The Study Of Periplaneta Americana Extracts On Hepatocyte Regenerationafter 70% Partial Hepatectomy In The Mouse

ObjectivesPost hepatectomy liver failure(PLD)remains to be the important causes of the perioperative status and postoperative prognosis ofpatients,especially those undergoing extensive hepatectomy such as hemihepatectomy.Development of medicine on promotion of hepatocyte regeneration have significant clinical value.Periplaneta Americana extracts(PAEs)was used broadly to enhance skin wound repair.Otherwise,it was proved to protect the live and the heart,have anti-inflammation and anti-fibrosis effects.We hypothesized that PAEs could have a promotive effect on hepatocyte regeneration.In this study,we aim to explore 1)the effect of PAEs on hepatocyte regeneration after 70%PH in the mouse;2)the signaling pathways underlying the promotive effects of PAEs on hepatocyte regeneration after PH with transcriptomic analysis;3)the mechanism of the promotive effects of PAEs on hepatocyte regeneration after PH.MethodsClarify the effects of PAEs on hepatocyte regeneration after 70%PH in the mouseC57/BL6J male mice were randomly divided into 5 groups,each with 25 mice:normal control(NC),surgery with saline(NS),surgery combined with low(200 mg/kg/d,PAEs 200),medium(400 mg/kg/d,PAEs 400),and high dose(800 mg/kg/d,PAEs 800)of PAEs treatment.Liver tissues were collected,and liver regeneration rate was calculated.Histological changes of the liver and the proportion of Ki67 positive cells were observed.The major components of PAEs was detected by high performance liquid chromatography.Explore the Signaling Pathways Underlying the Promotive Effects of PAEs on Liver Regeneration after PH with Transcriptomic AnalysisFresh liver tissues of mice from NC,NS,PAEs400 and PAEs800 groups were collected and high throughout RNA-seq method was applied.The data was used for the analysis of gene comparison,differential expression analysis,GO enrichment and KEGG pathway enrichment.Investigate the Mechanism of the Effects of PAEs on Hepatocyte Regeneration after Partial HepatectomyWestern blotting and immunohistochemistry were used to assess the expression of Cyclin D and factors of PI3K/Akt/mTOR signaling pathway in the liver tissue of each group.Liver Cell Line L02 was treated with PAEs,MK-2206(AKT inhibitor),Rapamycin(mTOR inhibitor),MK-2206 with PAEs and Rapamycin with PAEs.CCK8 was performed to compare the hepatocyte regeneration between NC and other groups.q-PCR,western blotting and immunofluorescence double staining were conducted to detect the changes of PI3K/Akt/mTOR signaling pathway.Flow cytometry was applied to assess the cell cycle changes of hepatocytes in each group.ResultsClarify the effects of PAEs on hepatocyte regeneration after 70%PH in the mouseThe weight and volume of the liver were increased 48 hours after 70%partial hepatectomy.The difference of liver regeneration rate was significant between PAEs800 and NS group,PAEs400 and NS group,PAEs800 and PAEs200 group.HE staining showed that hepatocytes were similar in size and arranged in neat rows in NC group,with few hepatocytes exhibiting two nuclei.While in NS group,some hepatocytes were swelled and disorganized,with more mitotic count and hepatocytes having two nuclei.After PAEs treatment,the number of hepatocytes having two nuclei was increased as the dose became higher,though the mitotic phases were decreased significantly.The percentage of Ki67 positive cells was increased after PAEs treatment,and it is the highest in the PAEs800 group.High performance liquid chromatography has shown the ratio of cytosine(5.91 mg/g),uracil(6.21 mg/g),cytidine(7.63 mg/g),uridine(5.97 mg/g),inosine(3.26 mg/g)and guanosine(1.89 mg/g).Explore the Signaling Pathways Underlying the Promotive Effects of PAEs on Liver Regeneration after PH with Transcriptomic AnalysisThere were 1092 differential expression genes between PAEs400/NS and PAEs800/NS.After KEGG pathway classification and enrichment on 159 genes filtered with Cellular Processes according to Q value<0.05,the signaling pathway involving the most differential expression genes with the most obvious fold changes was PI3K/AKT/mTOR signaling pathway.Our preliminary experiments have shown that Ki67 was highly expressed in PAEs group and the liver regeneration rate of this group is high.Therefore,we postulated that PAEs may enhance hepatocyte regeneration and liver regeneration through modulating PI3K/AKT/mTOR signaling pathway and cell cycle.Investigate the Mechanism of the Effects of PAEs on Hepatocyte Regeneration after Partial HepatectomyIn vivo experimental resultsThe expression of Cyclin D,AKT and PI3K in the liver tissue was significantly increased in the NS group compared with NC group.After PAEs intervention,the expression of Cyclin D,AKT and PI3Kwas significantly decreased compared with NS group.The expression of p-S6K and p-mTOR in the liver was significantly increased in the NS group compared with NC group.After PAEs intervention,the expression of p-S6K and p-mTOR was significantly decreased in the PAEs400 and PAEs800 groups compared with NS group.In vitro experimental resultsCCK8 result:PAEs could promote and accelerate hepatocyte regeneration and rescue the inhibition of MK-2206 and rapamycin on PI3K/Akt/mTOR signaling pathway.q-PCR result:The mRNA level of AKT,mTOR,Cyclin D and Ki67 was significantly increased in PAEs groups compared with NC group.After MK-2206 or rapamycin intervention,the mRNA level of these factors was significantly decreased.PAEs could partly rescue the reduction of these factors due to MK-2206 and rapamycin.Western blotting and immunofluorescence double staining result:The protein expression level of Cyclin D,Ki67,p-AKT(S473),p-AKT(T308),PI3K,p-mTOR(S2448),p-4E-BP1(T37/46)and p-S6K was significantly increased in the PAEs groups compared with NC group.The expression was significantly decreased after MK-2206 and rapamycin intervention,which could be partly rescued by adding PAEs.Flow cytometry result:PAEs could promote cellular regeneration through modulating the transition of L02 cell from G1 phase to S phase.Conclusions1.Prolonged administration of PAEs before seventy percent partial hepatectomy can significantly promote postoperative liver regeneration.2.PAEs may accelerate hepatocyte regeneration and thus liver regeneration through complex mechanisms.PI3K/AKT/mTOR signaling pathway may be an indispensable mechanism for regulating cell cycle,promoting hepatocyte regeneration and accelerating liver regeneration.