Genipin Alleviates CCl₄-induced Acute Liver Injury By Restoring Autophagy

Aim:The liver is the largest parenchymatous organ in the body,having important metabolic and clearance functions.Acute liver injury is a liver function abnormality that results from multiple reasons,such as viral infection,abuse of drugs or alcohol,ingestion of toxic substance and so on.Serious or continuous liver injury often gives rise to liver cirrhosis or liver failure and results in death.Autophagy is an essential cytoprotective system which is rapidly activated in response to various stimuli including inflammation and microbial infection.Many studies have shown that autophagy has a protective effect on acute liver injury.Genipin,an aglycone derived from an iridoid glycoside called geniposide,is a major component of the fruit of Gardenia jasminoides.It is well known to have anti-inflammatory,antibacterial and antioxidative properties.However,the contribution of autophagy to genipin-conferred hepatoprotection is still unclear.Methods:Acute liver injury was induced by CCl₄ in vivo and IL-6 in vitro,while it was ameliorated by genipin both in vivo and in vitro.Liver specimens were obtained to examine surface damage.Hepatic damage was quantified via measuring serum concentration of ALT and AST,and liver tissue was subjected to H&E staining.Expression of autophagy-related protein was assessed by Western Blot.The production of serum pro-inflammatory cytokines were determined using Milliplex.Transmission electron microscopy（TEM）was applied to observing the formation of autophagic vesicles.In vitro,AML12 cellular impairment was induced via incubating with IL-6.The production of autophagy-related proteins was compared by western blot,and the expression of pro-inflammatory cytokines was detected by RT-PCR.A fluorescent microscope was applied to observe labeled fluorescence after GFP-RFP-LC3 plasmid transfection.Results:1.In our study,genipin alleviated surface lesion of liver specimens after CCl₄ damaged.And it was statistically significant that genipin decreased serum concentration of ALT and AST after CCl₄ administration.Besides,the group which is deal with genipin obtained lower pathological scores of H&E staining.2.Compared with CCl₄ group,the serum levels of IL-1β,IL-6 and CCL20 significantly decreased after genipin treatment.At the same time,genipin significantly inhibited the activation of NF-κB signaling pathway.The levels of serum IL-33 and IL-17 in the CCl₄ groups were much higher compared with genipin treatment group,no statistical differences were observed.In addition,serum levels of IL-10 significantly increased compared with genipin treatment group.3.The expression of Atg12,Atg5,Atg7increased after CCl₄ challenging,and this increase was further enhanced by genipin.Moreover,genpin restored autophagic flux,which was indicated by increased expression of LC3-II and decreased p62 protein.However,the level of Beclin-1protein expression was not affected among any of the experimental groups.4.A further study on the upstream signaling pathway of autophagy revealed that the phosphorylation levels of mTOR significantly decreased compared with CCl₄ group.Moreover,genipin further increased the levels of p-p38 MAPK compared with CCl₄group,there were no differences in the level of p-JNK and p-ERK protein expression among any of the experimental groups.5.In addition,compared with the basal level of autophagic vacuoles in the control group,the number of autophagic vacuoles was markedly increased in the CCl₄ group and was further increased by genipin.6.To further observe the formation of autophagosomes and autolysosomes in the process of autophagy,AML12 were infected with GFR-RFP-LC3 plasmid.In the present study,the numbers of green and red dots were slightly increased after treatment with genipin.Moreover,treatment with 3-MA attenuated these effects of genipin.Conclusion:These findings suggest that genipin acts as a simple and applicable preconditioning intervention to protect against acute liver injury,and the protective mechanism may involve its ability to induce autophagy.These findings may provide new insights into the functional mechanism of genipin and an important framework for developing novel targeted clinical therapies for acute liver injury.