Abstract
The process of liver regeneration after partial hepatectomy is very complex and is associated with signalling cascades involving initiation signals, transcription factors, cytokines, growth factors, tissue remodelling and termination of growth related signals. To date the exact mechanism of liver regeneration remains poorly understood. Toll-like receptor 4 (TLR4) acts as a sensor for immune signals and plays a critical role in host defence. It is known that lipopolysaccharide (LPS) is one of the ligands for TLR4. Binding of LPS to TLR4 leads to activation of transcription factor, nuclear factor kappa B (NF-κB) via the intracellular adaptor molecule, myeloid differentiation factor 88 (MyD88), which in turn activates the production of proinflammatory cytokines, TNF-α and IL-6. Evidence suggests that LPS/TLR4 signalling may be involved in liver regeneration following partial hepatectomy, as delayed liver regeneration and impaired cytokine responses were observed in C3H/HeJ mice, a mouse that is hyporesponsive to LPS due to a point mutation in the tlr4 gene. In mice lacking TLR4 receptor (TLR4 knockout mice), the early phase of the regenerative response was found to be normal, indicating TLR4 might not be involved in the early phase of liver regeneration. However, the involvement of the TLR4 signalling in the late phase of liver regeneration has never been elucidated. Therefore, we investigated whether LPS/TLR4 signalling is critical for liver regeneration after partial hepatectomy.
In mice that are LPS-insensitive due to a mutation in the tlr4 gene (C3H/HeJ mice) or due to TLR4 receptor deficiency (TLR4 knockout/TLR4-/- mice) and in normal, LPS-sensitive mice (C3H/HeN and C57BL/6), hepatocyte proliferation and liver mass restoration, TNF-α and IL-6 production and NF-κB activation in the liver were assessed following partial hepatectomy. In addition, microcirculation and structural changes of the regenerating liver were examined in these mice following partial hepatectomy using the intravital fluorescence microscopy (IVFM). In the TLR4-/- mice, secondary gene expression changes deriving from the original signalling blockade was never considered. In light of this, hepatocyte proliferation and liver mass restoration following partial hepatectomy were examined in the LPS-sensitive mice (C3H/HeN) pretreated with anti-TLR4 monoclonal antibody (MTS510). Furthermore, in order to investigate the role of LPS in liver regeneration, hepatocyte proliferation and liver mass restoration were assessed following partial hepatectomy in the LPS-sensitive mice (C3H/HeN) treated with antibiotics, to restrict the availability of gram-negative bacteria in the gut (i.e. the main source of LPS).
In the antibiotic treated C3H/HeN mice, hepatocyte proliferation and liver mass restoration following partial hepatectomy were found to be impaired, indicating the importance of LPS in liver regeneration. In addition, hepatocyte proliferation after partial hepatectomy was significantly reduced in the anti-TLR4 antibody treated C3H/HeN mice compared to the non-treated group. In LPS-insensitive C3H/HeJ mice, hepatocyte proliferation and liver mass restoration were delayed, which were associated with delayed TNF-α and IL-6 responses, impaired early activation of NF-κB in the liver, altered red blood cell (RBC) velocity and delayed regenerative structural changes following partial hepatectomy. In LPS-insensitive TLR4-/- mice, normal hepatocyte proliferation and liver mass restoration were observed, which were associated with an early upregulation of the IL-6 response, rapid activation of NF-κB in the liver and increased RBC velocity and sinusoidal blood flow following partial hepatectomy. However, in the TLR4-/- mice, the TNF-α response was blunted, late phase activation of NF-κB in the liver was impaired and hepatocyte hypertrophy and hepatocyte clusters remained at the late phase following partial hepatectomy, which indicate a delay in hepatic tissue remodelling in the TLR4-/- mice.
In conclusion, the thesis has demonstrated that LPS/TLR4 signalling may be essential for liver regeneration following partial hepatectomy. The finding that TLR4 is involved in the hepatic tissue remodelling at the later phase of the regenerative process, most likely via TNF-α and subsequent late phase activation of NF-κB, is novel. In addition, the thesis has established differences between LPS-insensitive, C3H/HeJ and TLR4-/- mice, particularly in liver regeneration following partial hepatectomy.