Research On The Protection And Mechanism Of Imperatorin Against Acetaminophen-induced Acute Liver Injury And Its Liposomal Preparation

ObjectiveAdverse Drug Reactions(ADR)is an important cause of clinical disease and death.As one of the most serious adverse drug reactions,drug-induced liver injury(DILI)has already become the fifth leading causative factor of death in the world according to data released by the World Health Organization,and has caused more and more attention.Paracetamol(APAP)is one of the most widely used over-the-counter analgesics and antipyretics in the world.APAP is found to cause liver toxicity under excessive intake or excessive accumulation of drug within the body,and even lead to acute liver failure.Studies have revealed that APAP liver toxicity is related to the formation of electrophilic reactive metabolic intermediates,resulting in oxidative stress and mitochondrial function impairment,but the mechanism has not been fully clarified and no specific effective drugs are currently available.Traditional Chinese medicine Angelica dahurica has the functions of detoxification,anti-inflammatory,analgesia and anti-bacterial.Imperatorin(IMP)is the main natural active extract of 6,7-furan coumarins from Angelica dahurica.Recent studies have revealed that IMP are involved in regulation of multiple biological processes,including anti-oxidative stress,anti-inflammatory,anti-cancer,anti-fungal,and glycolipid metabolism and neuroprotection and immune regulation,as well as drug metabolism related enzymes and transporter activities.The potential effects of imperatorin on drug-induced liver injury and the mechanism have not been reported.In this he protective effect of IMP on APAP-induced acute liver injury was investigated from the aspects of liver tissue damage,oxidative stress,apoptosis,inflammatory response,with the molecular mechanism of hepatoprotective effect with IMP based on the FXR nuclear receptor signaling,and the establishment of imperatorin-entrapped long-circulation liposomes to improve drug bioavailability were studied.Methods1.Study on the protective effect of IMP on APAP-induced liver injury and its molecular mechanism.Male C57BL/6 mice(SPF grade)were randomly divided into 5 groups(n=6),including control group,APAP(300 mg/kg)liver injury model group,IMP low dose group(50 mg/kg/d),IMP high dose group(100 mg/kg/d),OCA positive agonist group(10 mg/kg/d).Each group was given IMP and OCA at the dose of gavage.IMP/OCA were administered by gavage for 5 consecutive days,while the normal control group were given the same volume of solvent gavage(0.5%CMC-Na).After starved for 12 hours,all groups were intraperitoneally injected with APAP(300 mg/kg)besides the normal control group given equal volume of normal saline.After 10h of APAP exposure,serum and liver tissues of mice were collected for serum biochemical indexes and histopathological analysis of liver injury.Hepatocytes apoptosis was detected by TUNEL method.And the survival rate of mice was also employed to evaluate the protective effect of IMP on APAP-induced liver injury.The primary mouse hepatocytes(PMHs)were isolated and in vitro cultured to establish an in vitro model of hepatocytes injury.IMP groups were pre-treated with 10 ? M,20 ?M,and then IMP treatment group and the injury model group were added with 10 mM APAP,the blank control group was added with an equal volume of DMSO solution.After incubation of 24 hours,cells were harvested for related index studies.Factors related to oxidative stress,apoptosis and inflammation were investigated.The phosphorylation level of JNK and the expression of Nrf2,Bcl2,Bax in liver tissues of each group were detected by Western blot.The serum GSH/GSSG ratio and MDA level in mice serum were tested by kits.The production of TNF-?,IL-1?,IL-6 and the transcription levels of inflammatory cytokines in liver tissues and PMHs were detected by ELISA and qRT-PCR,and the levels of arachidonic acid inflammatory mediators PGE2?LTB4?TXB2?20-HETE in liver tissues of mice were determined by LC-MS/MS.The cell viability of PMHs damage model with IMP pretreatment was detected by CCK-8 kit.FCMS and fluorescent staining method were adopted to detect the intracellular ROS generation,and the mitochondrial transmembrane potential(??m)was measured with JC-1 to elucidate the protective mechanism through comparative analysis.Transcriptome sequencing and Kyoto Encyclopedia of Genes&Genomes-base analysis were adopted for screening of significantly differentially expressed genes,together with the western blot,qRT-PCR,dual luciferase reporter gene and molecular docking experiments to further verify that IMP has an prominent activation effect on FXR nuclear receptor.2.The molecular mechanism of protection with IMP against APAP-induced liver injury was further investigated through FXR(Nr1h4)-/-mice and primary mouse hepatocytes.FXR-/-male mice were randomly divided into 4 groups(n=6),including control group,APAP(300 mg/kg)liver injury model group,IMP group(100 mg/kg/d),OCA positive agonist group(10 mg/kg/d).Pretreatment of IMP/OCA were given by gavage for 5 consecutive days.After the last administration and fasted for 12 hours,all groups were injected intraperitoneally with APAP(300 mg/kg),besides the normal control group given equal volume of normal saline.After 10 hours of APAP exposure,the serum and liver tissues were collected for biochemical indicators and histopathological analysis of liver injury.The apoptosis in situ was measured using the TUNEL method.By isolating FXR(Nr1h4)knockout PMHs,in vitro model of FXR-/-hepatocytes injury were established,and IMP/OCA groups were pre-treated with 20 ?M IMP,10?M OCA respectively.And then IMP/OCA treatment group and the injury model group were added with 10 mM APAP,the blank control group was added with an equal volume of DMSO solution.After incubation for 24 hours,cells were harvested for study on the protective mechanism with FXR.ROS levels in liver tissues of FXR+/+FXR-/-mice pre-treated with IMP were measured with in vivo small animal imaging system and Western blot was used to detect the expression of antioxidant and apoptosis-related factors Nrf2,Bcl2,Bax in liver tissues of FXR-/-groups.The serum GSH/GSSG ratio and MDA level in mice serum were tested by kits.ELISA and qRT-PCR were used to detect the serum production of inflammatory factors and the transcriptional level of inflammatory factors in FXR-/-mice liver tissue,LC-MS/MS was employed to determine the levels of arachidonic acid.CCK-8 kit was used to determine the cell viability of FXR-/-PMHs damage model with IMP pretreatment,intracellular ROS production were detected by FCMS and fluorescent staining.The mitochondrial transmembrane potential(??m)was measured with JC-1,and the transcriptional level of inflammatory factors in FXR-/-were measured with qRT-PCR.Curative effects of different pretreatment groups were analysed for verification of potential therapeutic target of related pathway.3.Construction of long-circulating imperatorin liposomes and in vivo pharmacokinetics study.With egg yolk lecithin(EPC)and cholesterol as membrane materials,DSPE-PEG2000 as surface active modifier and IMP as substrate,the preparation of long circulating IMP liposomes(IMP-PEG-LPs)was by thin film hydration.The Marvin laser light scattering apparatus was used to verify that the characterization indexes of liposome including encapsulation efficiency,drug loading,particle size and polymer dispersion index(PDI)meet the requirements of liposome preparation.Based on the elucidation of characteristic fragmentation pathway under electrospray ionization source,a liquid chromatography tandem mass spectrometry method for quantitative analysis of IMP with high specificity and sensitivity was established.In vivo pharmacokinetic experiments were performed on SD rats through tail intravenous injection of IMP/IMP-PEG-LPs,with the in vitro release test and erythrocyte hemolysis experiment to testify the bioavailability,stability and initial formulation security of imperatorin liposomes.Results1.Protective effect of IMP on liver injury induced by APAP in C57BL/6 mice.The results showed that compared with the APAP injury model group,high and low dose IMP pretreatment can increase the survival rate of mice(P<0.05).The serum ALT and AST levels were significantly reduced(P<0.05)and the pathological necrosis area of liver tissues were significantly reduced in mice with high dose IMP pretreatment compared with the injury model group(P<0.01).Thus,IMP can offer protection against APAP-induced liver damage.2.IMP pretreatment improves APAP-induced oxidative stress in C57BL/6 mice.High dose IMP pretreatment group significantly increased serum GSH/GSSG ratio(P<0.05)and reduced serum MDA content(P<0.01),and the transcriptional levels of SOD2 and Nrf2 mRNA in liver tissue tissues and PMHs were significantly increased(P<0.05).Western blot results showed that the expression level of Nrf2 in liver tissues of IMP pre-treated mice was increased,indicating that IMP could prevent liver tissues and hepatocytes damage by inhibiting oxidative stress caused by APAP.3.IMP prevents APAP-induced apoptosis in C57BL/6 mice liver.The results showed that compared with the APAP injury model group,the ROS levels detected with Flow Cytometry and fluorescence microscopy in high-dose pre-treated PMHs were significantly lower compared with the model group(P<0.05).Western blot results showed that the level of JNK phosphorylation in liver tissue was decreased,and the relative expression level of Bcl2/Bax protein was significantly higher compared with the model group(P<0.05).The result showed a reduction of TUNEL-positive cells and remarkable elevation of cell viability(P<0.01)in the IMP pretreatment group,while the mitochondrial membrane potential was increased,and the transcription level of Bcl2/Bax mRNA was obviously higher than that in the model group(P<0.05),indicating IMP could inhibit apoptosis of hepatocytes through mitochondrial pathway induced by APAP.Both in vivo and in vitro experiments suggested severe liver damage induced by APAP in mice lead to obvious oxidative stress,mitochondrial functional impairment and hepatocyte apoptosis.While high-dose IMP pretreatment can effectively protect against liver damage caused by APAP with ameliorating ROS-JNK activation caused by oxidative stress and regulating the mitochondrial function and hepatocytes apoptosis.4.IMP pretreatment inhibits the expression of inflammatory factors related to APAP-induced liver injury.The levels of TNF-?,IL-1?,IL-6 in mice serum and the transcription levels of TNF-?,IL-1?,IL-6 and COX-2 mRNA both in liver tissues and PMHs in the high dose IMP pretreatment group were significantly reduced compared with the injury model group(P<0.05).The levels of prostaglandin E2(PGE2),IeukotrieneB4(LTB4),thrombinB2(TXB2),and 20-hydroxyeico-satetraenoic acid(20-HETE)in high dose IMP pre-treated mice liver tissues decreased significantly(P<0.05),suggesting that IMP pretreatment can inhibit the inflammatory response induced by APAP.5.Validation of the molecular mechanism of protection of IMP against APAP liver injury through FXR(Nrlh4)-/-knockout mice and hepatocytes.After the FXR gene was knocked out,the serum ALT and AST levels,GSH/GSSG ratio,MDA,TUNEL-positive cells of apoptosis,and the area of liver histopathological necrosis of mice pre-treated with high dose IMP and OCA positive agonist showed no significant difference with the model group(P>0.05).Western blot results showed that there was no significant difference of the expression level of Nrf2 and Bcl2/Bax in liver tissue.In vivo small animal imaging of ROS in liver tissue showed that with FXR knockout,high dose IMP pretreatment lost the inhibitory effect on liver tissue ROS production.Transcription levels of TNF-?,IL-1?,IL-6,COX-2 mRNA and the expression of arachidonic acid inflammatory mediators in liver tissue of FXR-/-mice were not significantly different compared with the model group(P>0.05).FXR-/-PMHs pre-treated with IMP/OCA showed no significant difference in cell viability,ROS production,mitochondrial transmembrane potential(??m),and the transcription levels of SOD2,Nrf2 mRNA and cytokines with model group(P>0.05).The results above showed that IMP exerts hepato-protective effect through FXR activation.6.Characterization and pharmacokinetics of long circulating liposomes of imperatorin.The average particle size of the liposomes was 125.30±0.95 nm with Zeta potential of-24.64±0.67.The Polymer dispersion index(PDI)was 0.25±0.04,the Encapsulation rate(EE,%)was 6.27±0.77,and the Loading efficiency(LE,%)was 41.06±5.41 respectively.The results revealed that the liposome size was small and evenly distributed with proper sphericity(PDI<0.30).The encapsulation rate and loading rate were in line with the requirements of liposome loading.Comparison of in vivo pharmacokinetic parameters in SD rats between IMP and IMP encapsulated in Long-Circulating Liposomes showed that the bioavailability(AUC)of the liposome-entrapped imperatorin was significantly increased with the elevation of the area under the first-order moment curve(AUMC),Mean Residence Time in-vivo(MRT)(P<0.01)and significant reduction in total plasma clearance(CLz)(P<0.01).The average drug retention time(MRT)and terminal elimination half-life time(t1/2z)also increased,indicating the better efficacy and action time of the long-circulating liposomes.And the in vitro results showed that the IMP liposomes have modified sustaining of drug release and no significant hemolysis.Conclusion1.IMP pretreatment can significantly attenuate the acute liver injury induced by APAP overdose.2.The Mechanisms of protection by IMP against APAP induced acute liver injury is related to anti-oxidative stress,anti-apoptosis and inhibition of expression of inflammation-related factors.3.IMP exerts a protective effect against APAP-induced liver injury depending on activation of FXR pathway.4.Long-circulating liposomes of IMP(IMP-PEG-LPs)can effectively improve the bioavailability and prolong the in vivo duration of drug action.