| Part 1:Human brain death-mediated donor liver damage and poor prognosis of liver transplantation had the potential to be associated with excessive complement activation.Objective:Brain death donor(BDD)is the main source of liver transplantation donor organs.A small number of previous clinical studies suggested that brain death(BD)may cause donor liver damage and that the damage may be associated with excessive complement activation after BD.However,the effect of BD and its overactivated complement cascade pathway on the prognosis of donor liver and receptor liver transplantation was unknown.Therefore,through collecting and comparing the clinical data and changes in relevant physical and chemical testing indexes in two the different ways of BD liver and live liver transplantation,this paper intended to discuss the role of complement in liver damage caused by BD and early liver damage after transplantation,as well as the comparison of their postoperative survival and complications in liver transplantation from two different sources in ways that investigate the correlation between complement activation,BD-mediated liver damage and poor prognosis of BD,thereby providing clinical data for studying BD-mediated liver donor damage and its potential molecular mechanism.Methods:Relevant clinical data from patients undergoing liver transplantation from January 2017 to October 2020 were retrospectively collected.Also,the clinical blood and liver tissue samples matched with patients collected were divided into brain death liver transplantation(BDT)and living donor transplantation(LDT).Emphasis of laid on comparing the differences between liver damage and survival outcomes after transplantation.Hematoxylin-eosin(H&E)staining was used to analyze the hiopathological characteristics liver tissue of liver damage of liver donor tissue before and after transplantation,and immunohistochemical staining was used to analyze the deposition of C3d,liver damage marker CD68 in the liver donor tissue.The expression level of AST,ALT in different serum was detected by ELISA as a way to compare the differences of these indicators in the two groups,the incidence,and the survival of postoperative complications.Continuous data were applied to present mean average and standard difference,and T-test to compare difference.Median(min-max)was adopted to show categorical data;Mann-Whitney U to compare difference.Two groups of survival data differences were compared using Kaplan-Meier survival analysis.Results:Total 100 eligible patients were included.Among them,27 cases were included in BDT group and 83 cases in LDT group.According to survival analysis,6 of 27 BDT subjects died of liver failure within 37 days after transplantation,one of 27 LDT subjects died of primary inactive liver within one week after surgery and one of those died of liver failure caused by hepatic arterial embolism.Kaplan-Meier survival analysis suggests that the cumulative 1-year survival rate of BDT and LDT groups is 77.8%and 97.5%,respectively.LD group survival rate is higher than that of BD group(P<0.05).Compared with those in LDT group,patients in BDT group are likely to develop such postoperative complications as acute rejection,pulmonary infection,acute liver failure,hepatic encephalopathy,and intra-abdominal infection(P<0.05).The expression levels of AST and ALT of patients with BDT are higher than that of those of patients with ALT(P<0.05).Similarly,H&E staining of liver tissue of patients experiencing intertransplantation showed that the damage of BDT liver tissue was higher than that of the LDT liver(P<0.05).CD68~+monocyte line infiltration HE score of liver tissue in LDT group was lower than that in BDT group(P<0.05).Furthermore,in terms of evaluating complement deposition,the expression level of C3d in LDT liver was significantly lower than that in BDT liver,and the expression level of the expression level of was positively correlated with liver damage(P<0.05).Conclusion:Compared with living donor,BD causes severe donor liver inflammation and damage,and cause poor prognosis.BD-mediated liver damage and poor prognosis of BD donor liver transplantation were associated with the overaction of the complement system in human liver transplantation cases.Part 2:Mouse brain death-mediated donor liver damage and poor prognosis of liver transplantation are related to complement C3-C3a R signaling pathway.Objective:Through the analysis of the first part of clinical data,Compared with living donor,Brain death(BD)causeed severe liver inflammation and damage,and cause poor prognosis in donor.BD-mediated liver damage and poor prognosis of BD donor liver transplantation are associated with the overaction of the complement system in human liver transplantation cases.In addition,previous studies suggested that the complement activation-dependent pathway played an important role in liver damage and liver regeneration after mouse heatectomy.In order to further understanding the mechanism of BD on donor liver damage,we established a mouse brain death donor liver model to explore the specific molecular mechanism of relevant liver damage of complement activation-mediated BD.Methods:A wide-type mouse BD model of complement gene knockout background was established.Serum and liver tissue samples from different time nodes of various mouse models were collected.Hematoxylin-eosin(H&E)staining was used to detect liver tissue damage score and C3d;ELISA was adopted to detect the expression levels of inflammatory factors such as serum ALT,AST,C3a,MCP,IL-6,and TNF-a.TUNEL detection was used to analyze the degree of hepatocyte apoptosis after BD.Quantitative Real-time PCR was used to detect the m RNA expression levels of P-selectin,E-selectin,and ICAM-1.Western blot was used to detect the expression level changes of apoptotic pathway-related proteins of caspase-3,caspase-9,caspase-11,Bax,and Bcl-2.Gain and loss experiments were used to verify the role of the complement component in BD-related liver damage.Results:Compared with that in sham operation group,the arterial blood pressure of mice in BD model decreases significantly after 20 min,and finally stabilizes at about 60 mm Hg(P>0.05).The results of histomorphological analysis of liver tissue of BD mice showed that compared with that in sham operation group,the liver tissue damage score of BD mice was higher after 6h of BD induction(P>0.05),namely,liver damage in BD mice was increased.Additionally,HE score of Cd3 deposition in BD mice liver tissues increases(P<0.05),and the correlation analysis suggested that BD-related C3d deposition was positively associated with the degree of hepatic tissue damage,Similarly,the levels of ALT and AST were increased in BD donor serum markers(P<0.05).In addition,the apoptosis rate of hepatocytes in BD group was higher than that in sham operation group(P<0.05).Accordingly,the expression levels of pro-apoptotic proteins of Caspase-3,caspase-9,caspase-11,and Bax in BD mice were higher than those in sham operated mice(P<0.05),while the expression of anti apoptotic protein Bcl-2 was lower(P<0.05).That is,BD has mediated donor liver damage by promoting apoptosis.The expression levels of mice serum inflammatory markers such as MCP-1,IL-6,and TNF-αin BD group increased compared with that in sham operation group(P<0.05).Meanwhile,q RT-PCR results suggested that the expression levels of P-selectin,E-selectin,and ICAM-1m RNA in the liver tissue of mice in BD group also increased(P<0.05).Both suggested that BD can aggravate tissue damage by promoting the formation of an inflammatory microenvironment in the liver.Liver transplantation was performed after BD-induced 6h.C3a,C3d,HE scores from BD donor,serum ALT,serum AST,and liver tissue increased compared with that after LD donor liver transplantation(P<0.05).That was to say,initial BD-induced liver damage significantly enhanced subsequent liver damage and inflammatory cascade response after transplantation,while complement C3 may played an important role in BD-induced liver damage.C3d expression was missing or had a low level of expression in the C3 gene knockout background(C3-/-)BD mouse model,as compared to that in sham operation group and wild-type mouse models(P<0.05).The histological determination of C3 activation product(C3a)level and liver damage in the above models showed that the expression of C3a,AST,and ALT was missing or has a low expression level of expression in C3-/-BD mice model.However,after supplementing C3a,the expression levels of C3a,AST,and ALT in C3-/-BD mice returned to the original(P<0.05).The same results were also observed in C3a receptor(C3a R)knock background(C3a R-/-)BD mouse model(P<0.05).This showed that the complement system was at least partially regulated through the C3a-C3a R signaling pathway in the BD-mediated liver damage.Conclusion:BD had mediated donor liver damage by activating complement systems and promoting complement deposition,inflammatory microenvironmental formation,and apoptosis,while the C3a-C3a R signaling pathway participates in liver damage caused by BD.Part 3:Complement inhibitor CR2-Crry reduced brain-dead donor liver damage and liver cascade damage after transplantation in mice and improved the prognosis of liver transplantation.Objective:BD mediated donor liver damage and cascade damage of liver transplantation by activating complement systems and promoting complement deposition,inflammatory microenvironmental formation,and apoptosis.Furthermore,the complement system C3a-C3a R signaling pathway was involved in liver damage caused by BD.Can the targeted complement activation pathway improve the liver damage caused by BD and the prognosis after liver transplantation?Currently,no relevant reports are available yet.Therefore,we would prepare the inhibitor CR2-Crry through the targeted complement C3activation pathway to study whether the inhibitor of targeted complement C3activation pathway of CR2-Crry could reduce the BD-induced liver donor liver damage and inflammation,and whether it could reduce liver damage after BD for liver transplantation and improve the prognosis after donor liver transplantation,and further study the possible downstream signaling pathway mechanism.Methods:The purified complement inhibitor CR2-crry was prepared,and wild-type mice donor liver model and liver transplantation model were established.Serum and liver tissue samples from different time nodes of various mouse models were collected.Hematoxylin-eosin(H&E)staining was used to detect liver tissue damage score and C3d;ELISA was adopted to detect the expression levels of inflammatory factors such as serum ALT,AST,C3a,MCP,IL-6,and TNF-a.TUNEL detection was used to analyze the degree of hepatocyte apoptosis after BD.Quantitative Real-time PCR was used to detect the m RNA expression levels of P-selectin,E-selectin,and ICAM-1.Western blot was used to detect the expression level changes of apoptotic pathway-related proteins of caspase-3,caspase-9,caspase-11,Bax,and Bcl-2.Results:Compared with wild-type BD group mice treated with PBS alone,HE score of C3d deposition level and liver damage in the liver tissue of CR2-Crry-treated BD mice decreased(P<0.05).In addition,CR2-Crry also reduced the expression level of serum ALT,AST,C3a in BD mice(P<0.05).Consistent with the above results,expression levels of inflammatory factors such as MCP,IL-6,TNF-a could be reduced after the CR2-Crry targeted inhibitory complement activation pathway(P<0.05).The q RT-PCR results also suggested that CR2-Crry reduced the expression levels of the adhesion molecules P-selectin,E-selectin,and ICAM-1 m RNA(P<0.05).In terms of apoptosis,after being processed by CR2-Crry,the expression of pro-apoptotic proteins Bax,c-caspase 3,caspase 9and,caspase 11 was low compared with that in PBS processing group,but the expression of anti apoptotic protein Bcl-2 was increased(P<0.05).In accordance with the result,TUNEL analysis showed that CR2-Crry reduces hepatic apoptosis(P<0.05).That meaned CR2-Crry-targeted inhibitory complement activation pathway reduced BD-induced donor liver damage,inflammatory microenvironmental formation,and apoptosis,as well as reduce BD cascade damage after donor liver transplantation.Similarly,the liver damage,inflammatory reaction and hepatocyte apoptosis in BDR+CR2-Crry(donor and receptor)group were significantly lower than those in BDR+CR2-cry(receptor)group(P<0.05).Conclusion:The CR2-Crry targeted inhibitory complement activation pathway reduced BD-induced donor liver damage,inflammatory microenvironmental formation,and apoptosis,and cascade damage of BD donor liver-associated liver transplantation.Part 4:Activating pathway of complements PI3K/AKT/NF-κPI3K/AKT/m TORC2 reduced BD cascade damage for donor liver transplantation.Objective:BD had mediated donor liver damage and cascade damage of liver transplantation by activating complement systems and promoting complement deposition,inflammatory microenvironmental formation,and apoptosis.Meanwhile,the targeted inhibitory complement activation pathway could reduce the BD-induced liver donor liver damage,inflammation microenvironment formation,apoptosis,and cascade damage of liver transplantation associated with BD-induced donor liver.However,the downstream signaling pathway mechanism after complement activation remains unknown.To further explore the mechanism where the complement activation pathway regulated BD-induced transplanted liver cascade damage,we would further investigate the underlying downstream signaling pathway mechanisms via proteomics,bioinformatics,etc.Methods:Proteomic analysis was performed on liver tissues of LD receptor(LDR)+PBS,BD receptor(BDR)+PBS,LDR+CR2-Crry and BDR+CR2-Crry groups through LC-MS/MS to obtain differential proteins and key proteins.Bioinformatics analysis method was used to perform GO analysis and KEGG analysis of the selected differential expression protein gene to construct the protein-protein interaction network.Biochemical methods such as western blot and q RT-PCR were adopted to verify the final discovery of the differential protein and its signaling pathway.Related factor expression of liver tissue damage markers,complement composition,inflammatory factors,and apoptosis were detected based on the methods used in the above experiments.TUNEL method was adopted to detect apoptosis degree and analyze the survival of mice in each group.Results:A total of 4859 differentially expressed proteins were screened by proteomics analysis of protein spectra of LD and BD groups.By comparing the differences between LDR+PBS group and BDR+PBS group,185 differentially expressed proteins were identified,and 208 differentially expressed proteins were identified in LDR+PBS group and LDR+CR2 group.In addition,200differentially expressed proteins were detected in BDR+PBS group and BDR+CR2-crry group.The properties and commonness of the differentially expressed proteins obtained were further analyzed by Venn diagram.KEGG analysis showed that there are the same signaling pathways of PI3K/AKT and m TOR in LDR+PBS and BDR+PBS group,LDR+PBS and LDR+CR2 group,and BDR+PBS and BDR+CR2 Crry group.Compared with that in LDR+PBS group,the expression of PI3K in BDR+PBS group was decreased(P<0.05),and PI3K expression in LDR+CR2-Crry was higher than that in BDR+CR2-Crry group(P<0.05).That was to say,the activation of the complement is negatively associated with PI3K expression,suggesting that the complement system was involved in the regulation of the PI3K signaling pathway.Westernblotting detection of expression of middle and downstream proteins of PI3K enrichment pathway founded they were on AKT signaling pathway downstream of PI3K.The expression levels of p-AKT(Thr 308)and p-Bad in BDR+PBS group were lower than those in LDR+PBS group(P<0.05).The expression levels of p-AKT(Thr308)and p-Bad were increased after being processed by CR2-Crry.On the NF-κB signaling path downstream of AKT,the expression levels of p-P65 and p-IKKB in BDR+PBS group were higher than those in LDR+PBS group(P<0.05).The expression levels of p-P65 and p-IKKB were decreased after being processed by CR2-Crry.However,on the m TOR2 signaling path downstream of AKT,the expression levels of p-m TOR,p-SGK1,and p-Akt(Ser473)in BDR-PBS group were lower than those in LDR+PBS group(P<0.05).The expression of p-m TOR,p-SGK1,p-AKT(Ser47),and p-Rictor were higher after being processed by CR2-Crry.On the downstream m TOR1 signal pathway,there was no difference of p-70S6 and p-Raptor between the two groups(P>0.05).The survival analysis showed that the survival rate of all treatment groups was higher than that of BDR+PBS group(P<0.05).Conclusion:Complement activation is negatively associated with the PI3K signaling pathway.Complement activation mediates BD-induced cascade damage for corresponding liver transplantation by the PI3K/AKT/NF-B and PI3K/AKT/m TORC 2 signaling pathways.CR2-Crry activated the PI3K signaling pathway by targeting and inhibiting complement activation,causing activation of downstream AKT and m TORC2 phosphorylation and inactivation of the NF-κB pathway,thereby reducing liver apoptosis and inflammation.Hence,targeted inhibition of complement activation pathway can regulate PI3K/AKT/NF-B and PI3K/AKT/m TORC 2 signaling pathways to improve BD donor liver damage and prognosis of liver transplantation. |
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