat models were randomly divided into four groups,and every group was eight SD-Wistar rats:control group,CsA treated group(30㎎/㎏/d),SIN treated group(40㎎ /㎏ /d),SIN and half-value dose CsA treated group(SIN 40㎎/㎏/d+CsA 15㎎/㎏/d).The blank group was eight Wistar rats.Detected the hepatic function and renal function,Enzyme-linked immu- nosorbent assays was carried out to detect the level of IL-2,IL-10,sICAM-1.The expression of TGF-β1 protein in liver and TGF-β1mRNA in kidney used immunohistochemisty staining and RT-PCR,respectivly.The lymphocyte apoptotic index in header district of liver was detected using the TUNEL.The liver pathological lesion was scored with Williams system after HE staining.Result:⑴The damage of liver function in the control group was the most serious.The level of ALT,AST,TBIL,DBIL in this group were higher than other groups,but the level of BUN CRE in every group had no signif- icant difference.⑵The level of IL-2 of CsA-treated group,SIN-treated gro- up,SIN+CsA-treated group was lower than control group (P<0.05).⑶The SIN-treated group and SIN+CsA-treated group could stimulate the secretion of IL-10, but the CsA had no effect.⑷The level of sICAM-1 in the control group was the highest, and accompanied by less injury, sICAM-1 decreased gradually.⑸The expression of TGF-β1 protein in liver and TGF-β1mRNA in kidney had significant difference in five groups,and the expression of CsA-treated group was the highest.⑹The lymphocyte apoptotic index in header district of SIN+CsA-treated group was the most obvious than other groups(P<0.05).⑺The correlation was positive bet- ween the level sICAM-1 and liver pathological damage(r=0.875,P<0.01).Conclusion:⑴SIN could inhibit the Pro-inflammatory factor IL-2 ICAM-1,and stimulate the inhibition of inflammatory factor.SIN could restrain the inflammatory reaction.⑵SIN and CsA combination could enhance the lymphocyte apoptosis in header district, induced the immune- ological tolerance.⑶SIN and CsA combination could reduce CsA-reduced renal injury, and protected the kidney.⑷The level of sICAM-1 was a relia- ble indicator to monitor the rejection reaction and evaluate the antirejection therapy.PartⅢEvaluatated the hepatic pathological lesion of OLT by CDFI and IBS in ratsObjectie:Evaluated the hepatic pathological lesion and pharmacal therapeutical effect by CDFI and IBS after OLT in ratsMethod:We established the stable orthotopic liver transplantation model. The rat models were randomly divided into four groups,and every group was eight SD-Wistar rats:control group,CsA treated group(30㎎/㎏ /d),SIN treated group(40㎎ /㎏ /d),SIN and half-value dose CsA treated group(SIN 40㎎/㎏/d+CsA 15㎎/㎏/d).The blank group was eight Wistar rats. Measured hepatic IBS and the blood flow velocity of portal vein and inferior vena cava in the fourth and tenth day after OLT in rats.These rats sacrificed,then cut part of liver for pathological examination.Result:⑴In the fourth day:The portal venous flow velocity of control group was lower than other operative groups.The PV-velocity of CsA- treat- ed group and SIN+CsA-treated group were significantly faster,and the PV-velocity of SIN was slightly faster also.In the tenth day:the PV-velocity of all of operative groups slowed down significantly(P<0.05),but the velocity of CsA-treated group and SIN+CsA-treated group were still higher than blank control.The SIN-treated group and the blank group had no difference.⑵In the fourth day: the parameter of IBS in the control and SIN groups were higher than the other groups.And the CsA-treated and SIN+ CsA-treated groups were higher than blank group(P<0.05).In the tenth day:the parameter of IBS were lower than a week ago(P<0.05),but the co- ntrol group had no change(P>0.05).⑶Correlation analysis:The correlation was positive between IBS and liver pathological damage(r=0.814, P<0.01),but between liver pathological damage and portal vena velocity were negative correlation(r=-0.776,P<0.01).Conclusion:⑴Lower velocity of the portal venous could prompt the existence of acute rejection of OLT.⑵Detecting hepatic AII could diagnosis the level of liver damage after OLT.