Studies Of Mechanism And Hepatoprotection In The TACE-induced ALI

Primary liver cancer(PLC)is severely harmful to human health because of its high incidence and lethality.Transcatheter arterial chemoembolization(TACE)has become the first choice in the treatment of advanced PLC not available for surgery.The most common complication is acute liver injury(ALI)after TACE.Patients with severe liver injury are associated with ascites,jaundice,hepatic encephalopathy,and even acute liver failure,leading to treatment-related deaths.Therefore,how to reduce the incidence of ALI caused by TACE,improving quality of life and prolonging survival of patients,has always been hot and difficult in the treatment of advanced PLC.TACE includes hepatic arterial infusion chemotherapy and embolization,and the main mechanisms of TACE-induced ALI are as follows:(1)Hepatic arterial infusion chemotherapy induces drug-induced liver injury(DILI).Studies have confirmed that chemotherapeutic agents and their electrophilic products in the metabolic process can promote the production of a large number of reactive oxygen species(ROS)in mitochondria,which induces oxidative stress liver injury.(2)Embolic agents induce ALI.That superselective perfusion chemoembolization(SPC)may be difficultly completed because of tumour feeding vessels distortion and discount,and that hepatic sinusoids and rami anastomoticus may occur,result in an influx of embolic agents into the normal liver tissue,which leads to liver cell injury with ischemia and hypoxia.Thus producing a large number of ROS in liver tissue during ischemia,hypoxia,and ischemia-reperfusion further worsens the oxidative stress liver injury.The double-injury of normal hepatic tissue during arterial chemoembolization is closely related to the production of ROS,ischemia,hypoxia,ischemia-reperfusion,mitochondrial injury,and so on.The oxidative damage induced by excess ROS may be one of the important mechanisms of liver cell damage.Currently,the effect of ROS on TACE-induced ALI is still not clear.Doxorubicin(DOX)has a strong anti-tumor activity and a wild anti-tumor spectrum so that it is usually prescribed for arterial chemoemboliaztion in PLC.While it can be an effective anti-tumour agent,doxorubicin has strong cytotoxicity,which causes serious heart,liver,and kidney toxicity,especially for dose-dependent cardiotoxicity and irreversible cardiomyopathy.Its severe adverse events in individual patients make it difficult to popularize its efficient and broad-spectrum application.Therefore,it is of great significance to find the measures reducing the cardiac,hepatic and renal toxicity caused by DOX,and to increase the safety in its clinical application.The study demonstrates that ROS is the main toxic effect mechanism of DOX.Also,Magnesium isoglycyrrhizinate(MgIG)has been shown to have the effects of anti-inflammation,anti-oxidation,anti-apoptosis,and reduction in cytosolic Ca2~+.However,research on whether or not it improves the DOX-induced liver and heart injury during arterial infusion of chemotherapy has not been reported.Previous studies have confirmed that the application of hepatoprotective drugs after TACE can effectively improve the liver function of patients.However,there are many different types of hepatoprotective drugs.The choice and combined application of hepatoprotective drugs to promote the recovery of liver function after TACE is still not clear,and there are no medication standards or guidelines at home and abroad.Therefore,clear the correlation between different hepatoprotective regimens and liver function recovery as well as screening out the optimal hepatoprotective regimen for ALI induced by TACE have very important clinical significance.MgIG is a new multifunctional hepatocyte protective agent;MgIG can prevent DILI induced by chemotherapeutic drugs and has the potential for clinical application in ischemia-reperfusion.At present,the effect of MgIG combined with different hepatoprotective drugs and different hepatoprotective regimens on the treatment of ALI induced by TACE has not been previously reported.Conebeamcomputedtomographythree-dimensionalhepatic arteriography(CBCT-3DHA)provides a solid technical support for the comprehensive and accurate SPC.Currently,the application of CBCT-3DHA technology in PLC is still in its initial stages,lack of experience.Therefore,it has very important clinical significance to clear the application value of CBCT-3DHA in SPC.This study combined clinical samples with experimental animal models,the aims were as follows:1)After interventional operation of PLC patients,we investigated the ALI mechanism by analyzing MDA content and SOD,GSH-Px,ALT,AST,TBIL and DBIL levels in the preoperative and postoperative serum.2)After an animal model with liver and heart injury induced by DOX was established,the histopathological analysis of mice liver and heart was performed,and proteins Bax,Bcl-2,Caspase-3,and NF-κBp65were detected by Western blot,while apoptosis of myocardial cells were measured by TUNEL assay;the concentrations of MDA,CK,CK-MB,LDH,AST,ALT,SOD,and GSH-Px were analysed to better understand the protective effect and the mechanisms of MgIG on DOX-induced liver and heart injury.3)In this study,the effects of MgIG combined with different hepatoprotective drugs and regimens on TACE-induced ALI were analyzed by comparing the hepatic biochemical markers(ALT,AST,TBIL,and DBIL)under the preoperative and postoperative conditions.4)By comparing the liver biochemical indexes(ALT,AST,TBIL,DBIL)before and after operation,operation time,and contrast agent dosage,we analyzed the effects ofALI induced by conventional TACE(cTACE)and accurate TACE(aTACE)and the clinical application value of CBCT-3DHA-assisted aTACE in PLC.Part I The mechanism of TACE-induced ALI in PLC.Objective:We studied the mechanism of TACE-induced ALI by analyzing the content of MDA and the levels of liver biochemical indicators(SOD,GSH-Px,ALT,AST,TBIL,and DBIL)in serum before and after interventional operation,and their correlation with ALI,which provided theoretical basis for clinical hepatoprotective treatment.Methods:30 patients diagnosed with PLC by at least two kinds of imaging examinations,including CT,MRI,and ultrasonography,were selected.No hepatoprotective or any other medical pretreatment was given to patients.Venous blood of operative patients was collected and centrifuged in the 3 days before operation and at the 1st,3rd,5th days after operation.The levels of MDA in serum as well as SOD,GSH-Px,ALT,AST,TBIL,and DBIL were measured.Results:1 The content of MDA in the serum at the postoperative 1st,3rd,5th days was significantly higher than that in the preoperative 3 days.The difference was statistically significant(P<0.01).2 The levels of serous SOD and GSH-Px at the 1st day after operation was significantly higher than that before operation,and the difference was statistically significant(P<0.01).It produced opposite effect at the postoperative 3rd,5th days.However,the difference was statistically significant only at the postoperative 3rd day(P<0.01).3 The levels of serous ALT,AST,TBIL and DBIL were significantly higher in the postoperative measurements compared with preoperative measurements,and the difference was statistically significant(P<0.05).Conclusion:TACE-induced ALI was closely related to the production of ROS.The damage mechanism might be excessive ROS-induced oxidative damage resulted in serious dysfunction of hepatocyte membrane and mitochondria.Thus,increased oxidants and liver enzymes,as well as decreased antioxidants eventually caused oxidative stress liver injury.With the time went by,the performance of liver injury was different.Part II The mechanisms of magnesium isoglycyrrhizinate ameliorating doxorubicin-induced acute cardiac and hepatic toxicity in mice.Objective:This study investigated the effect and potential mechanisms of magnesium isoglycyrrhizinate(MgIG)in mice,by which cardiac and hepatic-toxicity caused by doxorubicin(DOX)was reduced.It provided theoretical basis for the treatment of ALI induced by TACE and the auxiliary treatment of liver and heart injury induced by antitumour drugs.Methods:The 50 Kunming mouse were subject to adaptive feeding and randomly divided into 5 groups,including a control group[Control,saline,intraperitoneal(i.p.)injection];drug-treated groups of low-dose MgIG(L-MgIG,10mg/Kg/d,i.p),middle-dose MgIG(M-MgIG,20 mg/Kg/d,i.p),and high-dose MgIG(H-MgIG,40 mg/Kg/d,i.p)as well as a model group(DOX,30 mg/Kg,i.p).Drug-treated groups accepted MgIG injection intraperitoneally every morning for 10 days.On the eighth day,both the drug-treated groups and the model group were administered DOX once;a control group was administered normal saline once.After 48 hours,the serum of all mice was separated for biochemical analysis after blood collection.The protective effect of MgIG was assessed directly by hepatic and cardiac histology detection and indirectly by serum levels of CK,CK-MB,LDH,AST,ALT,SOD,GSH-Px,and the concentration of MDA.The apoptotic hepatocytes and myocardial cells were measured using a TUNEL assay.Furthermore,apoptosis-related proteins Bax,Bcl-2,Caspase-3,and NF-κBp65were detected using Western blot.Results:1 The morphological changes of liver and heart histology:The hepatocytes became disordered and fatty,the nuclei were swollen,and the liver cells dissolved due to putrescence in the DOX group.Also,the cardiomyocytes showed a disordered arrangement,a destructive structure,some degeneration,and necrosis.In the MgIG pre-treatment groups,the heart and liver injury scores were statistically different compared to the DOX group(P<0.01).A significant difference was also seen in the heart and liver injury scores among MgIG pre-treatment groups,that is,the L-MgIG,M-MgIG,and H-MgIG groups(P<0.01).2 Comparison of serous biochemical indicators:We detected the activity changes of two hepatic-specific enzymes(AST and ALT)and three cardiac-specific enzymes(CK,CK-MB,and LDH)in serum to determine the protective effect of MgIG.Compared with the control group,levels of biochemical indicators in the DOX group obviously increased with statistical difference(P<0.01).Meanwhile,they were significantly lower in the MgIG pre-treatment groups than in the DOX group(P<0.01).3 Analysis of the levels of SOD and GSH-Px and the concentration of MDA:The three oxidative stress markers SOD,GSH-Px,and MDA in serum were measured to assess the oxidative stress level caused by DOX.Compared with the control group,SOD and GSH-Px activities were notably reduced,while the MDA concentration was remarkably increased in the DOX group.Pre-treatment with MgIG clearly increased the SOD and GSH-Px activities and reduced the MDA concentration.Both results were statistically significant(P<0.01).4 Expression levels of apoptotic markers including Bax,Bcl-2,Caspase-3,and NF-κBp65:Several apoptosis markers were expressed in normal liver and heart tissue,including a certain amount of Bax,Bcl-2,Caspase-3,and NF-κBp65,and the expression of Bax was significantly lower than Bcl-2.Compared with the control group,protein levels of Bax,Caspase-3,and NF-κBp65 were rapidly up-regulated,while the expression of Bcl-2 was significantly reduced in the DOX group(P<0.01,respectively).Also,comparing the DOX group with the MgIG pre-treatment groups,it produced the same results in a dose-dependent manner(P<0.01).5 TUNEL staining for apoptotic cells:TUNEL staining was used to assess the cardiac and hepatic cell apoptosis.The extent of apoptosis in hepatic and cardiac cells remained at a consistently low level in the control group,while TUNEL-positive tubular cells were dramatically increased in the DOX group compared with the MgIG pre-treatment groups(P<0.01,respectively).Moreover,MgIG treatment decreased the extent of apoptosis in a dose-dependent manner.Conclusion:Mg IG could ameliorate DOX-induced liver and heart injuries in mice.Its protective mechanism may be related to Mg IG enhanced the antioxidant capacity of liver cells and myocardial cells and inhibited cellular apoptosis.Part III Comparative study among different hepatoprotective schemes with MgIG in ALI induced by a TACE.Objective:The objectives of this study were to explore effects of combining different hepatoprotective drugs and schemes with MgIG in aTACE-induced ALI and to understand the relativity among different schemes and liver function recovery.It provided a theoretical basis for clinical hepatoprotective treatment.Methods:180 patients with PLC were randomly divided into 6 groups(A group,B group,C group,D group,E group,F group).Each group was given double-drugs combination therapy after aTACE.The A,B,C,D,E,and F groups individually received the following regimens:anti-inflammation drugs andlivercellmembrancerepairagent(Magnesium isoglycyrrhizinate+Polyene phosphatidyl choline injection),anti-inflammation drugs and detoxification drugs(Magnesium isoglycyrrhizinate+Reduced glutathione for injection),anti-inflammation drugs and cholagogic drugs(Magnesium isoglycyrrhizinate+Ademetionine 1,4-butanedisulfonate for injection),liver cell membrance repair agent and detoxification drugs(Polyene phosphatidyl choline injection+reduced glutathione for injection),liver cell membrance repair agent and cholagogic drugs(Polyene phosphatidyl choline injection+Ademetionine1,4-butanedisulfonateforinjection),and detoxification drugs and cholagogic drugs(Reduced glutathione for injection+Ademetionine 1,4-butanedisulfonate for injection).Levels of hepatic biochemical indicators(ALT,AST,TBIL,DBIL)were measured in the3 days preoperatively and at the postoperative 1st,4th,and 6th days.Results:1 No significant difference was seen in the levels of liver biochemical indicators(ALT,AST,TBIL,DBIL)among 6 groups in the 3 days before operation and at the 1st day after operation(P>0.05).2 A significant increase was noticed in the levels of ALT,AST,and TBIL,postoperatively at the 1st,4th,and 6th days compared with the preoperative 3days in the same group(P<0.01).3 Liver biochemical indexes in the A,B,and C groups were significantly lower than that in the D,E,and F groups at the 4th day after the intervention.Also,the C group was the lowest(P<0.01).However,there was no statistical difference when comparing A group with B group,Alao,no statistical difference was seen among the D,E,and F groups(P>0.05).4 Liver biochemical indexes in the A,B,and C groups were significantly lower than those in the D,E,and F groups at the 6th day after the intervention.The C and E groups were respectively lower than the A,B groups and the D,F groups,respectively(P<0.01).However,no statistical difference was found when comparing the A group with B group and comparing the D group with F group(P>0.05).Conclusion:The Mg IG was the optimal hepatoprotective drug in ALI after TACE.The increased efficacy is shown mainly through combination with Ademetionine 1,4-butanedisulfonate for injection and secondly through combination with Polyene phosphatidyl choline injection or reduced glutathione for injection.The levels of liver biochemical indicators were reduced to improve liver function.Part IV A comparative study of ALI induced by conventional transhepaticarterialchemoemboliaztion(cTACE)and accurate transhepatic arterial chemoemboliaztion(aTACE)in PLC.Objective:The objectives of this study were to study ALI induced by conventional transhepatic arterial chemoemboliaztion(cTACE)and accurate transhepatic arterial chemoemboliaztion(aTACE)and to discuss the application value of CBCT-3DHA aTACE in PLC.Methods:120 patients with PLC were randomly divided into the CBCT-3DHA aTACE group and the tablet-DSA cTACE group.Each group included 60 patients separately.Levels of hepatic biochemical indicators(ALT,AST,TBIL,DBIL)at 3 days after intervention and postoperatively at the 1st,3rd,and 5th days,as well as interventional operation time,X-ray radiation dose,contrast media dosage,rate of successful superselective intubation and complications of interventional therapy were recorded.Results:1 No significant difference was seen in levels of ALT,AST,TBIL,DBIL at 3 days before intervention between the aTACE group and the cTACE group(P>0.05).2 A significant increase was noticed in the serous ALT,AST,TBIL,and DBIL levels at the 1st,3th,and 5th postoperative days compared with that in the 3 days before intervention for the two groups(P<0.01).3 The hepatic biochemical indicators(ALT,AST,TBIL,DBIL)were measured higher in the cTACE group than the aTACE group at the 1st,3th,and 5th days after operation(P<0.01).4 Compared with the cTACE group,the operation time was significantly longer in the aTACE group(78.58±5.15min vs 64.48±9.82min,P<0.01);there was no statistical difference in X-ray radiation dose and contrast media dosage(P>0.05).A higher successful rate of superselective catheterization was noticed in the aTACE group(P<0.01).Conclusion:Acute liver injury is an unavoidable complication after TACE.However,CBCT-3DHA aTACE has achieved SPC under the condition of protection of normal liver tissue and obviously reduced the degree of liver damage.It does not increase X-ray dose and contrast media dosage despite its prolonged operation time.