The Role Of Hepatocyte Senescence During Hepatocarcinogenesis Under Chronic Liver Injury

Cellular senescence is a relatively stable state that characterized by irreversibly cell cycle arrest and loss of proliferation,and it alters microenvironment and tissue homeostasis.It has been proved by formal studies that cellular senescence is closely associated with tumor initiation,development and therapies.On the one hand,tumor cells exhibit immortalization as well as senescence obstacle,result in tangled cell cycle regulation.Immortalization,scilicet coming through cell cycle regulation,is a prerequisite of tumorigenesis.Meanwhile,arrested cellular senescence is the requisition of immortalization.The arrest on senescence-related signaling occurs in the early stage of tumorigenesis.On the other hand,apoptosis and cytocidal play a critical role in suppressing tumorigenesis.In addition,turning into senescence,arresting cell cycle and inhibiting cell division also act as tumorigenesis suppressing mechanisms.With the advances in cellular senescence research,the opinion that senescence acts as a natural barrier to tumorigenesis is accepted by researchers more and more widely.Hence,by probing the associations between cellular senescence and tumorigenesis,connecting the mechanisms underlying cellular senescence and tumorigenesis with anti-tumor therapies,we could get beneficial inspirations to facilitate researches on tumorigenesis and therapies.Liver cancer is a predominant lethal cancer in China.Its incidence keeps elevating in recent years.According to the statistical data,more than half of the global incidence is distributed in China.Hepatocarcinoma?HCC?takes up 80%incidence of liver cancer,mainly occurs in patients with chronic liver injury.Chronic liver injury is a complex pathological process,involving progressive destruction and regeneration of hepatic parenchymal cells that induced by various damage factors,like alcohol,virus infections,drug toxicity and hereditary metabolism disorder.These damage factors promote liver diseases progression via different mechanisms,and lead to hepatocarcinogenesis ultimately.By studying correlations between cellular senescence and hepatocarcinogenesis during chronic liver injury,we could get remarkable assistance for clarifying hepatocarcinogenesis mechanism and developing novel therapy strategies.This research could be divided into four parts.In the first part,we established an animal model of acute and chronic liver injury,and inspected the pathological changes during acute and chronic liver injury respectively.Fumarylacetoacetate hydrolase?Fah?knockout mice(Fah^-/-mice)is a tyrosinemia type I model established by Grompe et al.In this model,Fah deficiency leads to metabolic block of tyrosine and DNA-toxic metabolites accumulation,which lead to acute liver injury.Fah^-/-mice generally perished after 8 weeks without intervention.NTBC inhibits enzymes metabolizing tyrosine,suppress the generation of toxic metabolites and turns Fah^-/-mice into a normal state.But if NTBC is only administrated with 2.5%dosage,Fah^-/-mice underwent chronic liver injury.By treating two groups of 8 weeks Fah^-/-mice with withdrawal or 2.5%dosage NTBC,we respectively induced acute and chronic liver injury in two groups.After 8 weeks'treatment,in chronic liver injury group,treated with 2.5%dosage NTBC,we observed mild chronic pathological changes in the liver,like micro inflammatory necrosis foci,micro steatosis foci,mild nuclear polymorphism and hepatocyte swelling.Meanwhile,in acute liver injury group,treated with NTBC withdrawal,we observed severe acute liver injury,mainly characterized by massive nuclear polymorphism,diffuse necrosis,bile duct proliferation,severe hepatocyte steatosis and swelling.As unfinished division after cell cycle arrest leads to senescent cell enlargement,changes of cell volume can index cellular senescence.Applying hepatocyte area as cell volume index,we found that after 4 or 8 weeks,there's a significant hepatocyte area enlargement in Fah^-/-mice liver of the acute liver injury group.However,in the chronic liver injury group,we observed no obvious enlargement after 4weeks,and a relatively smaller enlargement compared to the acute liver injury group after8 weeks.This difference indicated a difference of hepatocyte senescence between two types of liver injury.To further confirm how hepatocyte underwent senescence during acute or chronic liver injury,we established composite senescence evaluation scheme.The scheme included SA-?-Gal staining,SAHF staining and expression measuring of p53 and p21.In order to identify the senescence pattern during acute and chronic liver injury,we harvested samples from the acute liver injury group at 0,2,4,6 and 8 weeks during treatment.Samples were tested with our evaluation scheme.In the acute liver injury group,we observed an elevation of all tested markers with the extending of treatment,which indicated that hepatocytes underwent senescence and exhibited stable senescent phenotypes.To deeper verify the presume that hepatocytes suffering chronic injury showed no senescence,we then harvested samples from the chronic liver injury group at 0,4,8 and12 weeks during treatment.Tests revealed that hepatocytes exhibited no stable senescent phenotypes.Combined the difference with distinct individual fate,we presume that hepatocyte senescence could only be induced when injuries crossed a certain threshold,and hepatocyte senescence was responsible for the high incidence of hepatocarcinogenesis in Fah^-/-mice suffering chronic liver injury.In the second part,to clarify whether hepatocyte senescence affected hepatocarcinogenesis during acute or chronic liver injury,we conducted experiments as follows:First,we evaluated acute and chronic injury phenotypes through liver function indexes.The evaluations confirmed acute liver injury of NTBC withdrawn Fah^-/-mice with serum indexes alterations,include significant elevation of ALT,AST and TBIL and significant degrade of ALB.Meanwhile,chronic liver injury of Fah^-/-mice treated with2.5%dosage NTBC was confirmed by slight elevation of ALT,AST and TBIL and slight degrade of ALB.Next,we counted how many tumors formed in two groups after treatment.Fah^-/-mice generally perished after 6 to 8 weeks'withdrawal,due to the accumulation of toxic metabolites.In order to extend the period of acute liver injury to match the tumor formation period of the chronic liver injury group,12 and 26 weeks for instance,we applied a new intervention,i.e.treat Fah^-/-mice with full dose NTBC for 3 days each time when mouse's body weight dropped to 70 to 75 percent of its original weight and then withdrawal again.By this intervention,the survival period of mice in the acute liver injury group extended to 12 and 26 weeks.After count and comparison,we found that no tumor formed in mice of the acute liver injury group and all mice of the chronic liver injury group bore tumor at 12 weeks.At 26 weeks,36%mice of the acute liver injury group bore tumor,the ratio is significantly lower than 100%of the chronic liver injury group.Meanwhile,the count and size of tumor-like nodes in mice of the acute liver injury group are significant smaller than those of chronic liver injury group.By the experiments fore mentioned,we can figure out that Fah^-/-mice undergo death for accumulation of toxic metabolites and severe liver injury without interventions.Short period NTBC administration rescued Fah^-/-mice from dying for liver failure,and activated hepatocyte senescence and senescence surveillance to efficiently suppressed hepatocarcinogenesis.Fah^-/-mice with chronic liver injury avoided severe liver injury and stable senescence phenotypes,abrogated the elimination of senescent hepatocyte and suffered hepatocarcinogenesis.Further,we gathered samples of different time points and ran RNA-seq analyzation.By evaluating alterations of senescence related signaling,HCC related signaling,cell cycle and proliferation related signaling,we confirmed former presume,i.e.hepatocyte senescence inhibition is accompanied by hepatocarcinogenesis,at transcriptome level.In the third part,to further investigate whether hepatocyte senescence inhibition is the main course of hepatocarcinogenesis,we induced hepatocyte senescence by aggravating liver injury of Fah^-/-mice,and observed whether hepatocarcinogenesis got suppressed.We picked Fah^-/-mice that had been treated with 8 weeks'chronic liver injury,and treated them with 4 weeks'withdrawal to induce hepatocyte senescence.After total 12 weeks'treatment,tumor formation of the livers was counted.We found that after this treatment scheme,hepatocytes of Fah^-/-mice re-exhibited senescence phenotypes,which means re-initiation of senescence.Still,the tumor formation ratio stay at lower level,only 14.29%at 12 weeks.Hence,hepatocarcinogenesis of Fah^-/-mice suffering chronic liver injury could be efficiently suppressed by inducing hepatocyte senescence through acute liver injury treatment.In the third part,we investigated the underlying mechanisms of cellular senescence suppression effect on hepatocarcinogenesis.As an intrinsic tumor inhibition mechanism,though the correlations between cellular senescence and tumorigenesis attracted more and more attention,the mechanisms through which hepatocyte senescence inhibited hepatocarcinogenesis remains equivocal.An important part of the mechanism is cellular senescence induced immune surveillance.By comparing the counts of immune cells in livers from acute and chronic liver injury group,we confirmed that hepatocyte senescence in Fah^-/-mice activated senescence related immune surveillance.For instance,senescent hepatocytes activated CD4⁺Th1 cells,CD4⁺Th1 cells then induced the activation and proliferation of NK cells and macrophages.In this manner,senescent hepatocytes got eliminated by cytotoxic effect,and hepatocarcinogenesis ultimately got suppressed.In this study,we applied Fah^-/-mice to mimic acute and chronic liver injury,and established a controllable hepatocyte senescence.As a hepatocarcinoma model,this model perfectlymimickedtherelationshipbetweenhepatocytesenescenceand hepatocarcinogenesis,clarified the major protect mechanism on hepatocarcinogenesis suppression is hepatocyte senescence.This model laid the theoretical basis for the suppression effect on hepatocarcinogenesis of hepatocyte senescence during chronic liver injury,and provided theoretical instruction and scheme for actualizing controllable hepatocyte senescence and senescence-inducing therapies for hepatoma cells.This study contains great biological and medical significance for inspecting relationships between cellular senescence and hepatocarcinogenesis,for illuminating mechanisms underlying hepatocarcinogenesis,and for consummating therapeutic strategies on hepatoma.