Synergistic Anti-aging Effect Of Agrocybe Cylindracea Polysaccharides With Lactobacillus Rhamnosus GG Strain

Agrocybe aegerita is a nutrient-rich edible fungus,and its fungal polysaccharides,as natural bio-antioxidants,could improve the oxidase system by D-galactose to induce in aging mice.As a beneficial bacterias,Lactobacillus rhamnosus GG(LGG),could enhance immunity,improve gastrointestinal function,and delay the process of aging.LGG could better grow due to the antioxidant effect of the polysaccharide.Besides,it provides a carbon source to promote for growth in vitro.The presence of polysaccharides could increase the proliferation of probiotics and that better regulate the function of the gastrointestinal tract in vivo.Based on the research that cell experiments,especial classical pharmacology,transcriptomics,metabolomics and other technologies through the Agrocybe aegerita polysaccharide(APS)and LGG intervention of hydrogen peroxide(H2O2)to effect cell growth,apoptosis,transcriptomics and proteomics molecular level analysis in aging-related genes;Behavioral observation to effects learning and memory,blood routine,liver Pathological and pharmacological analysis,metabolomic analysis of intestinal metabolites,etc in combination with D-galactose-induced aging mouse model.To investigate the anti-aging activity of APS and LGG in vivo and in vitro,we provide theoretical basis for the development of APS and LGG anti-aging nutrition and functional foods.1.First of all,Hydrogen peroxide to induce MRC-5 cell of senescence model was established to explore the role of APS and LGG in delaying senescence.The results showed that APS and LGG could maintain cell viability,reduced apoptosis,increased the antioxidant ability and delaied the disruption of mitochondrial membrane potential.Therefore,the APS+LGG group was significantly(P<0.05).The overall relative expression of Klotho,Nampt,Prx1,and Sirt1 mRNA was similar by RT-qPCR and Western blot analysis.Among them,the expression of mRNA and protein was significantly reduced in the H2O2 group,compared with the normal group.However,the protein expression of Sirtl gene was down-regulated.This indicated that the protein expression was not ideal.APS and LGG group showed up-regulation of mRNA expression levels in resveratrol group,and the APS+LGG group was significantly lower than the single group(P<0.05).The relative expression of protein(p16 and p21),model groups was significantly higher than the blank group(P<0.05).This indicated that the model was successful,but the effect of p53 was poor.In the sample group,both p16 and p53 were down-regulated,while the protein expression level(p21)was up-regulated indicated that the sample had a poorly expressed protein.2.Based on the above experiments that an aging model was established by D-galactose to investigate the effects of APS and LGG on behavioral observation,learning and memory.The studies showed that model group had significantly reduced food intake,weight gain,apathy,drowsiness,dark dull fur coats,and detachment,showing obvious aging characteristics.The treatment group was higher than the model group in the total number of times to cross the platform and to stay in the target quadrant,and the APS+LGG was better than that of the food alone.The aging group had poor escape latency and swimming speed,etc.But the effect was not significant(P>0.05),and it showed a decreasing trend.In addition,it was the positive cells in BDNF of D-galactose model group were significantly decreased in optical density,distribution density and target area compared with other groups(P<0.05)indicated that APS and LGG could enhance remember and delay the effects of aging.3.Aging model was established by subcutaneous injection of D-galactose to investigate the dietary intervention of APS and LGG on aging mice.The test results showed that APS and LGG could better affect the white blood cell(WBC),red blood cell(RBC),hemoglobin(HGB)and platelet(PLT)levels in mice,and the total antioxidant capacity(T-AOC).The antioxidant indexes such as catalase(CAT),glutathione peroxidase(GSH-Px),malondialdehyde(MDA)and superoxide dismutase(SOD)had a significant effect(P<0.05)in vivo.In addition,the levels of T-AOC,CAT,and SOD in male mice and the levels of CAT,GSH-Px,and MDA in female mice in the APS+LGG group were significantly better than those in the separate treatment group(P<0.05).It was the pathological section observation of the liver,and it was found that dietary intervention can improve the liver of the lesion and play a defensive role against oxidation and anti-aging.4.The high-throughput sequencing of 16S rRNA gene was used to study the effects of APS and LGG on the structure and function of gut microbiota in D-galactose-induced aging mice.The results showed that the content of short-chain fatty acids was significantly different between the model group and the blank group and the drug group(P<0.05).The total bile acid content in the model group was lower,but the difference was not significant(P<0.05).Alpha diversity analysis showed that the Ace,Chaol and Simpson model groups were significantly lower than the blank group(P<0.05),but there was no difference in Shannon(P<0.05).PCoA results showed that the APS group of female mice was less effective,the rest of the groups were between the model group and the blank group,indicating that the effect was better,but the distribution of dispersion degree was poor;in male mice,APS and LGG were in the model group.Between the blank group and the blank group,the experimental results were normal,and the composite group was similar to the resveratrol group,indicating that the APS+LGG had better PCoA values for the intestinal flora,but the degree of dispersion was not regular.Visual association analysis showed that female mice Helicobacter(OTU143)and WBC,CAT.Odoribacter(OTU500)and WBC,RBC,PLT,HGB,Bacteroidales(OTU215)and SOD,Bacteroidales(OTU6)and GSH-Px,Alloprevotella(OTU244)and SOD were correlated,and the difference was significant(P<0.05).In male mice,there was a correlation between Bacteroidales(OTU116)and T-AOC,Bacteroidales(OTU162)and GSH-Px,T-AOC,Alloprevotella(OTU244)and T-AOC,Bacteroidales(OTU3)and HGB.Bacteroidales(OTU135)and T-AOC,and the difference was significant(P<0.05).5.The mRNA expression levels indicated that APS and LGG significantly upregulated Sirt1 gene and significantly down-regulated the expression of p21 and p16 genes(P<0.01),and there was a significant difference in the regulatory results between male and female mice(P<0.05).The above results illustrate the mechanism that synergy interactions about APS and LGG could delay D-galactose-induced aging mice.