The Mechanism Of EYA-1in Stress Response And The Pharmacologic Action Of Compound L-carnitine In Caenorhabditis Elegans

PART1: EYA（eyes absent）belongs to an evolutionary conservative gene family,A highly conserved271-amino acid C-terminal region called EYA domain was foundin the products of human EYA family members. They were first identified inDrosophila as key co-regulators for eye development and subsequently they wereidentified in a number of species ranging from worms, fish, birds, mice and to humans.They are now implicated in processes as disparate as organ development, innateimmunity, DNA damage repair, angiogenesis, and cancer metastasis. In Drosophila,specific and recessive EYA mutations result in elimination of compound eyes inviable flies. In humans, EYA is also implicated in several diseases, such as themulti-organ developmental disorder branchio-oto-renal syndrome, congenital cataracts,and are overexpressed in numerous types of cancers. To further study the biologicalfunction of EYA-1, by using the C. elegans worm as a model system, we isolatedEYA-1of C. elegans and set up an effective feeding-based RNAi (RNA interference)against the gene.Our results indicated that C. elegans eyes absent ortholog EYA-1is required forstress resistance. Under heat stress, EYA-1knockdown shortened the mean lifespanby16.8%, which could be attributed to the decrease of heat shock protein-16.2(hsp-16.2) expression. Under oxidative stress, EYA-1knockdown could shorten themean lifespan by18.7%which could be attributed to its intracellular ROSaccumulation and the decrease of superoxide dismutase-3(sod-3) protein expression.In addition, our study also demonstrated that knockdown of EYA-1accelerated theonset of paralysis mediated by Aβ1–42proteotoxicity and polyQ in CL4176andAM140, respectively. Further studies suggested that knockdown of EYA-1could not inhibit daf-16nuclear accumulation in wild-type worms in response to stress. On theother hand, EYA-1deficiency did not further reduce stress resistance of daf-16mutants, which is stress sensitive. Quantitative real-time PCR results also showed thatthe expression of two daf-16target genes hsp-12.3and sod-3were down-regulated inEYA-1RNAi-treated worms under stress. All evidence indicated EYA-1is requiredfor stress resistance of worms, and it might act downstream of daf-16to regulatestress resistance-associated genes expression. We believe that these findings willprovide new insights into the role of EYA proteins. PART2: By using the C. elegans worm as a model system, we examined theeffect of L-carnitine, trimetazidine, compound L-carnitine on the lifespan andreproductivity of the worms. Our results showed that0.1%L-carnitine,0.0005%trimetazidine and compound L-carnitine did not change the lifespan of C. elegans. Onthe other hand，0.1%L-carnitine,0.0005%trimetazidine significantly reduced thebrood size in parental generation, F1and F2. However, compound L-carnitine has noeffect on the reproductivity of the worms. Worms undergoing DR utilize fat, anddiminish reproductive output. That is to say, animals with restricted dietary intakehave reduced broods. The accumulation of fat was visualized using Oil-Red-Ostaining. Fatty acid methyl esters (FAMEs) were analyzed by gas chromatography.The results indicated that0.1%L-carnitine,0.0005%trimetazidine reduces in both fataccumulation and overall fatty acid. However, compound L-carnitine has no effect onthe overall fat and fat acid. This study provides a theoretical basis for the pharmacologic action on compound L-carnitine.