The Protective Effects And Mechanisms Of Oridonin On Lipopolysaccharide/D-Galactosamine-Induced Acute Liver Failure

Backgroud:Acute liver failure is an uncommon but rapidly progressive inflammatory disease.Despite improvement in intensive care management,mortality continues to be 40%to 80%.Emergent liver transplantation is currently the only effective treatment for those patients who are unlikely to spontaneously recover.Therefore,it is of great clinical significance to explore a new strategy for the prevention and treatment of ALF.The acetylation,an important symbol of epigenetic,plays an important role in regulating gene transcription in response to cellular changes of the internal and external environment.Oridonin is a well-known tetracyclic diterpenoid isolated from the Chinese medicinal herb Rabdosia rubescens,also known as dong ling cao.Moreover,oridonin has been found to display anti-inflammatory effects.Thus,we propose that oridonin can inhibit hepatic inflammation and attenuate later hepatocyte apoptosis via regulating the activity of acetyltranseferase.Objective:（1）Assess the acetyltransferase inhibition activity of oridonin in vivo and in vitro.（2）Assess the protective effects of oridonin on LPS/D-Gal-induced ALF.（3）Explore the protective molecular mechanisms of oridonin on ALF.Methods:（1）Assess the effects of oridonin on the acetylation of H3,H4 and a-tublin in activated hepatic stellate cell JS1 as well as in LPS/D-Gal-induced ALF.（2）The mice were randomly divided into five groups（n=30 per group）:the control group（a）,the ALF group（b）,two oridonin treatment groups（c and d）,and the oridonin control group（e）.Among them,in group（c）:oridonin（0.2mg/0.5ml）was administered 1h prior to the LPS/D-Gal challenge,and in group（d）:oridonin（0.2mg/0.5ml）was administered every four days for a total of three doses,with the last dose given 1h before the LPS/D-Gal challenge.The potential effects of oridonin on the 48h survival rate in LPS/D-Gal-challenged mice were evaluated.Another set of mice were allocated to five groups（n=5 per group）as described and sacrificed after 6hours of LPS/D-Gal challenge.Liver histological changes and serum enzymes were analysed.Hepatocyte apoptosis and apoptosis-related proteins were determined.（3）Next-generation RNA-SEQ analysis was adopted to elucidate the underlying protective mechanisms of oridonin.We mainly focused on genes that were induced by LPS/D-Gal and then downregulated upon oridonin treatment.Hepatic MPO activity was analysed.The downregulated genes,as verified by real-time PCR and western blot,were subjected to GO and KEGG pathway analysis.Results:（1）Oridonin,an acetyltransferase inhibitor,decreased the acetylation level of H3,H4 and a-tublin in activated hepatic stellate cell JS1 as well as in LPS/D-Gal-induced ALF.（2）Pretreatment with oridonin improved the survival rate of LPS/D-Gal-challenged mice,consistent with the alleviation of histopathological abnormal changes and the suppression of plasma aminotransferases.Oridonin attenuated the LPS/D-Gal-induced hepatocyte apoptosis,as estimated by a reduction of pro-apoptotic signals,such as TNF-αand JNK.Furthermore,JNK-related mitochondrial pro-apoptotic proteins were also suppressed upon oridonin treatment.（3）278 genes induced by LPS/D-Gal and then downregulated upon oridonin treatment.GO analysis revealed that these genes were highly concentrated in immune response,chemotaxis and inflammatory response biological processes.Consistent with RNA-SEQ results,real-time PCR showed that the expressions of these genes were induced by LPS/D-Gal challenge and decreased upon oridonin treatment.What’s more,the hepatic MPO activity induced by LPS/D-Gal challenge was inhibited upon ordionin treatment.KEGG pathway analysis showed that the significant enrichment of these genes were in the TLR4 signaling pathway.Our results verified that the increased phosphorylated levels of upstream signal molecules in TLR4 signaling pathway,such as P-JNK,P-ERK,P-P38 and P-IκB were all significantly suppressed by oridonin.A further experiment revealed that stimulation with LPS/D-Gal also triggered the modifications including the phosphorylation in IRAK4(Thr³⁴⁵/Ser³⁴⁶)and acetylation in IRAK4(Lys³⁴).The modified pattern of IRAK4 in the presence LPS/D-Gal was significantly attenuated by oridonin treatment.Conclusion:（1）Oridonin manifested the acetyltransferase inhibitor activity.（2）Oridonin has protective effects on LPS/D-Gal-induced ALF in mice.Oridonin could inhibit hepatic inflammation and attenuate a later hepatocyte apoptosis signal.（3）The protective mechanisms of oridonin may be related to the interference of posttranslational modifications of IRAK4 including the phosphorylation of Thr³⁴⁵/Ser³⁴⁶ and the acetylation of Lys³⁴,resulting in the inhibition of IRAK4 kinase activity and the interaction between IRAK4 and My D88.