Study On The Anti-aging Effect Of Agarose Oligosaccharide On Drosophila Melanogaster And Mechanism

Agar gum extracted from marine red algae is widely used in food and other fields,but its complex structure of polysaccharide limits its digestive availability.Agar oligosaccharide(AOS)is a low-molecular-weight saccharide with a degree of polymerization of 2 to 10 obtained from the hydrolysis of agar polysaccharides and has a variety of biological activities such as antioxidant,antitumor,antiviral and immune regulation.In this study,the model organism Drosophila melanogaster was used as the research object to detect the anti-aging effect of AOS and explore its related mechanism of action.Furthermore,J774A1 and RAW264.7 cells were used as models to further verify the anti-inflammatory activity of AOS in vitro.The details are as follows:(1)The anti-aging effects of AOS including the longevity,climbing ability,reproductive capacity were examined for Drosophila respectively fed on the standard and experimental culture medium containing the high-,medium-and low-dose(0.25%,0.125%,0.0625%)of AOS.The results showed that within a certain concentration range,AOS effectively extended the average and maximum lifespan of fruit flies,improved climbing ability,and reduced reproductive capacity.The effect on female fruit flies is not significant.(2)Study of preliminary mechanism about AOS anti-aging effect.1)The activities of Cu,Zn-superoxide dismutase(Cu,Zn-SOD)and catalase(CAT)and the malondialdehyde(MDA)content in Drosophila were examined on the10th,25th and 40th days.The results showed that within a certain concentration range,AOS can significantly increase the activity of Cu,Zn-SOD and CAT and reduce the content of MDA in the male fruit flies.However,it has no significant effect on the antioxidant capacity in female Drosophila.2)The flies'midguts were removed on the 10th and 40th days for analyses of the intestinal microbial community by 16S rDNA sequencing.The results showed that AOS significantly reduced the diversity of intestinal microorganisms and the abundance of Proteobacteria and Firmicutes in the phylum level,but increased the abundance of Gluconobacter in the genus level.For the old female Drosophila,AOS significantly increased the?-diversity of the intestinal microbiota and decreased the abundance of Gluconobacter.3)Real-time PCR(RT-PCR)was used to quantitatively analyze the expression of four intestinal immunity-related genes:Drosocin(DRO),DUOX,IMD,and PIMS.Compared with the control group,the expressions of DRO and DUOX in males were not significantly different in each group,while the expression of PIMS was significantly reduced,and the expression of IMD was significantly increased.The expression of DRO was significantly reduced,the expression of DUOX was significantly increased,and the expression of PIMS and IMD was significantly decreased in the female elderly Drosophila of experimental group.(3)AOS could protect the Drosophila from the SDS or H₂O₂ induced stress injure via the intestinal microorganisms and immune regulation1)The fruit flies were respectively induced by sodium dodecyl sulfate(SDS)and hydrogen peroxide(H₂O₂)to produce the inflammative and oxidative intestinal model.The survival rate of fruit flies in each group was measured.And the ultrastructure of fruit fly midgut epithelium was observed by the transmission electron microscopy after the above treatment for 60 hours.The results showed that AOS can effectively prolong the survival rate of stressed Drosophila melanogaster in each group,and alleviate the damage of Drosophila midgut epithelial cells inspired by SDS or H₂O₂.2)16S rDNA gene analysis was used to detect the intestinal microbial composition of Drosophila intestine after 60 hours of inflammation.The results displayed that AOS increased the?-diversity of intestinal microorganisms of SDS-induced Drosophila and reduced the abundance of Klebsiella aerogenes.3)RT-PCR was used to detect the expression of intestinal immunity-related genes in Drosophila that induced inflammation for 60 hours.The results showed that AOS activated the expression of Att A,Cec C,Dfn,Dpt and 4E-BP;down-regulated the expressions of Relish,Dredd,FADD and m TOR;and inhibited the expression of autophagy-related genes AMPK?,Atg1,Atg5,and Atg8a.(4)Study on AOS anti-inflammatory activity in vitroLipopolysaccharide(LPS)was used to induce inflammation in mouse monocyte macrophages(J774A1)and mouse monocyte macrophage leukemia cells to study the anti-inflammatory ability of AOS in vitro.The results show that AOS can significantly reduce the production and release of Nitric oxide(NO),and inhibit the expression and secretion of Interleukin-6(IL-6)and tumor necrosis factor-alpha(TNF-?)at the protein level and m RNA level.