Antiaging Effect And Molecular Mechanism Of Ganoderma Lucidum Aqueous Extract On Tetrahymena Thermophila

Ganoderma lucidum has the function of enhancing immunity and delaying aging.It is a traditional anti-aging medicinal fungus in China.The revelation of the anti-aging effect and molecular mechanism of Ganoderma lucidum can provide theoretical and technical support for research on antiaging traditional Chinese medicine.G.lucidum fruiting body contains many active substances,and the extraction and pharmacological effects of monomers have been deeply studied.But the research on the composition and pharmacological mechanism of mixed aquedous extract obtained by traditional Chinese medicine boiling method is insufficient.In this study,the antiaging mechanism of G.lucidum aqueous extract was studied,and the main components of G.lucidum aqueous extract were clarified.Tetrahymena thermophila,a eukaryotic single-cell model organism,was selected as the experimental organism.The antiaging effects of the mixed aqueous extract and the main active monomers on the population and cells of T.thermophila were compared.The molecular mechanism by which the G.lucidum aqueous extract delayed the aging of T.thermophila was thoroughly studied.The main results are as follows:1.Main active components in G.lucidum aqueous extractThe preparation method of the G.lucidum aqueous extract was improved,and the polysaccharide content in the G.lucidum aqueous extract was 34% higher than that in past reports.The micro-component analysis in G.lucidum aqueous extract was analyzed by GC-MS,and 11 components were obtained with 55 compounds with greater than mass spectrometry library matching degree of 85%.It can be divided into 6 categories:alcohols,esters,aldehydes,ketones,alkanes and other derivatives.2.Anti-aging effect of G.lucidum aqueous extract on T.thermophilaBased on the uniform design of the experimental scheme,taking the concentration and addition ratio as the investigation factors.G.lucidum aqueous extract,G.lucidum polysaccharide,Ganoderic acid A,Gianoderal A and G.lucidum ergosterol were added to the culture medium of T.thermophila.The results showed that the antiaging effect of the G.lucidum aqueous extract on the population was better than that of any active The best scheme is to add 200 mg/m L G.lucidum aqueous extract to SPP medium at a rate of 20%,which can prolong the logarithmic phase of T.thermophila for 1 h,increase the population density by 92% and shorten the generation time by 20%.The observation of living cells showed that compared with the control group,the experimental group treated with G.lucidum aqueous extract had a more complete cell morphology in the logarithmic phase and decline phase.The results of transmission electron microscopy showed that G.lucidum aqueous extract improved the structural integrity of the mitochondrial membrane,and there were glycogen-like granules and nascent phagosomes in the cells during the decline phase.Both in logarithmic phase and decline phase,G.lucidum aqueous extract increased the activity of intracellular antioxidant enzymes(SOD,CAT,GPX)and ·OH clearance and reduced the accumulation of MDA.The comprehensive results showed that the G.lucidum aqueous extract mixed with a variety of active substances could delay the aging of T.thermophila and maintain the young state of cells.3.Transcriptomic analysis of the antiaging mechanism of G.lucidum aqueous extract on T.thermophilaAccording to the best scheme in the uniform design experiment,the G.lucidum aqueous extract was added to SPP medium,and the cells in logarithmic phase and decline phase were taken for RNA-Seq analysis.A total of 11,526 differentially expressed genes were obtained in logarithmic phase(up regulation: 6,208,down regulation: 5,318).There were 8,772 differentially expressed genes during the decline phase(up regulation: 4,721,down regulation: 4,051).GO enrichment analysis revealed that the differentially expressed genes in the two phases were closely related to the functions of "metabolic process","binding" and "membrane".The KEGG database was used to further analyze the main cellular signal transduction pathways of G.lucidum aqueous extract.The differentially expressed genes were mainly enriched in the "metabolic pathway",especially the "glutathione metabolic pathway".PPI network analysis showed that 83% of the node proteins belong to the phospholipid hydroperoxide glutathione peroxidase(PHGPx)protein family.4.The role of T.thermophila PHGPx protein family in anti-aging process of G.lucidum aqueous extract based on RNAi technologyTranscriptome analysis showed that Tt GPx1,Tt GPx7 and Tt GPx11 in the PHGPx-encoding genes were upregulated both in logarithmic and decline phases under the induction of G.lucidum aqueous extract..RNAi technology was used to interfere with the expression of Tt GPx1,Tt GPx7 and Tt GPx11.The positive cells were added with G.lucidum aqueous extract to observe the anti-aging effect.It was found that the anti-aging effect of G.lucidum aqueous extract on the positive cells with successful Tt GPx7 interference was weaker than Tt GPx1 and Tt GPX11 interference expression groups.This suggests that PHGPx plays an important role in the anti-aging effect of G.lucidum aqueous extract,and Tt GPx7 may be a potential target gene of G.lucidum aqueous extract for anti-aging of T.thermophila.In conclusion,G.lucidum aqueous extract mixed with a variety of active substances can better enhance the antioxidant capacity of T.thermophila,and has a more significant effect on maintaining cell viability and delaying cell aging.The mechanism is closely related to the regulation of the glutathione metabolic pathway,especially the up-regulated expression of the gene encoding PHGPx.This study provides new data for the application of G.lucidum aqueous extract in the field of anti-aging,and also provides new ideas for the follow-up research on cellular anti-aging mechanism.