| Objectives:Previous study in our laboratory has demonstrated that methyl 3,4-dihydroxybenzoate(MDHB)might prolong the lifespan of C.elegans partly via W06A7.4 gene.However,the underlying molecular mechanisms of MDHB in lifespan extension are not yet thoroughly understood.In this study,we exploited the C.elegans model system to explore exact mechanism of life-span-extending activity of MDHB in C.elegans.Methods:1.Wild type(WT)C.elegans were exploited to analyze the lifespan-extension effects of MDHB in C.elegans through lifespan assay,and to analyse the effects of MDHB on fertility,lipofuscin accumulation and activity of superoxide dismutase(SOD)in C.elegans.2.The C.elegans of W06A7.4 gene mutation were gained via a CRISPR-Cas9 gene editing technology,which were used to investigate the effects of the W06A7.4 gene on the lifespan and on the lifespan-extension of MDHB in C.elegans.3.TRAP-ELISA telomerase assay kit was used to examine the effects of MDHB on telomerase content of C.elegans.4.The C.elegans of daf-2(e1370),daf-16(mu86)and daf-2(e1370);daf-16(mu86)mutants were respectively exploited to investigate whether MDHB lifespan prolonged effect is with the help of the daf-2/daf-16 and insulin/insulin-like growth factor-1 signaling(IIS)pathway.daf-2(e1370)mutant can alter the domain of the daf-2 receptor tyrosine kinase,block the daf-2 signal transmission,and the C.elegans with daf-2(e1370)mutant would display an extended lifespan,while daf-16(mu86)mutation results in deletion of most of the daf-16 coding sequence including all of the forkhead domain and show shortened lifespan compared to wild type C.elegans.daf-2(e1370);daf-16(mu86)mutant would make both daf-2 and daf-16 defective.5.Testing expression of specific genes in C.elegans: CF1553(sod-3::GFP)worms and CL2070(hsp-16.2::GFP)worms,stably expressing a sod-3::GFP and hsp-16.2::GFP fusion protein respectively,were used for quantitative analysis of GFP expression in worms exposure to MDHB by a fluorescence microscope.Besides,we visualized the effects of MDHB on the nuclear localization of daf-16 using a specific transgenic TJ356(daf-16::GFP)strain that includes a daf-16::GFP reporter gene integrated in the genome.Futhermore,real-time PCR was used to detect the effects of MDHB on the expression of daf-2 and daf-16 in C.elegans.Results:MDHB extended the lifespan of wild type adult C.elegans by 19%(<0.01),superior to positive drug resveratrol.MDHB could inhibit the proliferation of E.coli OP50 bacterial.MDHB pretreated C.elegans,cultured in alive or in death E.coli,displayed a lifespan extention by 18.41±0.323 days(+19%,<0.01)and 19.76±0.327days(+17%,<0.01)compared to the corresponding normal controls,respectively.When compared with the wild type C.elegans,the lifespan of C.elegans with W06A7.4 mutants,W101,W203 and W213,has reduced by 16%(<0.05),16%(<0.05)and 11%(p<0.05),separately.Our results showed that MDHB could extend the lifespan of these W06A7.4 mutants' C.elegans(W101,W203 and W213 strains),for W101,by +11.6%(p<0.05),W203,+7.9%(<0.05)and W213,+9%(p<0.05).But the lifespan increased by MDHB in the C.elegans with W06A7.4 mutants were less than that in wild type C.elegans(+19%,<0.05).MDHB could extend the lifespan of wild type C.elegans without reducing its fecundity.Compared to untreated controls,intestinal lipofuscin level was reduced by 23.2% in MDHB-treated C.elegans(p< 0.01),and by more than 20% in resveratrol treated C.elegans( < 0.01).Furthermore,we found that MDHB pretreated C.elegans showed higher SOD activity and telomerase activity compared to untreated controls.MDHB extended the lifespan of the adult nematodes with daf-16(mu86)mutant by +7%(p<0.05),but less than the increase caused by MDHB in wild type C.elegans(+19%, < 0.01).Surprisingly,the MDHB-extended lifespan was completely abolished by the daf-2(e1370)mutation and daf-2(e1370);daf-16(mu86)mutation(p<0.05).Besides,MDHB pretreated C.elegans showed lifespan increase under oxidative stress conditions(<0.05).Finally,MDHB protected wild type C.elegans from oxidative damage while did not enhance the nematodes with daf-16(mu86)mutant oxidation resistance(>0.05).MDHB promoted daf-16 into cell nucleus,and enhanced the expression of sod-3 and hsp-16.2 that played a positively role in C.elegans against stress and longevity..Conclusions:1.MDHB could extend the lifespan of wild type adult C.elegans without reducing their fecundity,better than the positive control drug resveratrol.2.MDHB may extend the lifespan of C.elegans by several mechanisms,including upregulating W06A7.4 gene expression,improving the telomerase activity,enhancing the capability of antioxidation and acting as a modulator of the daf-2/daf-16 pathway in C.elegans. |
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