Tetrahedral Framework Nucleic Acid Promotes Liver Regeneration In Mice And Its Mechanism

Background and Objective:The liver is responsible for many complex physiological functions such as metabolism,synthesis,and detoxification.The normal performance of liver function is the prerequisite for healthy survival of the body.Acute liver injury may occur when the liver is acutely damaged by various pathogenic factors such as viruses,drugs,chemical poisons,trauma,and surgical resection.In severe cases,it may even induce acute liver failure and cause death.Due to the high mortality of acute liver failure,liver transplantation is often needed to save life.However,liver transplantation is scarce and expensive.In order to reduce the proportion of patients who develop from acute liver injury to acute liver failure,and reduce the mortality,new drugs or materials are sought to promote liver regeneration and repair lost liver tissue as soon as possible,Thus maintain stable liver function.This is an important treatment strategy for acute liver injury.Nanomedicine is an emerging interdisciplinary subject that uses nanomaterials to prevent,diagnose,treat diseases and promote health.In the past two decades,nanomaterials have developed vigorously in the medical and health fields,such as drug targeted therapy and drug delivery,medical imaging,in vitro diagnosis,implant materials,tissue regeneration.DNA nanomaterials have become one of the most promising nanomaterials because of their stable structure,good biocompatibility and easy programming modification.Tetrahedral framework nucleic acid（TFNA）is a tetrahedral-shaped nucleic acid nanomaterial that is automatically assembled by four DNA strands with different sequences according to the principle of base complementary pairing.Some studies have reported that TFNA can enter cells autonomously through endocytosis;other studies have suggested that TFNA can promote the proliferation of various cells,such as chondrocytes and fibroblasts.However,there is currently no relevant research on whether TFNA can promote the proliferation of hepatocytes and liver regeneration to treat acute liver injury or liver failure.Therefore,the main purpose of this study is to use AML12 mouse hepatocytes and primary mouse hepatocytes as cell models,APAP/CCl₄-induced acute liver injury mice and 70%hepatectomy mice as animal models to study whether TFNA can promote mouse hepatocyte proliferation in vitro cell culture model and its corresponding mechanism,and whether TFNA can promote mouse hepatocyte proliferation,Promote liver regeneration in vivo model and its regulatory mechanism,in order to provide a new drug for treatment of acute liver injury or liver failure.Materials and Methods:According to the different research methods and content,this research is divided into three parts:（1）Synthesis,characterization and biological characteristics of tetrahedral framework nucleic acid:Firstly,the TFNA needed for the experiment was prepared according to standard procedures and its characterization were verified;secondly,AML12 hepatocytes were co-cultured with TFNA in vitro for 12 hours,and then cellular immunofluorescence and flow cytometry were used to explore the ability of TFNA to enter cells autonomously.Finally,the metabolic process of TFNA in living nude mice was initially explored through Micro-CT,which laid the foundation for subsequent experiments.（2）Tetrahedral framework nucleic acid promotes the proliferation of primary hepatocytes and AML12 cells and its mechanism:on the one hand,the primary hepatocytes of C57BL/6 mice were First isolated according to the two-step collagenase in situ perfusion method;then the cells were identified by glycogen PAS Staining and CK18 immunofluorescence detection;and CCK-8 cell proliferation assay,Cell cycle detection by flow cytometry were used to determine whether TFNA can promote the proliferation of primary mouse hepatocytes.On the Other hand,AML12 cells were first co-cultured with PBS（control group）or different concentrations of TNFA（experimental group）for 12 hours,and then CCK-8 cell proliferation assay,Ed U immunofluorescence detection and flow cytometry of cell cycle were used to study whether TFNA can promote the proliferation of AML12cells.Finally,the expression of key genes and proteins in Wnt/β-catenin signaling pathway and Notch signaling pathway were detected by Quantitative real-time PCR（q-PCR）and Western blot respectively.（3）tetrahedral framework nucleic acid promotes liver regeneration in mice and its mechanism:the mouse models of acute liver injury induced by CCl4,APAP and70%hepatectomy were established respectively.First of all,after the above model was successfully established,the mice were intraperitoneally injected with the same dose of normal saline（control group）or different concentrations of TFNA（experimental group）respectively,and the body weight（before liver taken）and liver weight（after liver taken）of mice was recorded.the eyeball blood of mice was collected for ALT and AST tests;the liver of mice was taken for paraffin section,and HE staining,immunohistochemical staining of Brd U and Ki67 were carried out to study whether TFNA can promote liver regeneration of model mice.Then,RNA and protein samples were extracted from the liver tissues of 70%hepatectomy model mice,and the m RNA full transcriptional group was sequenced to find out the differential genes between the experimental group and the control group,the key genes and proteins of Cell cycle and p53 signal pathways were verified by q-PCR and Western blot.Results:The main results are as follows:（1）Judging from the observation results of nano-size Zeta potentiometer,capillary electrophoresis,PAGE gel electrophoresis and atomic force microscope,we have successfully synthesized the TFNA needed for the experiment.The results of cellular immunofluorescence and flow cytometry showed that TFNA can enter AML12 cells in large quantities,while single-stranded DNA can hardly enter the cells.In addition,the metabolic process of TFNA in living nude mice was preliminarily explored by Micro-CT:after TFNA was injected through tail vein,it was mainly distributed in the liver about 30 minutes later,and then metabolized by kidney about60 minutes later.（2）In this study,the primary hepatocytes of C57BL/6 mice with sufficient cell number and good activity were isolated;the extracted cells were confirmed to be primary hepatocytes by glycogen PAS staining and CK18 immunofluorescence detection.Then,the results of CCK8 cell proliferation detection and flow cytometry of cell cycle proved that TFNA can help mouse primary hepatocytes maintain normal phenotype and promote their proliferation in vitro.In addition,the results of CCK-8cell proliferation assay,flow cytometry of cell cycle and Ed U cell proliferation fluorescence imaging,showed that TFNA could also promote the proliferation AML12 cells.Through q-PCR and western blot experiments,it was found that the mechanism of TFNA promoting the proliferation of AML12 hepatocytes is mainly by activating the Wnt/β-β-catenin signaling pathway and the Notch signaling pathway,up-regulating the expression of Cyclin-D1,accelerating the cell cycle,and promoting the proliferation of hepatocytes at last.（3）In this study,the chemical liver injury model induced by CCl₄,the drug-induced acute liver injury model induced by APAP and the classic 70%hepatectomy model were established.First of all,the ratio of liver weight to body weight showed that the speed of liver regeneration in the TFNA group was faster than that in the control group at 36 h;blood samples were taken to detect the liver function of mice,the results suggested that TFNA could significantly reduce the peak values of ALT and AST at 24 hours compared with the control group;and the HE staining of liver tissue sections indicated that TFNA could significantly reduce the range of inflammatory injury.The outcomes of immunohistochemical staining of Ki-67 and Brd U demonstrated that the proportion of proliferated hepatocytes in the TFNA group was significantly higher than that in the control group.It is confirmed that TFNA can promote the proliferation of hepatocytes and accelerate the process of liver regeneration in mice.Furthermore,the corresponding molecular mechanism was explored by transcriptome sequencing,q-PCR and western blot experiments.The results revealed that TFNA could accelerate the cell cycle and promote liver regeneration by activating cell cycle signal pathway,and activate the p53 pathway to terminate the process of liver regeneration in time.Conclusion:In summary,this project proved that TFNA can activate the Wnt/β-catenin and Notch signaling pathways,and finally activate the downstream cell cycle signaling pathways,accelerate the cell cycle,promote the proliferation of mouse liver cells,and thus promote liver regeneration;And activate the P53 pathway to maintain the robustness and fidelity of the liver regeneration process.Therefore,TFNA is expected to treat acute liver injury represented by APAP-induced liver injury through its characteristics of promoting liver regeneration,prevent it from progressing to acute liver failure,and thereby reduce mortality.