雷帕霉素对肝移植术后胆管细胞再生的影响

Role of rapamycin in cholangiocytic regeneration after liver transplantation

目的探讨白介素6（IL-6）／信号转导及转录激活因子3（STAT3）通路是否参与肝移植术后胆管上皮细胞（BEC）再生过程，以及免疫抑制剂雷帕霉素是否通过影响STAT3活化从而抑制BEC增殖。方法将大鼠随机分为5组：正常肝移植组（OLT）-1h、OLT-12h组（供肝分别冷保存1、12h后行原位肝移植术）、抗sIL-6R组（OLT-12h组术前1h静脉注射可溶性抗IL-6受体抗体16．7μg／k，术后每日给予相同剂量直至观察结束）、RPM组（OLT-12h组术前3d腹腔注射雷帕霉素0．05mg／kg，术后每日给予相同剂量直至观察结束）、sham组（假手术组），样本检测时间点为术后1、3、7、14d。测定血清碱性磷酸酶（ALP）、谷氨酰转肽酶（GGT）含量以评价胆道功能，并进行肝脏组织学检查；采用酶联免疫吸附法检测肝组织IL-6含量；实时定量RT—PCR法检测BEC内IL-6mllNA表达；Western印迹法检测BEC内磷酸化STAT3（p-STAT3）、细胞周期蛋白（eyclin）D1的蛋白表达水平；电泳迁移率变动分析法（EMSA）检测BEC内STAT3的DNA结合活性；免疫组化法测定BEC增殖情况。结果与sham组相比，OLT．1h组术后1、3d，血清GGT、ALP轻度、短暂升高：其中GGF分别为（69±6）U／L、（34±4）U／L，ALP分别为（86±9）U／L、（45±3）U／L，BEC损伤轻微。肝组织及BEC内IL-6含量、P—STAT3及cyclinD1表达略增加：其中肝组织IL-6含量分别为（273±20）ng／g、（159±18）ng／g，在BEC内IL-6mRNA表达分别为0．40±0．04、0．234-0．04，BEC内P—STAT3表达分别为0．420±0．023、0．230±0．040，而eyclinD1表达分别为0．580±0．023、0．420±0．015；STAT3的DNA结合活性也相应短暂增加，与sham组之间的积分吸光度（A）比值为38±10、22±7；BEC再生不明显。相反，OLT一12h组术后1d，血清GGT、ALP即明显升高，分别为（108±9）U／L、（189±14）U／L，BEC损伤严重，至术后14d才恢复正常。肝组织及BEC内IL-6含量增加：其中肝组织IL-6含量分别为（659±28）ng／g、（446±23）ng／g，在BEC内IL-6mRNA表达分别为0．73±0．06、0．54±0．04。BEC内P—STAT3和cyclinDl蛋白表达上调，其中BEC内P—STAT3分别为0．72±0．04、0．58±0．06，eyclinDl蛋白表达分别为0．88±0．04、0．74±0．07，STAT3的DNA结合活性明显增加，sham组的A值分别为45±16、31±12；BEC再生活跃。OLT．12h组经抗sIL-6R和RPM处理后，p-STAT3和cyclinDl表达明显降低，STAT3的DNA结合活性及BEC再生受到明显抑制，术后14d仍可见细胞损伤的形态学表现，ALP、GGT明显高于sham组水平。不同的是sIL-6R还能显著降低OLT-12h组肝内IL-6的表达，而RPM对IL-6的表达并无明显影响。结论IL-6／STAT3信号通路介导肝移植术后BEC的再生／修复过程，雷帕霉素通过抑制STAT3活化从而影响BEC的再生，可能对肝移植术后胆道功能的恢复产生不利影响。

Objective To investigate the role of IL-6/STAT3 pathway in the proliferation of cholangiocytes after liver transplantation and determine whether or not rapamycin （RPM） depresses the regeneration of cholangiocytes by blocking the activation of STAT3. Methods Rats were randomized into OLT-1 h and OLT-12 h groups （supplied livers preserved for 1 or 12 h）, anti-sIL-6R group （rats of the OLT-12 h group injected intravenously with 16.7 Ixg/kg anti-rat sIL-6R antibody at 1 hour pre-operation and daily post-operation）, RPM group （rats of the OLT-12 h group injected intraperitoneally with 0. 05 mg/kg RPM for 3 days pre-operation and daily post-operation） and sham group （ transverse laparotomy and closure without liver manipulation）. At 1, 3, 7, 14 d post-operation, the IL-6 concentration in liver homogenate and cholangiocytic proliferation were detected by ELISA （enzyme linked immunosorbent assay ） and histochemistry respectively. The expressions of IL-6 mRNA, phophorylated-STAT3 and cyclin D1 protein in cholangiocytes were determined by real-time RT-PCR （reverse transcription-polymerase chain reaction） or Western blot. The DNA binding activity of STAT3 was determined by electrophoretic mobility shift assay. The serum concentrations of ALP （alkaline phosphatase ） and GGT （^-glutamyltransferase） were also measured. Results The minimal expressions of IL-6, p-STAT3, cyclin D1 and DNA binding activity of STAT3 were detected in OLT-lh group. And a slight increase of IOD （integral optical density） ratio （38 ± 10 and 22 ± 7 ） indicated a mild cholangiocytic proliferation. The concentrations of GGT were （69 ± 6）U/L, （ 34 ± 4） U/L and ALP （ 86 ± 9 ） U/L, （45±3 ） U/L. The expression of IL-6 in liver homogenate were （273 ± 20） ng/g, （ 159 ± 18 ） ng/g and 0. 40 ± 0. 04, 0. 23 ± 0. 04 in cholangiocytes. The expressions of P-STAT3 were 0. 420 ±0. 023 and 0. 230 ±0. 040 in eholangioeytes and cyclin D1 0. 580 ±0. 023 and 0. 420 ±0. 015 respectively. Cholangiocytes responded to extended cold preservation with severe bile duct injures and marked increases in IL-6 secretion, p-STAT3 and cyclin D1 protein expression and DNA binding activity of STAT3, followed by compensatory cholangiocytic regeneration. Meanwhile biochemical index and morphology indicated that bile duct injury recovered at 14 d post-operation. The IOD ratios were 38 ± 10 and 22 ± 7 respectively. The expressions of IL-6 were（ 659 ±28 ）and（446 ± 23 ）ng/g in liver homogenate and 0. 73 ± 0. 06 and 0. 54 ± 0. 04 in cholangiocytes. The expression of P-STAT3 were 0. 72 _± 0. 04 and 0. 58 ± 0. 06 in cholangiocytes and cyclin D1 0. 88 ± 0. 04 and 0. 74 ± 0. 07 respectively. However, anti-slL-6R inhibited the cholangioeytic proliferation and reduced the expressions of IL-6, STAT3 and eyclin D1. The DNA binding activity of STAT3 with cellular injury and the increases of serum ALP or GGT were also abrogated by the administration of anti-slL-6R. With similar results, the RPM treatment had insignificant effects on the expression of IL-6. Conclusion The IL-6/STAT3 pathway initiates the cholangiocytic regeneration after liver transplantation so as to accelerate the biliary recovery. However RPM represses the cholangiocytic regeneration by inhibiting the STAT3 activation. It may have a negative effect on the healing and recovery of bile ducts in grafts with extended cold preservation.