Protective Effect Of Cationic Antimicrobial Peptides Against Acute Liver Failure Induced By D-GalN/LPS In Mice

Background and Objective Liver failure is a serious liver disorders such as liversynthetic function,detoxification,excretion and biotransformation functions that caused bymany factors,leading to severe decompensated in liver function,appearing jaundice,coagulation disorders,hepatic encephalopathy and ascites as the main manifestation of aclinical syndrome.Liver failure has rapid progression, many complications, poor prognosisand high mortality.At present, although orthotopic liver transplantation and artificial liversupport therapy are effective means of treatment in liver failure, but the donor liverdeficiency, complex operation, and other shortcomings of rejection after surgery, make itdifficult to widely promote for the former; the latter is also facing the problems of thetension of plasma source and the poor treatment for advanced liver failure.So looking for anew treatment against liver failure is imperative.Secondary liver injury whose core isintestinal endotoxemia is the important reason leading to rapidly progress indisease.Therefore,it is a hot subject for exploring the treatment of endotoxin neutralizationand blocking the progression of liver failure caused by endotoxemia.AMPs are a class of small gender peptides with net positive charge, which is widelydistributed in living organisms. The study found, AMPs have strong ability of endotoxinneutralization, and could inhibit the release of inflammatory cytokines. Based on this, thestudy investigated the feasibility of AMPs against liver failure by in vitro experiments andanimal models in mice.Methods We first studied the efficiency of endotoxin neutralization by differentkinds of AMPs in vitro.Detected the neutralization of endotoxin on four kinds ofAMPs(PY,Ts,LL-37,Th)by LAL assay;Detected cytotoxicity effects of AMPs onRAW264.7by CCK-8assay;Evaluated hemolysis activity of four kinds of AMPs by Enzyme standard instrument; Evaluated the rate of hepatocyte apoptosis on AMPs by flowcytometry; Established the mice model of acute liver failure induced by D-GalN/LPS andobserved the survival rate of the model mice and pathological changes of the livertissue,then detected Caspase-3expression of apoptotic-related protein byimmunohistochemistry and Western blot.Conclusion1. Effect of neutralizing endotoxin:PY,Ts,LL-37,Th have strongbinding to endotoxin, and there is a certain dose relationship, with the increasing of AMPsconcentration, the rate of binding was significantly increasing; PY and LL-37havestronger neutralizing capacity compared to Th and Ts; Further inter-group comparison,there was statistically significant difference between different concentrations(P <0.05);there was statistically significant difference between fourAMPs in any of1、10、100umol/L(P <0.05).2. The study of cytotoxicity:,Ts,Th have weak cytotoxicity, no obvious relationshipin the dose; LL-37has a dose relationship, and only at relatively high concentration (>100umol/L) showed significant cytotoxicity; PY has obviously dose-dependent incytotoxicity,at high concentration (>40umol/L), there is strong cytotoxicity,and in lowconcentration only has weak cytotoxicity; below10umol/L，four group have higher cellviability. Further inter-group comparison, there was statistically significant differencebetween PY, Ts, LL-37Th in different concentrations(P <0.05), and with decreasingconcentration gradually,the cytotoxic of AMPs was gradually reduced; there wasstatistically significant difference between any concentration in fourAMPs(P <0.05).3. The study of hemolysis activity:Four kinds of AMPs have weak hemolysis below40umol/L;and in relatively high concentrations (>80umol/L) still showed slight hemolysisactivity, while LL-37showed obviously hemolysis activity. Further inter-group comparison,there was statistically significant difference between four kinds of AMPs in differentconcentrations(P <0.05), and with decreasing concentration gradually,the hemolyticactivity of AMPs was gradually reduced; there was statistically significant differencebetween any concentration in fourAMPs (P <0.05). 4. The protective effect of the primary hepatocyte apoptosis in mice:Compared withthe control group, four kinds of AMPs have inhibition for hepatocyte apoptosis, and theinhibition of PY was the most obviously; compared with the control group, there werestatistically significant difference in four kinds of AMPs respectively(P <0.05);At the sameconcentrate of different AMPs inhibit hepatocyte apoptosis are showed: PY> LL-37> Ts>Th; Ts, Th, The hepatocyte apoptosis rate of LL-37are much lower in80umol/Lconcentration; while the hepatocyte apoptosis rate of PY was much lower in40umol/Lconcentration than other groups.5. Survival of mice models of liver failure:PY, LL-37, Ts, Th have some influencefor survival rate in liver failure mice, and there is a dose relationship; PY and LL-37in3mg/kg, as well as Ts and Th in6mg/kg have higher survival rate in liver failure mice;during1.5mg/kg and6mg/kg, the survival rate was increasing in Ts, Th with the doseincreasing.6. Histological examination:HE staining: four kinds of AMPs respectively comparedwith the control group, the degree of liver tissue injury was as follows: PY1.5> controlgroup> PY6> PY3; Immunohistochemistry was used to detect the expression ofCaspase-3(cleaved) from liver tissue,the results can be seen, Caspase-3expression incontrol group was significantly higher, but not or little expression in normal liver tissue;The levels of Caspase-3expression showed that PY1.5>control group> PY6> PY3>normal group.7. Western blot:the gray area ratio were as follows：PY1.5>control group>PY6>PY3>normal group; Compared with control group,there were statistically significantdifference in normal group,PY3,and PY6(p <0.05);there were statistically significantdifference between PY1.5and PY3,as well as between PY1.5and PY6.Conclusion By comparing the effect of neutralizing endotoxin,cytotoxic,hemolyticactivity and inhibition of LPS induced primary hepatocyte apoptosis in mice on PY, Ts,LL-37and Th in vitro experiments,and found that PY has low cytotoxicity and strongendotoxin neutralization. Further studied the protective effect of PY against acute liver failure induced by D-Galn/LPS in mice,and found that a specific dose of PY cansignificantly improve the performance of liver tissue in liver failure mice, inhibit theapoptosis-related protein expression of Caspase-3and significantly improve the survivalrate of mice.