| Obesity accelerates the aging process,triggers age-related diseases earlier,or aggravates the disease degree.It is of great significance to explore the possibility of delaying natural aging and resisting premature aging caused by obesity.Caenorhabditis elegans(C.elegans)has become the preferred model for research on aging and obesity due to its short life cycle,rapid reproduction and high genetic homology with human beings.Through previous experiments,it was found that the crude polysaccharide of Guangxi sweet tea had the potential of anti-aging and regulating glucose and lipid metabolism.In this study,using C.elegans as a model,polysaccharide fraction with the best anti-aging activity in sweet tea leaves was screened and its structure and function were identified.At the same time,the anti-aging effect and mechanism of sweet tea polysaccharide on normal diet nematodes and obese nematodes constructed with high glucose diet were systematically studied.Firstly,the crude polysaccharide of sweet tea was separated and purified by hot water extraction,step alcohol precipitation and DEAE-52 ion exchange column chromatography,STP-60a was screened as the best anti-aging fraction of sweet tea polysaccharide.The primary structure of STP-60a was characterized by HPGPC,FT-IR,GC-MS,periodate oxidation and Smith degradation,methylation and NMR.STP-60a was a relatively homogeneous component,the polysaccharide purity was 94.21%,and the average molecular weight was 9.16×10~4Da.STP-60a was mainly composed of rhamnose,arabinose,glucose,galactose and glucuronic acid with a molar ratio of1.00:4.12:0.80:10.05:1.68.The main chain of STP-60a was composed ofβ-L-Rhap-(1→,→3)-β-D-Galp-(1→,→4)-β-D-Glcp-UA-(1→,→3,6)-β-D-Galp-(1→,→6)-β-D-Galp-(1→,→3)-4-OAc-β-L-Arap-(1→and→3)-α-L-Araf-(1→,the branch chain consisted ofα-L-Araf-(1→and→3)-α-D-Glcp-(1→.The morphological characteristics of STP-60a were analyzed by Congo red test,scanning electron microscope and atomic force microscope.STP-60a was an irregular sheet with smooth surface and some concave structures.It was in the form of irregular polymer particles in water,but did not have a typical triple helix structure.In the simulated digestion model in vitro,the average molecular weight and reducing sugar content of STP-60a only changed significantly during the simulated gastric juice digestion process,indicating that STP-60a was easily degraded in a gastric juice environment.STP-60a showed excellent antioxidant and immunomodulatory activity,which could eliminate DPPH,ABTS and hydroxyl free radicals,and promote the proliferation of RAW264.7 cells,enhance the phagocytosis of neutral red and the secretion of NO,TNF-αand IL-1β.The effects of STP-60a on delaying the senescence of C.elegans and the signal pathways regulating the longevity of C.elegans were explored.STP-60a significantly prolonged the longest life span and average life span of normal diet nematodes,reduced the accumulation of lipofuscin,and improved many physiological indicators(exercise ability,pharyngeal pump rate,stress resistence).STP-60a increased the activity of SOD,CAT and GSH-Px,decreased the content of ROS and MDA,and affected the redox system of nematodes.Using q RT-PCR and transgenic nematodes to study the key pathway,the results showed that STP-60a prolonged the life span of normal diet nematodes mainly through insulin,mitochondrial respiratory chain and autophagy pathway.STP-60a significantly increased the expression of three transcription factors(DAF-16,SKN-1 and HSF-1)and their target genes(sod-3,ctl-2,prdx-3,gst-4,gst-7,hsp-12.6,hsp-16.1,hsp-16.2)downstream of insulin signaling pathway in normal diet nematodes,and enhanced the accumulation of DAF-16 and SKN-1 in the nucleus.The deletion mutation of daf-16,skn-1 and hsf-1 prevented STP-60a from prolonging the life span of nematodes.At the same time,STP-60a could improve the mitochondrial function of normal diet nematodes,increase the activity of mitochondrial respiratory chain complex I and complex II,regulate the expression of mitochondrial respiratory chain genes(mev-1,clk-1,isp-1),and induce mitochondrial unfolded protein reaction.In addition,STP-60a activated the autophagy system of nematodes in the form of insulin and mitochondrial pathway dependence,upregulated the expression of autophagy related genes(unc-51,lgg-1,bec-1,vps-34),enhanced the autophagy level,and ultimately prolonged the life span of normal diet nematodes.The obesity model of C.elegans was constructed by using high glucose diet,and the key pathway of STP-60a in delaying the aging of C.elegans was studied.Obesity accelerated the aging of nematodes,while STP-60a significantly prolonged the longest life span and average life span of the obese model nematodes,reduced the content of lipofuscin,alleviated the oxidative stress state,and improved several physiological indicators(exercise ability,pharyngeal pump rate,number of offspring,and anti stress ability).The results of oil red staining experiment,DHS::GFP transgenic nematode visual analysis and TG content determination showed that STP-60a could reduce the fat accumulation of obese model nematodes.By studying the key enzymes and genes in lipid metabolism pathway,it was found that STP-60a inhibited the expression of fasn-1,fat-5,fat-6 and fat-7 genes,weakened the enzyme activity of ACC and FAS,and thus reduced the synthesis of fatty acids;STP-60a activated the expression of acs-2,ech-1.1,kat-1 and atgl-1 genes,enhanced the enzyme activity of LPS,and thus improved fatty acid decomposition andβ-oxidation.In addition,STP-60a regulated the expression of lipid conduction(sbp-1,nhr-49),insulin(daf-2,daf-16,skn-1,hsf-1)and autophagy(unc-51,lgg-1,bec-1)pathway genes in obese model nematodes,the deletion mutation of lipid signal transduction factor sbp-1 or nhr-49,and the inhibition of insulin and autophagy signal pathways made STP-60a unable to reduce the lipid content and prolong the life span of obese model nematodes.These results suggested that STP-60a regulated lipid metabolism and prolonged the life span of obese model nematodes through lipid transduction,insulin and autophagy pathways.In this study,STP-60a,a polysaccharide fragment with the best anti-aging activity,was isolated and purified from Guangxi sweet tea leaves,and its molecular structure,morphological characteristics,and digestive characteristics were identified.At the same time,it was found that STP-60a could delay the senescence of nematodes fed on normal and high glucose diets,and the potential action pathway was revealed.This study will provide a theoretical basis for the application of sweet tea polysaccharides as anti-aging products,and open up a new avenue for rational utilization of sweet tea resources. |
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