Expression And Regulation Of Connective Tissue Growth Factor In Liver After Brain Death

Liver transplantation(LT)is an effective method for the treatment of end-stage liver disease and liver cancer.Brain-dead donors are the major source of grafts for liver transplantation.Brain death(BD)involves the irreversible and permanent loss of function of both the cerebrum and the brainstem,caused by brain trauma,cerebral hemorrhage and other reasons.The release of inflammatory factors,hemodynamic instability,and "catecholamine storm" caused by brain death can lead to poor organ perfusion,tissue ischemia and hypoxia,progressive damage to liver cells and severe liver dysfunction.However,the mechanism of brain death caused liver damage has not yet been fully understood.Therefore,studying the mechanism of liver damage after the brain-dead can provide theoretical basis and potential intervention targets for improving the function of brain-dead donor organs and reducing the damage.Due to the shortage of donor livers,marginal graft,including elderly donor livers,steatosis,livers with prolonged ischemia time,are also considered for transplantation.The accurate assessment of brain-dead donor livers will also improve the prognosis of liver transplantation and efficiency of organ utilization.We used transcriptomic sequencing to detect the molecular changes in the brain-dead liver.Through transcriptomic sequencing we found that the expression of connective tissue growth factor(CTGF)in the liver of brain-dead donors was significantly decreased.CTGF is a member of the CCN(Cellular communication network factor)protein family.CCN is a secreted extracellular matrix(ECM)related glycoprotein,which plays an important role in a variety of cellular events,including extracellular matrix production,cell adhesion,cell proliferation and growth,apoptosis,cell survival,etc.CTGF was first found in vascular endothelial cells.It is also expressed in activated hepatic stellate cells,but the expression is low in hepatocytes.Previous studies have shown that CTGF is closely related to liver disease and liver damage.The expression and regulatory mechanism of CTGF in the liver from brain-dead donors have not been reported.In this study,we detected the expression of CTGF in livers from brain-dead donors and verified the transcriptomics results.We established model of brain death in rats and collected liver samples at different time points after brain death to explore the dynamic changes of and location of CTGF in the livers after brain death.In order to study the regulation of CTGF,we used vascular endothelial cell lines and liver stellate cell lines to study related signal pathways in vitro.It is reported that activating transcription factor 6(ATF6)is activated in vascular endothelial cells(HUVEC)under hypoxia.ATF6 is one of the important proteins in the endoplasmic reticulum stress pathway which closely related to cell oxidative stress and apoptosis.Transcriptome sequencing and previous studies have shown that the endoplasmic reticulum stress pathway in the liver is activated in the state of brain death.Therefore,we speculate that ATF6 may have a regulatory effect on CTGF in vascular endothelial cells.The regulation between ATF6 and CTGF has not been reported before.We use human umbilical vein endothelial cells(HUVEC)to study the regulatory relationship between ATF6 and CTGF.The expression of CTGF in hepatic stellate cells increased significantly and was regulated by the TGF-β pathway during the process of liver fibrosis in some studies.The Hippo/YAP pathway can also regulate the expression of CTGF.However,the expression and regulation of CTGF in hepatic stellate cells after brain death have not been reported.Transcriptomics analysis results show that there are many differential genes enriched in the Hippo/YAP pathway in brain death liver.Therefore,we studied whether the expression of CTGF is regulated by the Hippo/YAP pathway in the liver stellate cell line LX2.During the research,we found that the expression of CTGF in the livers with non-alcoholic steatosis after brain death was significantly increased.We detected the expression of CTGF in the blood of brain death donors and analyzed clinical data to explore the potential of CTGF in predicting nonalcoholic fatty liver disease in brain-dead donors.In this study,we explored the expression,regulation and potential of clinical application of CTGF in the livers after brain death through the following five parts.Part 1:Transcriptomics analysis and CTGF expression in livers after brain deathObjective:To screen out key molecules that may play an important role in liver after brain death by transcriptomic sequencing and bioinformatics analysis.To clarify the expression of CTGF in liver after brain death.Methods:The liver biopsy specimens from brain-dead donors were used as the experimental group,and the normal liver tissue around hepatic hemangioma were used as the control group.Analyze differential gene expression through highthroughput second-generation sequencing technology.Predict the pathway enriched with differential expression genes through enrichment analysis.Analyze the expression of CTGF in the sequencing and verify it by transcription-quantitative polymerase chain reaction(RT-qPCR).Results:1.The sample correlation analysis showed that the samples in each group were highly correlated.Compared with the control group,the experimental group had 4367 mRNAs with a difference of more than 2 times and P<0.05.2.Through GO enrichment analysis,it was found that pathways related to cell adhesion molecules,regulation of arterial blood pressure,ROS response,hypoxia-induced apoptosis signaling pathway,endoplasmic reticulum stress-induced apoptosis signaling pathway,cholesterol biosynthesis process,steroid metabolism process,the enrichment of differential genes in ischemia-related pathways are highly enriched.3.Through KEGG enrichment analysis,it was found that pathways related to liver metabolism and secretion and pathways related to endothelial cells are also highly enriched.4.Transcriptome sequencing results showed that CTGF expression is down-regulated in liver specimens from brain-dead donor.5.RT-qPCR and immunohistochemical verified that CTGF expression was down-regulated in the livers from brain-dead donor.Conclusions:1.Compared with the control group,the experimental group had 4367 mRNAs with a difference of more than 2 times and P<0.05.Pathways related to blood pressure regulation,hypoxia injury,metabolism and secretion,cell interactions,non-parenchymal cells,and endoplasmic reticulum stress-related pathways are highly enriched in differentially expressed genes.These pathways may play important roles in liver injury caused by brain death.3.The expression level of CTGF in the liver reduced in livers from brain-dead donors.Part 2:The dynamic expression and location of CTGF in rat livers after brain deathObjective:To clarify the dynamic expression and cell location CTGF in rat livers after brain death.Methods:Lewis rats were used to establish brain death model by intracranial compression.After brain death the rats were sacrificed at different time points to collect liver tissue samples,and the expression of CTGF was detected by immunohistochemistry,Western Blot,RT-qPCR and other techniques.Through immunofluorescence experiments,we can study whether CTGF co-localized with CD31,and whether CTGF co-localized with α-SMA.The expression of TGF-β was detected by RT-qPCR and immunohistochemistry.Results:1.The results of immunohistochemistry,western blotting,and RT-qPCR showed that CTGF increased in a short period of time after brain death and then decreased dynamically in rat livers.2.The immunofluorescence results showed that CTGF andα-SMA have co-localization,CTGF and CD31 have co-localization in the liver of brain-dead rats.3.The expression of TGF-β in the liver of brain-dead rats is low with no change.Conclusion:1.CTGF has a dynamic process of increasing first and then decreasing after brain death.2.CTGF located in both vascular endothelial cells and hepatic stellate cells in livers from brain-death donor.3.The TGF-β pathway may not be involved in the regulation of CTGF expression in livers after brain death.Part 3:The expression and regulation mechanism of CTGF in vascular endothelial cells and the protective effect of CTGF on hepatocytesObjective:To study the expression and regulation mechanism of CTGF in vascular endothelial cells and the protective effect of CTGF on liver cells under hypoxia and reoxygenation.Methods:Human umbilical vein endothelial cells(HUVECs)were cultured in vitro and stimulated by hypoxia-reoxygenation to detect the expression of CTGF and ATF6.Expression of CTGF were measured after in HUVEC treated with AA147,an ATF6 agonist.Expression of CTGF were measured after ATF6 knocked down in HUVECs by using small interfering RNA.Binding site of ATF6 and CTGF promoter region were predicted by using data from database.Binding site of ATF6 and CTGF promoter region were verified by dual luciferase reporter assay and Chromatin immunoprecipitation.L02 cells were treated by recombined CTGF and hypoxiareoxygenation condition.Cell viability and reactive oxygen accumulation were measured in L02 cells.Results:1.The expression of activated ATF6 and CTGF increased at 1 hour after hypoxia-reoxygenation condition in HUVECs.2.The expression of CTGF were increased in HUVEC treated by ATF6 agonist.3.The expression of CTGF were decreased in Si-ATF6 treated HUVECs.4.The results of the dual luciferase reporter assay showed that the fluorescence intensity of the mutated CTGF promoter luciferase plasmid was significantly lower than that of the wild-type CTGF promoter luciferase plasmid.Chromatin immunoprecipitation results showed that the enrichment fold of CTGF promoter in the ATF6 coprecipitation group was 4.3 times compared with the negative group.5.In hypoxia-reoxygenation condition,cell viability of LO2 cells was increased after CTGF treatment,and the accumulation of reactive oxygen species ROS were decreased after CTGF treatment.Conclusion:1.The expression of activated ATF6 and CTGF in HUVECs were increased in hypoxia-reoxygenation condition.2.Activated ATF6 has a positive regulatory effect on CTGF.3.Activated ATF6 plays as a transcription factor by combining with the promoter region of CTGF.4.CTGF protects liver cells from hypoxia-reoxygenation damage by reducing the accumulation of reactive oxygen species.Part 4:The expression and regulation of CTGF in hepatic stellate cellsObjective:To explore the expression and regulation mechanism of CTGF in hepatic stellate cells under hypoxia-reoxygenation condition.Methods:The human hepatic stellate cell line LX2 was cultured in vitro and stimulated by hypoxia-reoxygenation condition to explore the expression of CTGF.The expression of CTGF,YAP,p-YAP,p-Smad2/3 were analyzed by Western blot.Explore the expression regulation of CTGF under hypoxia-reoxygenation conditions in LX2 cells treated by small interfering RNA and YAP inhibitor(verteporfin).The nuclear translocation of YAP cells was observed by immunofluorescence.Results:1.In LX2 cells,the expression of CTGF was significantly increased after 3 hours of hypoxia and reoxygenation for 1 hour;the expression of CTGF was decreased after 6 hours of reoxygenation.2.After 3 hours of hypoxia and 1 hour of reoxygenation,the expression of YAP protein increased significantly,the expression of YAP in the nucleus increased,the expression of phosphorylated YAP protein decreased significantly,and the expression of phosphorylated Smad2/3 protein has no significantly change.3.The expression of CTGF decreased after YAP knocked down by small interfering RNA(Si-YAP).4.After using Verteporfin inhibit YAP protein function,CTGF expression decreased.The expression of CTGF decreased after treated by Verteporfin.Conclusion:1.Under hypoxia-reoxygenation condition,the expression of CTGF in hepatic stellate cells was increased.2.The expression of CTGF in hepatic stellate cells under hypoxia-reoxygenation conditions is regulated by Hippo/YAP pathway.Part 5:The relationship between CTGF and non-alcoholic fatty liver disease of brain-dead donorsObjective:To explore the expression of CTGF in steatosis liver tissue after brain death.To study the relationship between CTGF expression and nonalcoholic fatty liver disease(NAFLD)in brain-dead donors,and to explore the possibility of CTGF as a biomarker for predicting NAFLD activity in brain-dead donors.Methods:NAFLD model in rats was established by high-fat diet,and the model of brain death model was established by increasing intracranial pressure.Using rat and human liver specimens to study the expression of CTGF in steatotic liver after brain death.Blood samples and liver tissue samples from brain-dead donor were used to explore the possibility of CTGF in blood predicting NAFLD in brain-dead donors.Results:1.In rat and human liver samples,the expression of CTGF and TGF-β in steatosis liver tissue were significantly increased after brain death.2.The concentration of CTGF in blood samples from brain-dead donors was positively correlated with the expression of CTGF in liver tissues.3.CTGF concentration in blood correlates with donor NAFLD activity score.4.The expression of CTGF in the blood from brain-dead donors with medium/highly active NAFLD was significantly higher than that of donors with low/non-active NAFLD.The area under the receiving operator characteristic curve of prediction of moderate/high-activity NAFLD was 0.903.Conclusion:1.The expression of CTGF and TGF-β in steatosis liver tissues increased significantly after brain death.2.The concentration of CTGF in the blood form brain-dead donors can predict the NAFLD activity in brain-dead donors,and it is expected to be biomarker for predicting the activity of NAFLD in brain-dead donors.Conclusion1.There are dynamic changes of CTGF expression in the liver after brain death,which increases first and then decreases.2.CTGF can be detected in liver vascular endothelial cells after brain death.ATF6 upregulate CTGF by binding to the promoter region of CTGF in vascular endothelial cells.CTGF protects liver cells from hypoxia-reoxygenation damage by reducing the accumulation of reactive oxygen species.3.CTGF can be detected in hepatic stellate cells after brain death.The expression of CTGF in stellate cells is regulated by the Hippo/YAP pathway.4.The expression of CTGF in the non-alcoholic steatostic liver is significantly increased after brain death.Blood CTGF can be considered as a potential biomarker to predict NAFLD in brain-dead donors.