Study On Anti-aging Mechanism Of Lycium Barbarum Leaves Flavonoids Based On HUVECs And Caenorhabditis Elegans Model

Lycium barbarum leaves are widely used as vegetables and functional tea in China.The content of flavonoids in Lycium barbarum leaves was higher than that in Lycium barbarum fruits.The combination of D101 resin and polyamide resin(PR)were used to purify Lycium barbarum leaves flavonoids(LBLF),and flavonoids content of 90.98%was obtained in purified product.In this study,human umbilical vein endothelial cells(HUVECs)and Caenorhabditis elegans(C.elegans)were used as model organisms to evaluate the protective effects of LBLF on oxidative stress damage and anti-aging,and to preliminary explore its molecular mechanism.(1)The D101 rein was used for static adsorption and desorption for 24 h.The preliminary purified product of LBLF was obtained,and then the further optimal purification conditions were screened through PR.The loading solution passes through the PR column at a concentration of 3 mg/mL,a flow rate of 2 BV/h,and a volume of 1 bed volume(BV),and then eluates at 2 BV,80%ethanol,and 2.5 BV/h.Preliminary identification of LBLF with UV-Vis spectroscopy(UV-Vis)and fourier transform infrared spectroscopy(FT-IR)showed that LBLF has ultraviolet characteristic absorption peaks of flavonoids and the resolution of FT-IR spectra is increased,the peak shape was basically similar to rutin.The main flavonoids in LBLF analyzed by high performance liquid chromatography(HPLC)were rutin,chlorogenic acid and kaempferol,and the content of rutin is the highest,accounting for 69.71%.Ultra-performance liquid chromatography-mass spectrometry(UPLC-MS)technology identifies 11 components in LBLF,mainly including rutin,chlorogenic acid and kaempferol.(2)The H₂O₂-induced oxidative damage model of HUVECs was used to explore the protective mechanism of LBLF on H₂O₂-induced oxidative damage of HUVECs.The safe dose of LBLF was 100 ?g/mL based on the carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h-tetrazolium(MTS)method.The oxidative stress damage model of HUVECs was established with different concentrations of H₂O₂,and the optimal concentration of H₂O₂ was 150 ?M.Determination of LBLF on H₂O₂-induced HUVECs reactive oxygen species(ROS),superoxide dismutase(SOD),glutathione peroxidase(GSH-PX),catalase(CAT)activity and malondialdehyde(MDA)content.The LBLF significantly reduced the content of ROS(P<0.05)and MDA(P<0.01),and significantly increased the activities of SOD,GSH-Px and CAT(P<0.05)in HUVECs,compared with H₂O₂ group.The mechanism of LBLF on H₂O₂ induced HUVECs gene expression and signal pathway were analyzed by RNA sequencing(RNA-Seq).Under the condition of H₂O₂ oxidative stress,the protective mechanism of LBLF on HUVECs was by up-regulating the expression of EPO and HO1 genes downstream of the mitogen-activated protein kinase(MAPK)signaling pathway.(3)Caenorhabditis elegans was an aging model to explore the anti-aging effects of LBLF.After different treatments,the Caenorhabditis elegans lifespan,movement ability,stress ability,the mRNA expression levels of antioxidant enzymes and aging-related genes were measured.LBLF prolonged the average lifespan by 15%better than metformin and rutin,and improved the movement ability of C.elegans in the middle of growth.At the same time,LBLF can significantly extend the survival of C.elegans under oxidative stress and increased the mRNA expression levels of antioxidant enzymes sod-2 and gcs-1.LBLF up-regulated the expression of skn-1 and daf-16,especially significantly up-regulated the expression of skn-1(P<0.05),indicating that LBLF increased the expression level of skn-1 gene to extend lifespan of C.elegans and improve the ability to resist pressure.