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Targeted knockdown of PI3K components with small interfering RNA (siRNA) inhibits liver regeneration following partial hepatectomy
      Introduction: Hepatectomy is associated with rapid regeneration, macrophage infiltration, and Kupffer cell hyperplasia and hypertrophy within the remnant liver; signaling pathways responsible for this process have not been clearly delineated. Phosphatidylinositol-3 kinase (PI3K), consisting of p85α regulatory and p110α catalytic subunits, regulates multiple cellular processes, including cell growth, cell survival, and leukocyte migration. We have previously demonstrated that inhibition of the PI3K pathway significantly inhibits stimulated growth of the pancreas and intestinal mucosa. The purpose of this study was to determine the effect of selective p85α and p110α subunit inhibition on hepatic regeneration following partial hepatectomy. Methods: Swiss-Webster mice (n=90) underwent 70% hepatectomy or sham operation and then randomized to receive either non-targeting control (NTC), p85α, or p110α siSTABLE siRNA (20μg, iv, qd). Mice were sacrificed over a time course (48h, 72h, or 7d) following operation. Bromodeoxyuridine (BrdU), a measure of DNA synthesis, was given ip 3h prior to sacrifice. Livers were harvested for weight (wet and dry), histology, immunohistochemistry, and extracted for RNA, DNA, and protein. Expression of F4/80, a macrophage marker, proliferating cell nuclear antigen (PCNA), and BrdU were assessed by immunohistochemistry. In addition, H&E, Periodic Acid Shift (PAS), and Oil Red O stains were performed. Results: (i) Targeted inhibition of p85α and p110α subunits significantly decreased early liver regeneration as noted by decreased remnant weight, BrdU incorporation, and PCNA staining. Real time PCR and Western blotting demonstrated knockdown of p85α and p110α mRNA and protein in mice treated with the respective siRNA; Western blot analysis also demonstrated decreased phosphorylation of the downstream PI3K signaling protein Akt in p85α and p110α siRNA-treated mice relative to NTC-treated mice. (ii) Infiltrating macrophages and resident Kupffer cells were markedly decreased in the remnant liver of mice treated with p85α siRNA as noted by a paucity of F4/80 staining cells; treatment with p110α siRNA also led to a decrease in F4/80 staining cells. (iii) H&E staining revealed an unexpected morphology of regenerating hepatocytes in the livers of mice treated with both p85α and p110α siRNA compared to NTC; subsequent PAS and Oil Red O staining showed this altered morphology to be aberrant glycogen and lipid storage in proliferating hepatocytes. Conclusions: PI3K/Akt pathway activation plays a critical role in early liver regeneration after resection. Interestingly, our findings suggest that PI3K inhibition decreases macrophage infiltration and Kupffer cell hyperplasia, and alters glycogen and lipid distribution in proliferating hepatocytes. We conclude that inhibition of the PI3K/Akt pathway not only attenuates hepatocyte regeneration but also has marked metabolic consequences which include increased glycogen deposition and steatosis in the remnant liver.
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