Rapamycin Inhibits Adaptive Bile Duct Proliferation In Response To Ischemia

As technical improvement in organ transplantation field,more and more researches concentrated on postoperative long-term survival and better quality of life.Side-effects of immunosuppressant,which is the most important element in post-operative therapy,may harm postoperative survival and life quality.In recent years,numerous researches have been carried out to explore new-type immunosuppressant,new anti-rejection strategies and optimal dosage adjusting according to blood concentration of drug.Calcineurin inhibitor (CNIs),fundamental agent in a varity of anti-rejection strategies,increased incidence of renal dysfunction and neural disorder postoperatively.In 1999,rapamycin was permitted into immunosuppressant therapy after renal transplantation.As a mTOR specific inhibitor, rapamycin binds to its intracellular receptor FKBP12 to form FKBP—rapamycin compounds,which conjugate to FRB domain on mTOR moleculi,inactivate mTOR,block downstream signal activation of p70S6k and 4EBP1,hamper transcription of key mRNA answered for transformation from G₁ to S phase,and finally arrest cells in G₁ phase and inhibit cell proliferation.Since its initiation of clinical use,rapamycin caused less burden to renal function in contrast to CNIs.In combination with CNIs,or even in replacement of CNIs,rapamycin gradually occupied the fundamental place in postoperative anti-rejection. Recently,it has been reported that rapamycin had anti-tumor activity and lowered malignancy incidence after transplantation.What's more,rapamycin prevented coronary arteries from re-stenosis after coronary arterioplasty.In a animal model,rapamycin reduced extracellular matrix deposit,improved liver function and increased survival.Some clinical data indicated that rapamycin contributed to treatment of proteinuric glomerulopathy,polycystic renal pathy and refractory uveitis.However,as increasing clinical use of rapamycin,its side-effects attracted more and more concern.Besides arthralgia,extremity edema,insufficient wound healing and hematologic changes,such as anemia,hypercholestemia,thrombocytopenia and leukocytopenia,rapamycin caused pulmonary toxity and angioedema according to updated data.Further more,some papers held that rapamycin harmed self-repair of marginal renal donor,inhibit activation of p70S6k and put obstacle to repair of injured renal tubuli.It seemed that anti-proliferative nature of rapamycin could cause negative effect on tissue repair.In field of liver surgery,operation,transplantation,organ reservation and intervention may cause injury to liver tissue.As a compensatory mechanism,hepatocytes and cholangiocytes proliferate.Whether rapamycin inhibits proliferation,hampers self-repair of liver tissue remains unclear.In the present,paper,effect of rapamycin on adaptive bile duct proliferation in response to ischemia will be explored.Partâ… Effect of Rapamycin on Adaptive Bile Duct Proliferation in Response to IschemiaAimObserve intra-hepatic adaptive bile duct proliferation after complete deprivation of bile duct arterial supply.Explore the effect of rapamycin on the proliferation of bile ducts.MethodMale SD rates were randomly assigned into 4 groups:ischemia(n=32), ischemia+rapamycin(n=32),sham(n=28) and sham+rapamycin(n=28).In ischemia groups,complete deprivation of bile duct arterial supply was performed while in sham groups,open-close operation was carried out.Daily intake of rapamycin(2mg/kg) was given in rapamycin groups and the same volume of saline was given in non-rapamycin groups.On day 1,3,7 and 14,fresh live tissue were obtained,followed by H-E and Ki-67 immunohistochemical staining.In high magnification fields,15～20 portal areas were examined per animal.In H-E sections,interlobular bile duct within portal areas and ductuli at periphery of portal areas were counted separately and average number of bile ducts per portal area were calculated.In Ki-67 sections,Ki-67 positive and negative bile duct cells were counted and ratio of Ki-67 positive to negative bile duct cells per portal area were calculated.Statistic analysis was performed by SPSS 13.0 software,results showed as X±S_X. Kruskal-Wallis H test was used for comparison of multiple independent samples and the following pair-wise comparisons were performed by rank transformation and ONE WAY ANOVA.Statistic difference was considered when P＜0.05.ResultsFor H-E sections,there were no obvious bile duct proliferation in sham and sham+rapa group,while bile duct proliferation were observed obviously in ischemia group, including proliferation of interlobular bile duct within portal areas and ductuli at periphery of portal areas.Compared with sham,a sharp increase of interlobular bile duct number was found within 2 weeks postoperatively.When rapamycin was administrated,the increase shrunk and obvious differences appeared on day 7 and 14 in contrast to ischemia group (P=-0.034 and 0.026,respectively).Compared with sham,number of peri-portal ductuli increased in ischemia group.When rapamycin was given,the increase also shrunk and resulted statistic differences on day1 and 7(P=0.042 and 0.009,respectively).Ki-67 stained sections showed there was no obvious positive staining within portal area in sham group,while a large number of Ki-67 positive bile duct cells were found in ischemia group. On postoperative day 3,ratio of Ki-67 positive to negative bile duct cells reached peak level,which descended by 37.74%(P=0.002) due to rapamycin administration.ConclusionRapamycin inhibits expression of Ki-67 antigen in bile duct epithelium,inhibits interlobular bile duct proliferation within portal areas and as for peri-portal ductuli, rapamycin may also put obstacle to their proliferation.Partâ…¡Effect of Rapamycin on Liver Function and SurvivalAimExplore the effect of rapamycin on liver function,body weight and survival of experimental rats.MethodMale SD rates were randomly assigned into 4 groups as previously described.In ischemia groups,complete deprivation of bile duct arterial supply was performed while in sham groups,open-close operation was carried out.Daily intake of rapamycin(2mg/kg) was given in rapamycin groups and the same volume of saline was given in non-rapamycin groups.Body weight was measured before operation,postoperative day 7 and postoperative day 14.On day 1,3,7 and 14,blood sample(2～3ml) were obtained, followed by liver function examination including TBIL,ALP,GGT and ALT.Statistic analysis was performed by SPSS 13.0 software,results showed as X±S_X. Kruskal-Wallis H test was used for comparion of multiple independent samples and the following pairwise comparisons were performed by rank transformation and ONE WAY ANOVA.Kaplan—Meier survival analysis was performed followed by log-rank test. Statistic difference was considered when P＜0.05.ResultsSerum TBIL in ischemia group ascended sharply postoperatively.When rapamycin administrated,TBIL of bile duct ischemic rats reached higher levels on day 7(P=0.005) and day 14(P=0.003) compared with ischemia group.Serum ALP of ischemia group increased from day 1 to day 7 postoperatively and on day 14,increasing tendency ceased. In ischemia+rapamycin group,serum ALP ascended continuously,without any remission even on day 14,when ALP was higher than ischemia group(P=0.012).Based on measurement of body weight,in ischemia group,experimental rats experienced weight loss after operation.While in ischemia+rapamycin group,animals suffered more weight loss than in ischemia group(P＜0.01).Kaplan—Meier survival analysis showed compared with sham,there was no statistical decrease of cumulative survival in ischemia group,while decrease in ischemia+rapamycin group was significant(P＜0.05).ConclusionAfter ischemia injury,liver experienced cholestasis.Rapamycin exaggerated intra-hepatic cholestasis,deteriorated liver function and affected negatively on postoperative nutrition and survivalPartâ…¢Effect of Rapamycin on VEGF mRNA,p70S6k and phosphorylated p70S6kAimExplore the effect of rapamycin on expression of VEGF mRNA and content of p70S6k and phosphorylated p70S6k in liver tissue of experimental rats.Method Male SD rates were randomly assigned into 4 groups as previously described.In ischemia groups,complete deprivation of bile duct arterial supply was performed,while in sham groups,open-close operation was carried out.Daily intake of rapamycin(2mg/kg) was given in rapamycin groups and the same volume of saline was given in non-rapamycin groups.On day 1,3,7 and 14,fresh liver tissue were obtained,followed by VEGF immunohistochemical staining,real-time PCR(dye method) and Western-blot to detect content of VEGF mRNA,p70S6k and phosphorylated p70S6k.Statistic analysis was performed by SPSS 13.0 software,results showed as X±S_X. Kruskal-Wallis H test was used for comparion of multiple independent samples and the following pairwise comparisons were performed by rank transformation and ONE WAY ANOVA.Statistic difference was considered when P＜0.05.ResultsIn ischemia group,obvious VEGF positive staining was found within potal areas, which mainly located at bile duct epithelium and vessel endothelium.While in sham or sham+rapamycin groups,less obvious positive staining was found.Right after surgey, VEGF mRNA increased sharply in ischemia group and reached peak level on day 3 postoperatively,which was 7.57 folds to sham(P＜0.001).On day1,3,7,and 14,content of VEGF mRNA in ischemia group was signifantly higher than in sham(P＜0.001).During rapamycin administration,VEGF mRNA of ischemia+rapamycin rats fluctuated in a similar manner to ischemia group,with a peak level on day 3,which was only 54%of that in ischemia group(P=0.005).On day1 and day 7,VEGF mRNA was significantly lower in ischemia+rapamycin group than in ischemia group(P＜0.05).According to Western-blot, p70S6k was up-regulated in ischemia group,with the peak level 4.43 folds to sham (P＜0.001).Simultaneously phosphorylated p70S6k was up-regulated,with a peak level 9.64 folds to sham(P＜0.01).Giving rapamycin to bile duct ischemia rats,though there was a increase of p70S6k content,the peak was only 50%of that in ischemia group (P=0.004).On day1,7 and 14,p70S6k was lower in ischemia+rapamycin group than in ischemia group(P＜0.005).In addition,there was no obvious increase of p70S6k when bile duct ischemia rats were administrated with rapamycin.On day 3 and 7,obvious difference was found between ischemia+rapamycin and ischemia group(P＜0.005).Conclusion Rapamycin inhibits VEGF expression at transcriptional level,down-regulated p70S6k and its phophorylated formation.These results provided explanation to negative effect of rapamycin on adaptive bile duct proliferation in response to ischemia.