| Objective: To explore the anti-aging effects and mechanism of Dendrobium nobile Lindl alkaloids(DNLA)on Caenorhabditis elegans(C.elegans).Methods: Wild type C.elegans were cultured synchronously to L4 larvae and were randomly divided into four groups as follows: control group,DNLA 0.035 ?g/m L,0.35?g/m L and 3.5 ?g/m L group.Firstly,the effects of different concentrations of DNLA on the lifespan in C.elelgans were observed.Nematodes were incubated for 72 h at 20°C,the heat stress assay(35°C)and paraquat-induced oxidative stress assay were conducted to examine whether the optimal concentration of DNLA could improve the anti-stress ability and extend the lifespan of C.elegans.Microscopic observation of the effects of DNLA on life-related physiological indicators such as reproductive capacity,mobility,pharyngeal pumping rate and lipofuscin fluorescence intensity of nematodes.Real-time fluorescence quantitative PCR(RT-q PCR)was used to assay the effect of DNLA on the m RNA expression levels of aging-related genes,such as the FOXO in C.elegans(daf-16),superoxide dismutase(sod-3),catalase(ctl-1),heat shock protein(hsp16.2)and a homologue of the mammalian nrf2(skn-1).The C.elegans strain of TJ356(daf-16::GFP)were employed to detect the nuclear localization of daf-16 gene by fluorescence microscope.The kit detects the activity of SOD enzyme.Lifespan test of the CF1038(daf-16 deletion)and EUI(skn-1 deletion)mutants were respectively exploited to observe the life extension effect of DNLA on C.elegans after daf-16 and skn-1 gene deletion.Results: In the lifespan assays,the lifespan of wild-type nematodes was prolonged after DNLA administration,and the best concentration of DNLA was 0.35 ?g/m L.The average life expectancy of the C.elegans in the control group was 24.62 days,the average life expectancy of C.elegans in the DNLA group was 27.61 days,and the lifespan was extended by 12.14%.DNLA could also prolong the lifespan of C.elegans under heat stress(35°C)and 2 m M paraquat-induced oxidative stress,and improve the ability of nematodes to resist stress.Compared with the paraquat group,survival curve were shift to right in the paraquat+0.35 ?g/m L DNLA group,and the life-cycle were extended.The average lifespan of DNLA group was increased from 9.15 days to 10.10 days,which was extended by10.38%.In the heat stress experiment,the effect of 0.35 ?g/m L DNLA was more significant,and the average lifespan of C.elegans was increased from 13.33 days to 18 days,an increase of 35.03%.Compared with the control group,the reproductive capacity and pharyngeal pumping rate of the 0.35 ?g/m L DNLA group showed no apparent change,but the mobility was significantly enhanced.The m RNA transcription levels of daf-16,sod-3,hsp-16.2,ctl-1,and skn-1 were up-regulated,after 0.35 ?g/m L DNLA administration for 3 days.TJ356 strain detected daf-16 nuclear localization showed that daf-16::GFP fluorescence localized to cytoplasmic nematodes was reduced to 19.64% after administrated with 0.35?g/m L DNLA,while the intermediate distribution of nematodes was increased from 31.24% to 61.70%,the proportion of nematodes located in the nucleus was also increased to 18.37%.SOD activity was increased by 40.87% after administration of DNLA.After treated the mutant nematode CF1038 and EUI with DNLA,the lifespan of C.elegans strains did not change significantly compared with the control group.Conclusion: Dendrobium nobile Lindl alkaloids can delay the senescence of C.elegans,and its anti-aging mechanism may be associated with up-regulation of daf-16 and skn-1genes and increased anti-oxidative stress. |
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