Anti-aging Effect Of Agaricus Blazei Polypeptide And Its Mechanism

Objective: Isolate and purify the active components of Agaricus blazei polypeptide and explore its anti-aging effect and its mechanism.Methods: The peptide was extracted by ammonium sulfate precipitation and enzymatic hydrolysis,and purified by DEAE-32,Cellulose CM-52 and Sephadex G-25 column chromatography to obtain ABp;continued to use Superdex 30 column chromatography to obtain ABp-S2 and analyze the components of ABp-S2Composition: ICR mice were randomly divided into blank control group(Con),model group(Mod),piracetam group(Pir)and Agaricus blazei polypeptide group(ABp),and subcutaneous injection of D-gal 300mg/kg in the back of the neck was established.Mouse senescence model;Morris water maze and dark avoidance test were used to test the learning and memory ability of mice;enzyme-labeled assay was used to detect the contents of MDA,T-AOC,CAT and ROS in mouse serum;HE and immunofluorescence staining were used to observe hippocampus Neuronal structural changes;functional annotation and enrichment analysis of transcriptomics sequencing(RNA-seq)to analyze differential expression of genes;q RT-PCR to verify m RNA expression of significantly different genes;Western blot to detect Keap1,Nrf2,HO-1.P53,Hsph1,Apoe,Trim32 protein expression levels.Based on previous anti-aging research,MTT method was used to detect the repair rate of senescent cells by different peptides in ABp-S2.NIH/3T3 cells were divided into blank control group(Con),model group(Mod),Agaricus blazei polypeptide low-dose group(ABp-RQR-L),Agaricus blazei polypeptide medium-dose group(ABp-RQR-M),Agaricus blazei polypeptide high In the dose group(ABp-RQR-H),CAT,MDA,ROS,and T-AOC activities in extracellular fluid were detected by enzyme-labeled assay.The content of IL-1?,IL-6 and TNF-? in extracellular fluid was detected by ELISA.?-galactosidase staining was used to observe the cell morphology and the percentage of positive cells.Western-blot method was used to detect the expression of Keap1,Nrf2,HO-1,TLR4,and NF-?Bp65 cytokines and the expression of Nrf2 and HO-1protein factors after application of ML385 blocker.Results:After purification and separation by DEAE-32,Cellulose CM-52 and Sephadex G-25 column chromatography,the single peak was ABp.The components obtained after repurification by Superdex 30 column chromatography consisted of three peptides ABp-RRQ,ABp-RQR,and ABp-TRAR;ABp-RQR was the main active ingredient;compared with the Mod group,the water labyrinth of mice in the ABp group and The dark avoidance latency is shortened and the number of errors is reduced(P<0.05);CAT and T-AOC in serum are significantly increased,and ROS and MDA content are decreased(P<0.05);HE staining results show that neurons in the ABp group are ranked compared to the Mod group Tighter,reduced number of nuclear condensed cells;compared with the Mod group,ABp group screened 295 differential genes,79 up-regulated genes,216 down-regulated genes;q RT-PCR verification results showed Hsph1,Trim32,HK1 in brain tissue of ABp group,Hn RNPA1,Grik5 m RNA expression increased significantly,Apoe,Atp1a3,Stxbp1,Mapk8ip1 m RNA expression decreased significantly(P<0.05).Western blot results showed that the expression of Keap1 and P53 protein in brain tissue of ABp group decreased,and the expression of Nrf2,HO-1,Hsph1,and Trim32 protein increased(P < 0.05).Compared with the Mod group,in each group of ABp-RQR,the activities of SOD and CAT in the extracellular fluid of senescent NIH/3T3 were enhanced,the contents of MDA and ROS were reduced,and the contents of IL-1?,IL-6 and TNF-? were decreased(P<0.05);?-Galactosidase staining showed that the number of SA-?-gal positive senescent cells in the Mod group was significantly increased and the cells became large and irregular in shape.The ABp-RQR groups were regular in shape and the number of positive cells was reduced compared with the Mod group(P < 0.05);And the expression of Nrf2,HO-1,TLR4,NF-?Bp65increased,and Keap1 expression decreased in senescent cells(P<0.05).After ML385 intervention,the expressions of Nrf2 and HO-1 cytokines in ABp-RQR group were weakened(P<0.05).Conclusion: ABp can improve oxidative stress damage in aging model mice and play an anti-aging role;the specific mechanism may be related to the expression of related factors Apoe,Hsph1,Trim32,Atp1a3,Stxbp1,Mapk8ip1,Hn RNPA1,Hk1,Grik5 m RNA expression;ABp-RQR can improve the senescence of NIH/3T3 cells induced by D-gal;the specific mechanism may reduce the oxidative-inflammatory response by regulating Nrf2/ARE and TLR4/NF-?Bp65 signaling pathways to produce anti-aging effects.