Multicenter Investigation Of Non-biological Artificial Liver System For Acute Liver Failure And Mechanism Of Rotenone Protect Against Liver Failure

Acute Liver Failure(ALF)refers to a large area of hepatocyte necrosis or severe liver function damage caused by various causes.Compared with adult ALF,the cause of pediatric acute liver failure(PALF)is complicated.It is an acute onset,rapid progress,and extremely high mortality.It is still one of the treatment challenges faced by pediatric intensive care.The current non-biological artificial liver treatment has greatly improved the survival rate of ALF patients in the treatment of acute liver failure,but there is still much controversy about the specific methods.Acetaminophen(APAP)is widely used in pediatric clinical antipyretic analgesics.Excessive intake of APAP can cause ALF.Rotenone is mainly derived from the roots and stems of the Lonchocarpols and Derris species and is a strong inhibitor of the mitochondrial respiratory chain(MRC)complex I.In this study,in order to determine the role of mitochondrial complex I in APAP-induced liver injury,we treated mice with the mitochondrial complex I inhibitor rotenone before exposure to APAP and found that rotenone has significant protection against APAP hepatotoxicity.effect.AAP-induced ALF is protected by inhibiting abnormal mitochondrial activity and subsequent mitochondrial oxidative stress and inflammatory response.Purpose: 1.Construct a multi-center joint clinical research collaboration network to carry out a non-biological artificial liver support system for the treatment of acute liver failure in children.Compare the effects of two different blood purification methods on children with acute liver failure.2.Animal experiments observed the protective effect of rotenone on acute liver failure induced by acetaminophen in mice.3.To explore the molecular mechanism of the protective effect of rotenone on acute liver failure induced by acetaminophen in mice.Method: 1.Construct a multi-center combined clinical research collaboration network,a non-biological artificial liver support system in children with acute liver failure,a prospective,multicenter,randomized,controlled clinical study In conjunction with the PICU of 27 domestic top three hospitals including the ICU of Beijing Children’s Hospital,a multi-center joint clinical research collaboration network was constructed to apply the unified diagnostic criteria and randomized randomization method to classify the selected cases into two groups.Group: Continuous static-venous hemodiafiltration(CVVHDF)+ plasma exchange(TPE)+ standard medical treatment(SMT);Group B: If necessary,CVVHDF + TPE + SMT treatment protocol for comparison.A prospective,multicenter,randomized,controlled clinical trial of a non-biological artificial liver support system in children with acute liver failure was performed to compare the therapeutic effects of two different intensities of blood purification therapy on acute liver failure in children.2.Animal experiments,observe the protective effect of rotenone on acute liver failure induced by acetaminophen in mice(1)Exploring the appropriate dose of acetaminophen to construct a stable animal model of acute liver failure caused by acetaminophen poisoning in BALB\c mice.(2)The rats were fed with rotenone diet.Compared with the normal diet group,the serum function of liver was measured by ELISA,and the liver injury was observed by pathology.The effect of the therapeutic dose of rotenone on the liver of mice was observed.(3)7-8 weeks old BALB\c mice were randomly assigned to groupA: control group(Vehicle),group B: acetaminophen liver failure(APAP),group C:rotenone pretreatment + APAP liver failure(APAP+ Rot).ELISA method was used to measure acute liver injury indicators and pathological observation of liver injury.3.To explore the molecular mechanism of rotenone on the protective effect of acetaminophen on acute liver failure in mice.(1)qRT-PCR method was used to determine the mtDNA level of liver in three groups of animals;(2)Western-Blot method and qRT-PCR method were used to determine the oxidative stress index of liver tissue,and rotenone treatment was used to reduce the oxidative stress induced by APAP;(3)qRT-PCR method was used to measure inflammation in liver tissue.Response indicators,observation of rotenone treatment to reduce acute inflammation caused by APAP;(4)Western-Blot method and qRT-PCR NADPH oxidase 4 expression;(5)Mitochondrial complex III azoxystrobin treatment failed to improve APAP-induced liver toxicity in mice.Results 1.This study finally included 188 cases of PICU from 15 tertiary hospitals in China.Severe sepsis is still the most important cause of acute liver injury in children.The main cause of drug-induced liver damage is APAP accounting for 12.2% of all cases.Compared with two different blood purification methods,the 28-day survival rates were 58.33% and 55.32%,and the 90-day survival rates were 52.08% and 46.81%,respectively.No statistically significant difference was found.ALT level in the continuous treatment group decreased significantly on the first day after the artificial liver began treatment.On the 5th day,ALT and AST levels in the two groups were significantly lower than those in the continuous treatment group.On the 5th and 7th day of treatment,the serum bilirubin level of the CRRT treatment group decreased significantly when necessary in the continuous treatment group.There was no significant difference in improvement in coagulation between the two groups.Blood examination between the two groups found that the levels of Hb and PLT in the continuous blood purification treatment group decreased significantly,and there was a statistically significant difference between the two groups.There was no significant difference in mechanical ventilation time and ICU staying time between the two.There was no difference in the pediatric critical illness score(PCIS)and the Glasgow score(GCS).Comparing the total hospitalization cost and the cost of ALSS treatment during the treatment of the two groups of patients,the total cost of ICU hospitalization in the continuous CRRT group increased significantly,and the cost of artificial liver treatment increased significantly.2.The toxicity test of rotenone found that rotenone at 250 PPM had no significant effect on liver pathology and blood biochemical ALT and AST in mice.A stable mouse model of APAP liver injury was successfully established by intraperitoneal injection of 300 mg/kg APAP solution.APAP treatment resulted in liver center lobular necrosis after 24 hours of APAP administration,and rotenone pretreatment significantly attenuated cell necrosis.Plasma ALT of Vehicle group,APAP group and APAP+ Rotenone group were 82.86±68.49 IU/L,17825±7731 IU/L,2125±3191 IU/L,respectively.AST levels of the three groups were 230.0±348.1 IU/ L,11950 ± 6240 IU / L,1282 ± 1906 IU / L.The levels of GSH in liver tissue of the three groups were 8.157 ± 1.211 μmol/mg,4.42 ± 1.386 μmol/mg,and 7.257 ± 1.141 μmol/mg,respectively.Rotenone pre-treatment significantly extenuated the elevation of ALT and AST caused by APAP and significantly inhibited GSH consumption.3.qRT-PCR was used to detect the mtDNA copy and 13 mitochondrial genes in the liver tissue of the Vehicle,APAP and APAP+ Rotenone groups.The expression levels of mtDNA in the three groups were 1.0±0.256,0.68±0.131,1.053±0.261,and the rotenone significantly improved.APAP-induced downregulation of mtDNA in hepatocytes.APAP administration resulted in a significant decrease in mRNA levels of SOD1 and SOD2 in liver tissue,and a significant decrease in SOD2 protein expression.Rotenone treatment reduced down-regulation of SOD2 induced by APAP.The increased oxidative stress marker MDA in the liver can also be blocked by rotenone pretreatment.APAP-induced up-regulation of inflammatory cytokines by IL-6,IL-1β and MCP-1 can be blocked by rotenone pretreatment.APAP significantly down-regulated the protein and mRNA levels of NOX4 in mouse liver,which was significantly reversed by rotenone treatment.Similar to AAP treatment alone,large AMLX pretreatment group also showed massive hepatocyte necrosis,and elevated ALT and AST plasma levels were not blocked by AZOX.Conclusion 1.Acute liver failure in children remains an important challenge for PICU.Compared with the artificial liver model of CRRT+TPE when necessary,more blood purification treatments can significantly improve the patient’s liver function,but it does not significantly improve the patient’s final survival rate,and does not shorten the patient’s hospital stay and relying on the time of respiratory support,increases the patient’s need for blood transfusions and significantly increases the patient’s hospitalization costs.2.Mitochondrial complex I rotenone can improve liver function and mitochondrial abnormalities in mice ALF induced by APAP.3.Rotenone treatment can protect acute liver injury induced with APAP in mice,by blocking APAP-induced liver oxidative stress,down-regulating inflammatory response,up-regulating NOX4 expression.The mitochondrial complex III azoxystrobin treatment has no such effect.