Hepatotoxicity And Molecular Mechanism Of Pyrrolizidine Alkaloids

Pyrrolizidine alkaloids（PAs）are common plant secondary metabolites,which has serious hepatotoxicity,pulmonary toxicity,nephrotoxicity,cardiotoxicity,neurotoxicity,carcinogenicity and teratogenicity.Hepatic sinusoidal obstruction syndrome（HSOS）occurs when humans intake herbal or herbal infusions containing PAs for a long time.There is currently no effective clinical treatment for HSOS.Therefore,a comprehensive assessment of the hepatotoxicity of PAs for HSOS patients treatment is crucial.Intermedine（Im）is one of PAs with a high content in comfrey,which belongs to retronecine-type monoester.The unsaturated double bond in the structure is active,and it has hepatotoxicity after metabolic activation.At present,there are few studies on the cytotoxicity and toxicity mechanism of Im,and there is also a lack of study on joint cytotoxicity and toxicity mechanism.In this paper,the toxic effects of eight PAs on four animal cells were studied,and the cytotoxicity evaluation method was used to elucidate the toxicity and toxicity mechanism of Im on hepatocytes.And we further used the method of cytotoxicity evaluation to explore the joint hepatotoxicity and toxicity mechanism of Im and lycopsamine（La）.In order to provide basic data for the safety evaluation and risk assessment of PAs,the main research results are as follows:1.To reveal the cytotoxicity of eight PAs to four animal cells:The cytotoxicity of intermedine,intermedine N-oxide,lycopsamine,lycopsamine N-oxide,retrorsine,retrorsine N-oxide,senecionine and senecionine N-oxide on mouse primary hepatocytes,human hepatocytes（Hep D）,mouse hepatoma Cells 22(H₂₂)and human hepatoma cells（Hep G2）were detected by CCK-8 experiment.The results confirmed that eight PAs had inhibitory effect on the proliferation of four cells,among which the half maximal inhibitory concentration of Im on mouse primary hepatocytes,human hepatocytes（Hep D）,mouse hepatoma Cells 22(H₂₂)and human hepatoma cells（Hep G2）,namely IC₅₀ values,were 49.4μg/m L,71.7μg/m L,48.5μg/m L and 56.6μg/m L,respectively,and the IC₅₀ values of the other seven PAs on the four types of cells were 30-90μg/m L.2.To explore the reason why Im inhibited the proliferation of Hep D cells by using the cytotoxicity evaluation method.The treatment concentrations of treatment is 0,20,50,75,and 100μg/m L,and the treatment time was 24 h.According to the results of cell morphology observation,cell colony formation,wound healing and apoptotic cell detection experiment,Im induced Hep D cells apoptosis to inhibit cell proliferation.3.To clarify the mechanism on hepatotoxicity of Im.Reactive oxygen species fluorescent probe DCFH-DA,mitochondrial membrane potential probe JC-1 and biological transmission electron microscopy（TEM）were applied,these results showed that Im induced Hep D cells death by activating intracellular oxidative stress,mitochondrial membrane potential drop,and destroying mitochondrial structure.The results of western blot assay confirmed that Im triggered Bax expression to increase mitochondrial membrane permeability,release cytochrome c（Cyt c）in mitochondria into the cytoplasm.Then,Cyt c combined with apoptotic protease activating factor-1（Apaf-1）),leading to the activation of caspase-9,thereby activating the caspase-3 pathway and triggering apoptosis on Hep D cells.4.It was confirmed that the hepatotoxicity of mixed PAs was greater than that of single PA.In vitro,CCK-8 experiments confirmed that the low concentration（5μg/m L）of intermedine,intermedine N-oxide,lycopsamine,lycopsamine N-oxide,retrorsine,retrorsine N-oxide,senecionine and senecionine N-oxide mixture has significant inhibitory effect on Hep D cell viability,compared with single PA.These results indicated that low dose PAs mixture has high cytotoxicity and high safety risk.The mixture of Im and La was selected as the target to further explore the toxicity and mechanism of Im and La mixture on Hep D cells according to the method of cytotoxicity evaluation.5.It was confirmed that the hepatotoxicity of Im and La combination was greater than Im,and that the PERK/e IF2α/ATF-4/CHOP pathway in endoplasmic reticulum and the caspase-3 pathway in mitochondrial was involved in hepatocyte apoptosis induced by Im and La.According to the results of CCK-8 experiment,compared with Im or La,the mixture of Im and La significantly inhibited the proliferation of Hep D cells.In vitro,it was confirmed qualitatively and quantitatively that Im and La mixture induced Hep D cells apoptosis by inhibiting cell proliferation,colonization and migration ability,according to the results of cell morphology change,cell colony formation,wound healing,Annexin V/PI double staining assay and flow cytometry assay.Furthermore,reactive oxygen species fluorescent probe DCFH-DA,mitochondrial membrane potential fluorescent probe JC-1,mitochondrial localization detection probe Mito-Tracker Red CMXRos,calcium ion fluorescent probe Fluo-4AM and endoplasmic reticulum localization probe ER-Tracker Green demonstrated that Im and La mixture initiated intracellular oxidative stress,decrease in mitochondrial membrane potential,mitochondrial dysfunction,calcium ion imbalance and endoplasmic reticulum stress-induced apoptosis.At the same time,the biological electron microscope images showed that the mixture of Im and La severely damaged the mitochondrial structure,and the results of western blotting experiments confirmed that the caspase-3 pathway of mitochondrial apoptosis and the PERK/e IF2α/ATF-4/CHOP pathway of endoplasmic reticulum stress were both involved in Im and La co-induced hepatocyte apoptosis.Taken together,monoester-type PAs（Im）can induce hepatotoxicity and induce hepatocyte apoptosis by triggering oxidative stress reactive oxygen species burst and mitochondrial apoptosis pathways in hepatocytes.Furthermore,the complexation of two monoester PAs（Im and La）was able to significantly induce hepatotoxicity and induce hepatocyte apoptosis by triggering intracellular endoplasmic reticulum stress,reactive oxygen species burst,and mitochondrial apoptosis pathways.