| Silibinin is a natural polyphenolic flavonoid,isolated from the seeds of the milk thistle of Silybum marianum(L.)Gaertn.Silibinin has been widely used as a clinical medicine for liver diseases.Alcoholic liver disease has become one of the main causes of liver injury,and its prevention and cure are important medical tasks.In the body,ethanol is metabolized in the liver by alcohol dehydrogenase,which transforms ethanol into a toxic compound acetaldehyde.This study investigated the protective role of silibinin in ethanol-or its metabolite acetaldehydeinduced apoptosis in human carcinomatous liver HepG2 cells and immortalized liver HL7702 cells,focusing on the elucidation of the underlying mechanism in vitro.MTT results showed that ethanol and acetaldehyde exhibited potent cytotoxicity in liver cells,with 50-150 mM for ethanol and 100-400 μM for acetaldehyde,respectively,while treatment with silibinin(2.5 μM)significantly improved cell viability.Silibinin treatment significantly decreased ethanol-or acetaldehyde-induced apoptosis by western blotting.Mitochondria are important organelle structures that generate energy in cells.Mitochondrial dynamics is an equilibrium process maintained by mitochondrial division and fusion.We examined whether mitochondrial fusion/fission dynamics are involved in ethanol or acetaldehyde-induced cell injury.The results indicated that silibinin inhibited excessive abnormal mitochondrial fission caused by ethanol or acetaldehyde,leading to the change in the balance of mitochondrial dynamics.Treatment with mitochondrial division inhibitor 1 significantly reduced apoptosis induced by ethanol or acetaldehyde,and silibinin further enhanced this protective effect.Collectively,our results identify that ethanol or acetaldehyde induces damage in HepG2 and HL7702 cells by activating DRP1-related mitochondrial fission,while silibinin exhibits protective effects by inhibiting mitochondrial fission.With the study of alcoholic liver injury,we found that ethanol and acetaldehyde significantly reduced the cell viability(cell viability about 50%),while the apoptosis rate only reaches about 25%.We then sought to determine the type of cell death involved in ethanol-or acetaldehyde-treated cases.Inhibitors for apoptosis(ZVAD),necroptosis(necrostatin-1,nec-1),and ferroptosis(ferrostatin-1,fer-1)were applied to HepG2 or HL7702 cells treated with ethanol or acetaldehyde.The results showed that all inhibitors increase the cell viability,and among them,ferroptosis inhibitor fer-1 gave the maximal protection in HepG2 and HL7702 cells,suggesting that ethanol-and acetaldehyde-induced cells injury is accompanied by apoptosis,necroptosis,and ferroptosis,in which ferroptosis plays the major role in the cell death.ROS levels,MDA,and iron ions in ethanol-or acetaldehyde-treated cells were reduced after treatment with Fer-1 or silibinin.Since ferroptosis is a form of death dependent on iron ions,we examined the expression of iron transport receptors(TFR1)and Ferritin.Results showed that ethanol or acetaldehyde alone obviously increased TFR1 but reduced Ferritin,indicating increased cellular iron stress,but these changes were attenuated by silibinin.To determine the degradation of Ferritin,we examined the co-localization of Ferritin and lysosome.The results showed that the co-localization of Ferritin and lysosome was higher in the ethanol or acetaldehyde group than in control group,but reduced by treatment with silibinin.Treatment with ethanol or acetaldehyde induced conversion of LC3-Ⅰ to LC3-Ⅱ and reduced p62(an autophagy substrate)as indicated by western blot results.However,administration with silibinin reduced the conversion of LC3-Ⅰ to LC3-Ⅱ and increased p62.Immunofluorescence results showed that treatment with 3-MA or CQ(an inhibitor of autophagy)reduced colocalization of Ferritin and lysosome in ethanol or acetaldehyde-treated cells,resembling the results with silibinin-treatment in immunostaining experiments.These results confirmed the involvement of autophagic degradation of Ferritin in ethanol-or acetaldehyde-caused ferroptosis.Ferroptosis is associated with severe impairment of mitochondrial morphology,bioenergetics,and metabolism.We examined mitochondrial morphology under the electron microscope.Results showed that the numbers of mitochondria are significantly increased in the ethanol and acetaldehyde groups,but reduced by silibinin treatment.Mitophagy not only controls the number of mitochondria,but also maintains the quality by clearing the dysfunctional mitochondria.Western blot results showed that ethanol or acetaldehyde treatment reduced the expression of mitophagy-related proteins PINK1 and Parkin in mitochondrial fractions.Treatment with silibinin rescued the mitochondrial levels of PINK1/Parkin in ethanolor acetaldehyde-treated.The expression of Ferritin was further reduced and ROS level was increased by PINK1 or Parkin-knockdown,indicating that the absence of PINK1 or Parkin accelerates ethanol-or acetaldehyde-induced ferroptosis in liver cells.Our results showed that ethanol or acetaldehyde caused damaged in HepG2 and HL7702 cells,and then we established an ethanol-induced SD rat liver injury model,and investigated the pharmacological role of ferroptosis in vivo.In vivo,the results indicated that ethanol induced autophagy,elevated ROS levels,and increased oil droplet formation in rat liver,but these were reduced by treatment with silibinin or fer-1 which exerted a better protective effect.The oil droplet was accumulated in the ethanol group indicating that ethanol may have interfered with normal lipid metabolism,leading to liver cellular damage.Ethanol promoted degradation of Ferritin by autophagy,inhibited antioxidant protein GPX4 expression and mitochondrial autophagy,while silibinin and fer-1 attenuated the above experimental changes.Furthermore,silibinin and the ferroptosis-associated proteins(GPX4,ferritin,transferrin,and TFR1)had a stronger combination in molecular docking.The evidence fully supports our hypothesis that inhibition of ferroptosis is involved in the protection of silibinin against ethanol-induced liver injury.In conclusion,we clarify that ethanol or acetaldehyde causes hepatocyte damage by regulating mitochondrial and iron metabolism,while silibinin protects liver cells by regulating mitochondrial function and iron metabolism.This study reveals the underlying pathological mechanisms and the potential therapeutic strategies for alcoholic liver injury. |
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