[Role of rapamycin in cholangiocytes regeneration after liver transplantation].

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 µg/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 cholangiocytes proliferation were detected by ELISA (enzyme linked immunosorbent assay) and histochemistry respectively. The expressions of IL-6 mRNA, phosphorylated-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 (γ-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-1h group. And a slight increase of IOD (integral optical density) ratio (38 ± 10 and 22 ± 7) indicated a mild cholangiocytes 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 cholangiocytes 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 cholangiocytes 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-sIL-6R inhibited the cholangiocytes proliferation and reduced the expressions of IL-6, STAT3 and cyclin 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-sIL-6R. With similar results, the RPM treatment had insignificant effects on the expression of IL-6.

Conclusion: The IL-6/STAT3 pathway initiates the cholangiocytes regeneration after liver transplantation so as to accelerate the biliary recovery. However RPM represses the cholangiocytes 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.