The Mechanism Of Liver Injury Induced By High L-carnitine Ingestion And The Regulatory Effect Of Quercetin In Mice

Recent findings showed that L-carnitine(a traditional nutritional methyl-donor foods)can be metabolized by the gut microbiota and liver to trimethylamimine N-oxide(TMAO),leading to the vascular damage.The findings were successively reported in Nature(2011),Nature Med.(2013)and Cell(2015)by SL Hazen(a research group in USA).Furthermore,we have preliminarily found that L-camitine plays a strong harmful role in the liver,and dietary quercetin exhibit antagonistic effects against the damage.However,the speciifc mechanism of hepatoxic damage of L-camitine and its regulation remains fully unclear,and therefore,the investigation is an urgent need.With this in mind,3%high L-carnitine drinking water was used to feeding mice for 12 weeks to investigate the mechanism of liver injury and the protective effect of quercetin by using LC-MS/MS,16S rRNA,fermentation in vrtio,transcriptome technique and other modern molecular biology techniques.This study will also provide the theoretical basis for the scientific knowledge on nutrition and safety of L-carnitine foods.The may results were as follows:(1)Hence a simpler and faster performance liquid chromatography-tandem mass spectrometry method was developed and validated.The quantitative analysis was achieved within 6 min by using Agilent 6460C UPLC-MS/MS with 10%methyl alcohol isocratic elution.Furthermore,method verification result showed that the method correlation coefficient was 0.9997,0.9999 and 0.9878,and the detection limit was 0.121,8.063 and 0.797 ?g/L,and the CV values of the retention time and peak area of analytes was less than 0.331%and 3.280%,respectively.Quantitative recoveries of TMAO,TMA and DMA were in the range of 94.2%-101.0%,and the RSD values were lower than 5.17%.The verification results indicating that the method was more simple,convenient and rapid than that of previously reported methods.(2)After established the determenation of L-camitine metabolites,the effects of high L-carnitine ingestion on the liver function and metabolites distribution in mice were investigated.The physiology and biochemistry index results showed that high L-carnitine ingestion could induce liver injury,which was proved by the increases of serum AST and ALT activities,production of inflammatory liver cytokines(IL-1,IL-6,TNF-?,and TNF-?),lipid metabolism(TC,TG,HDL,and LDL)disorder,decline of antioxidant ability(SOD,GSH-Px,MDA,and RAHFR)and obvious cell necrosis and cytoplasmic vacuoles.In addition,high L-carnitine ingestion could induce the accumulation of TMAO,TMA and DMA in mice.This study put forward the negative effects of high L-camitine on mice and it will provide more theoretical basis for the scientific knowledge on the nutrition and safety of L-carnitine.(3)Recent studies have emphasized the significant role of gut microbiota on inducing liver injury,therefore the mechanism of high L-carnitine induced liver injury on the levels of microbita was investigated by using 16S rRNA sequencing,fermentation study in vitro and additional animal tests.16S rRNA sequencing results showed that the abundances of Anaerobiospirillum,Helicobacter,Akkermansia_muciniphila(AKK)and Coriobacteriaceae had a significant increases in high L-carnitine treated mice.Meanwhile,the abundance of Bacteroidales_S24_7group decreased when the mice were treated with high L-carnitine.In order to verify the role of AKK in the hepatotoxicity induced by high L-carnitine in mice,a fermentation validation experiment in vitro was designed and the results showed that L-carnitine could be metabolized to TMA in vitro by AKK,indicating that AKK plays a key role in the liver injury of high L-carnitine induced.In addition,The supplement of AKK exacerbates the hepatotoxicity induced by high L-carnitine ingestion in mice,validating the role of AKK in the hepatotoxicity induced by high L-carnitine ingestion.The specific manifestations of AKK aggravating high L-carnitine induced liver injury in mice are as follows:further increases of transaminases,more severe inflammation,further damage of the antioxidant ability and further accumulation of TMAO in systemic circulation.(4)Excluding the disorder of microbiota constitution,another underlying mechanism may be related to the differential expression of genes because they have been reported to have a big relationship with some physiological and pathological processes.We here assessed gene expression in liver using transcriptomics to investigate the key biological processes and pathways regulated by high intake of L-carnitine.After mapping 51.67 GB clean RNA-SEQ reads of all mice against the Mus musculus genome,we detected 23,952 genes.High L-carnitine intake induced 298 genes differential expression compared with chow diet,most of which were enriched in liver injury-related KEGG pathways,i.e.AMPK,PPAR and bile secretion(the accumulation of Cyp7al,Sult2a7,Abcb1a,Slc4a5,Abcc4,Scd1,Pltp,Ccnd1 and the decrease of Slcolal,Fabp1,Cpt1b,Fabp5,Lepr and Fbp2).Consistently,high intake of L-carnitine elevated genes related to the GO terms via upregulating genes expression.Specifically,lipid biosynthetic process,steroid biosynthetic process,cellular response to chemical stimulus were regulated.(5)The regulatory of hepatotoxicity induced by high L-carnitine in mice is an important research field as well.Thus high L-carnitine mice were treated with 150,300,450 mg/kg of quercetin and the physiological and biochemical index results showed that quercetin could effectively antagonism liver injury induced by high L-carnitine ingestion,which was proved by the decrease of transaminase levels,the remission of inflammatory response,the repair of antioxidant system,the acceleration of lipid metabolism and the decrease of TMAO levels in systemic circulation.The research provided an important theoretical basis for the study on the regulation of hepatic toxicity induced by high L-carnitine ingestion in mice.In conclusion,this article put forward that high L-carnitine could induce hepatotoxicity in mice and the mechanism was investigaed from the level of gut microbiota and the genes.At the same time,the study also showed that quercetin could effectively antagonism liver injury induced by high L-carnitine ingestion.This study will provide more theoretical basis for the prevention of sub-healthy condition.