Apoptosis Response And Related Regulation Mechanism During Rat Liver Regeneration

Liver is one of the few organs which have the capacity to regenerate rapidly after injury. 7-10 days after 70%partial hepatectomy,liver recovers to its original mass and volume. LR is a complex process under precise regulation from numerous proliferative and inhibitory factors.The present study mainly focuses on the proliferative mechanism involved in LR.Factors such as hepatocyte growth factor(HGF),tumor necrosis factorα(TNFα),epidermal growth factor(EGF),transforming growth factorβ(TGFβ),IL-6, insulin and noradrenaline(NA) play important role in initiation of LR.Inhibitory regulation mechanism such as LR termination and structure/function reorganization remain to be studied.Clarifying the mechanism of LR termination and structure/function reorganization from the point of inhibitory mechanism not only help to comprehensive understanding LR,but also is of great significance for liver disease,such as for exploring the path mechanism and treatment of liver tumor.Role of apoptosis in LR termination and structure/function reorganization receives more and more attention recently.In theory,as an inhibitory mechanism in vivo,apoptosis might participate in clearing vicious cell due to rapid proliferation during LR,reorganizing structure/function of liver tissue and terminating proliferation process at suitable occasions. Recent studies further demonstrated the importance of apoptosis in LR.At the early stage of LR,activity of apoptosis executing enzyme caspase-3 was significantly increased. Apoptosis related factors such as Fas,TGFβand Bcl-2 were induced or inhibited during LR.These studies preliminary indicate that LR was accompanied with apoptosis response. At the same time,most of these studies focused on early stage of LR and some of them were paradoxical.There still is not conclusive opinion about whether or how apoptosis response was changed during LR,especially at the late stage,activity of apoptosis enzyme caspase during LR,role of important organelle and the related signal pathway such as mitochondria and endocytoplasmic reticulum,apoptosis related genes participating in LR termination and structure/function reorganization,if possible how is the related regulation mechanism.All of the above lack sufficient support from experiment.In the present study,using 70%rat partial hepatectomy model and special agent,we studied the change of apoptosis response and expore the possible mechanism involved in. The present study is summarized as following:Part One:Change of apoptosis response during LRDue to the minor percentage of apoptosis cells in the regenerating liver,we selected severa index to evaluate change of apoptosis response objectively.We firstly observe LR index(LRI),LR degree(LRD) and serum AST/ALT levels which can reflect the state of LR and possible liver function disturbance.We found that at 1 d after PH,mass of the liver started to grow significantly and reached maximal value at 7 d. Levels of serum AST/ALT began to increase at 3 h after PH,reaching maximal at 1 d and recovering to normal after 3 d.We found that the acticity of apoptosis executing enzyme caspase-3 increased at 3 h after PH,decreased to normal soonly,and increased again at 7 d and 9 d.The activity change pattern of caspase-8,9,12 and calpain was consistent with that of caspase-3.Among them,the activation amplitude of caspase-9 was maximal,and that of caspase-12 and calpain was relatively lower.Serum levels of TNFαwere increased at the early and late stage of LR.The above results indicate that apoptosis is closely connected with LR.At the early and late stage of LR,apoptosis response was increased significantly.We postulate that the early increase of apoptosis may exert complex impact on the initiation of LR,while the increase at late stage may participate in termination and structure/function reorganization.The results also indicate that extrinsic and intrinsic pathway both contribute to activation of caspase-3.Part Two:Screening of apoptosis modulating genes during LRRat 10k cDNA microarray was used to identify apoptosis modulating genes during LR(3 h,6 h,1 d,3 d and 7 d),and RT-PCR was used to verify the result.As a result,over 200 apoptosis modulating genes including several components in KKS,RC3,ACBP and SOD2 were found to be discrepantly expressed.The number of discrepantly expressed genes at 3 h,6 h,1 d,3 d and 7 d was 60,124,64,81 and 64 respectivly.RT-PCR verified the result of microarray.Part Three:Role ofmitochondrial permeability in activation of caspaseMitochondrial permeability pore play important role in intrinsic pathway of apoptosis activation,opening of mPTP eventually lead to the release of apoptosis-inducing factors and activation of caspase-9.Due to ROS is one of the most commen factors leading to the opening of mPTP,we examined content of NO,activitis of NOS and GSH-Px during LR. As a result,we found that at the early stage,content of NO was increased,at the same time activity of GSH-Px was decreased.The result indicated that at the early stage,liver was under stage of oxidative stress.At the late stage,content of NO was increased,and activity of GSH-Px was increased at a later time until to 11 d after PH.The result indicates that at the late stage accompanied the increase of ROS,and ability of anti-oxidation was increased compensatorily.To further verify the role of mitochondrial permeability pore in activation of caspase,specific inhibitor of mitochondrial permeability CsA was used.As a result,we found that CsA treatment decreased activity of caspase-3 and 9 at early and late stage,at the same time generation of ROS was inhibited.Part Four:Role of kupffer cell in activation of caspaseSpecific blocking agent of liver kupffer cell Gd was used to clarify the role of kupffer cell in activation of caspase during LR.As a result,we found that Gd treatment significantly decreased serum TNFαlevel and activities of caspase-8,at the same time the activation of caspase was partly inhibited.These results indicate that during LR,KCs might contribute to actiation of caspase-3 through pathway mediated by TNFαand caspase-8.Part Five:Expression and preliminary study of the role of KKS during LR1.Expression of components in KKS during LR.The result of microarray indicated that several components of KKS were discrepantly expressed during LR especially at the late stage.Together with literature,we postulated that KKS may be closely associated with LR termination and structure/function reorganization.So,real-time PCR was used to verify the result of the microarray.As a result,we found expression of HK was increased during the whole process of LR and that of PLK was increased at the late stage.These results indicated that more BK and HKαwere generated.Expression of downstream moleculars of KKS such as bEGF and tropomyosin were decreased at the late stage,at the same time,content of NO was significantly increased at the late stage.Based on these result,we postulate that at the late stage ofLR,BK and HKαmay play a pro-apoptosis role through different signal pathway.2.Preliminary study of the role of KKS during LRTo further study the role of BK in the activation of caspase at the late stage of LR, angiotensin converting enzyme inhibitor captopril(CA) was used.As a result,we found that CA treatment increased activity of caspase at the late stage and aggravated oxidative stress.These results indicated that at the late stage of LR,high concentration of BK may contribute to activation of caspase through NO and other signal moleculars,and eventually contribute to LR termination and structure/function reorganization of liver tissue.All above results were summarized as follows:1.Apoptosis response was significantly increased at the early and late stage of LR.The early temporal increase may result from operation induced stress,while at the late stage the increase was independent on stress and may contribute to LR termination.Extrinsic and intrinsic pathway both contribute to activation of caspase-3.2.Over 200 genes were found to be closely connected with LR,among them over 60 genes including several components of KKS,SOD2 and stathmin may contribute to the increased apoptosis response and LR termination and structure/function reorganization.3.Mitochondrial permeability pore exert important impact of the activitation of caspase during LR.At the early and late stage of LR,oxidative stress may participate in activation of caspase by inducing opening of mitochondrial permeability,and eventually contribute to LR termination and structure/function reorganization.CsA inhibited the activation of caspase-3 and 9 by specificly blocking the opening of mitochondrial permeability pore, and content of ROS was decreased at the same time.4.Extrinsic pathway mediated by KCs play important role in activation of caspase during LR.At the late stage,KCs participate in activation of caspase-3 by TNFαand caspase-8.5.At the late stage of LR,increased concentration of BK in KKS may contribute to actication of caspase and oxidative stress,and eventually play important role in LR termination and structure/function reorganization.