Role Of Hepatocyte PPARα In Partial Hepatectomy-induced Hepatocyte Proliferation And The Underlying Mechanisms

Beckground Partial hepatectomy(PHx)commonly used to treating end-stage liver disease such as hepatocellular carcinoma(HCC)and cirrhosis.After PHx surgery,the body’s metabolic response changes to meet the energy needs for postoperative liver regeneration.Peroxisome proliferator-activated receptor(PPARɑ)is a nuclear receptor that regulates fatty acid oxidation as its main function.Meanwhile,many studies have also shown that PPARɑis a potential mitogenic factor.Although it has been reported that PPARɑgene knockdown can attenuate liver regeneration after PHx,its specific mechanism of action remains unclear.Our previous studies showed that specifically knocking out hepatocyte PPARɑcan inhibits PPARɑagonists WY-14643 induced hepatocyte proliferation.But the defficiency of myeloid cells PPARɑdid not have similar effects.These results suggests that the expression of PPARɑon different cell types has different effects on liver regeneration.In this study,hepatocyte-specific PPARαdeficient mice were used to investigate the role of hepatocyte PPARαin PHx-induced hepatocyte proliferation underlying mechanisms.Objectives To investigate the role of hepatocyte PPARαin PHx-induced hepatocyte proliferation underlying mechanisms using hepatocyte-specific PPARαdeficient mice.Methods1.PHx surgery was performed on hepatocyte-specific PPARαdisruption mice(Ppara~△HepHep mice)and littermate mice(Ppara~fl/fll/fl mice).2.Mice were injected with BrdU intraperitoneally(5-Bromo-2,-deoxyuridine,BrdU)(5mg/ml,50mg/kg body weight)two hours before killing the mice to drawing into the proliferating cells for subsequent staining.The harvested liver tissue was fixed in 10%formaldehyde solution for 24 hours,dehydrated and embedded in paraffin,and cut into4μm for BrdU staining.The levels of liver regeneration in different groups of mice were compared by calculating the BrdU positive rate(BrdU+hepatocytes/total number of hepatocytes).3.Partial sections were used for hematoxylin and eosin(H&E)staining to observe the pathological changes of the liver.4.Partial sections were used for glycogen PAS staining to measure the contents of liver glycogen.5.Some liver tissues were fixed in 4%paraformaldehyde,embedded into optimal cutting temperature compound(OCT),and cut into 10μm for oil red O staining to measure liver lipid accumulation.6.Real-time quantitative PCR(q-PCR)was used to detect the hepatic gnenes mRNA level in different groups of mice.7.Western blot(WB)was used to quantitatively detect the expression of PCNA,CyclinD1 and PPARαprotein in different groups of mice liver tissues.8.Microarray analysis was performed by CNKINBIO company,China.Results1.2/3 PHx animal model was constructed successfully2/3 PHx operation and Sham operation were performed on WT mice,and liver tissues were harvested at indicated time points.Q-PCR results showed that liver cyclin D1 m RNA levels were obviously increased at 24,32 and 40 h after PHx compared with Sham operation mice.Brd U immunochemistry staining show signicantly increased hepatocyte proliferation in mice after PHx compared with Sham operation mice.These results were consistent with previous reports,indicating that 2/3 PHx animal model was constructed successfully.2.Hepatic PPARα protein expression were significantly increased in mice after PHx at 12 and 32 h compared with sham operation mice.Western blot results showed that the protein level of PPARα in liver increased significantly at 12 and 32 h after PHx compared with Sham group.3.Verifying mouse knockout efficiencyTo validate the knockout efficiency of hepatocyte-specific PPARα knockout mice,q PCR and western blot experiments were performed and the results show that the knockout efficiency difference was more than 90%,which met the experiments requirement.4.Hepatocyte specific deletion of PPARα inhibits liver regeneration in mice after PHxBrd U staining and H&E staining show that the Brd U positive hepatocytes and mitotic index were significantly decreased in Ppara△Hep at 32 h after PHx,the ratio of liver weight to body weight also decreased at 32 h after PHx compared with Pparafl/fl mice.These results indicated that hepatic PPARα can promotes hepatocyte proliferation after PHx.5.Decreased expression of cell cycle related gene in Pparα△Hep miceq-PCR and western blot results show both decreased cyclin D1 m RNA levels at 12,24 and 32 h;and decreased Pcna m RNA at 32 h in Pparα△Hep mice.The same changes of CYCD1 and PCNA protein expression were confirmed using western blot.6.Hepatocyte specific deletion of PPARα decrease DNA damage and repair gene expression in mice after PHxq-PCR show that the DNA damage injury and repair genes,such as Chek1,Mcm2/5,Prkdc and Rad51 m RNA level were significantly decreased in Pparα△Hep mice compared with control mice at 24 h after PHx.These indicated that PPARα can promotes the process of DNA damage and repair in liver.7.Increased hepatic lipid accumulation in hepatocyte specific PPARα deletion miceHepatic oil red O staining and triglyceride measurement indicated increased hepatic lipid accumulation Pparα△Hep mice,Pparα△Hep mice also showed reduced fatty acid β-oxidation gene expression.8.Decreased hepatic glycogen content in in hepatocyte specific PPARα deletion miceGlycogen PAS staining and liver glycogen content test results showed that hepatic glycogen content were increased in Ppara△Hep mice,accompanied by decreased gluconeogenesis and glycolytic levels.Conclusion1.Hepatic PPARα promotes liver regeneration by regulating cell cycle related gene expression and promoting hepatic lipid metabolism and glucose metabolism to meet the large energy meet for cell proliferation.