Preliminary Study On The Anti-aging Mechanism Of Ganoderma Atrum Polysaccharide Through Autophagy Alleviating Mitochondrial Dysfunction

Ganoderma atrum is a precious Chinese herbal medicine,belonging to basidiomycetes poraceae,which has been used in China for thousands of years.It has been recorded as early as Compendium of Materia Medica that black ganoderma has the effects of invigorating qi and calming nerves,prolongation and longevity,and has extremely high medicinal value.The main active ingredient of Ganoderma atrum is polysaccharide.Many modern studies have shown that Ganoderma polysaccharides have a variety of biological activities,such as anti-oxidation,anti-aging,enhancing immunity,regulating blood sugar and lipid,etc.Aging,usually refers to a variety of physiological,metabolic and functional degenerative changes appearing with the growth of age.Cells are the basic unit of the body,and the aging of the body is caused by the accumulation of senescent cells.The occurrence and development of many age-related diseases are related to cellular senescence.Therefore,to elucidate the mechanism of aging and explore the causes of age-related diseases,we must start from studying the mechanism of cellular senescence.Meanwhile,with the gradual aging of our society,the study of aging biology and anti-aging has more important scientific significance and social value.In our previous experiments,Ganoderma atrum polysaccharide（PSG）was found to significantly alleviate the senescnece and DNA damage of A375 cells.In order to explore its potential mechanism,we performed RNA-Seq analysis and found that PSG upregulated autophagy and mitochondrial pathway compared with the model group.Further experiments showed that PSG alleviated mitochondrial dysfunction by inhibiting the accumulation of reactive oxygen species（ROS）and the loss of mitochondrial membrane potential（MMP）.PSG also enhanced the expression of transcription factor EB（TFEB）in nucleus and enhance lysosomal function to activate autophagy.Finally,ROS scavenger NAC and autophagy inhibitor Bafilomycin A1（Baf A1）were used to explore the relationship between ROS,mitochondrial dysfunction and autophagy,as well as the anti-aging mechanism of PSG.The main conclusions are as follows:1.Establishment of hydrogen peroxide（H₂O₂）induced senescent model.H₂O₂is a common inducer of cellular senescence.The cells were treated with different concentrations of H₂O₂solution,and SA-β-Gal method was used to detect cellular senescence.Finally 50μM was choosed for modeling.2.PSG alleviated the senescence of A375 cells.Utilizing SA-β-Gal method to detect the degree of cellular senescence.The results showed that under the treatment of PSG,cellular senescence was alleviated,and the degree of remission was concentration dependent.At the same time,the cellular senescence related DNA damage index H2A.X and the expression of p53、p21 was also inhibited by PSG.3.PSG affected gene expression in senescent A375 cells.RNA-Seq and bioinformatics analysis were conducted to detect the effect of PSG on the gene expression of senescent A375 cells.There were 2964 differentially expressed genes between PSG treatment group and model group,of which 1116 genes were up-regulated and 1848 genes were down-regulated.GO enrichment analysis showed that autophagy,cell cycle,longevity regulation and mitochondrial related pathways were significantly up-regulated in PSG treatment group.At the same time,cell cycle arrest,DNA damage response,p53 signaling pathway,cellular senescence and TNF signaling pathway were significantly down-regulated.GSEA enrichment analysis also showed that PSG treatment group down-regulated p53,SASP and other senescence related pathways.4.PSG activated autophagy in senescent A375 cells.Based on the results of immunofluorescence and Western Blot,it was found that PSG up-regulated the expression of autophagy marker proteins LC3 and down-regulated p62.Lysosome is a key organel in the autophagy process.When the functions of autophagic lysosome transcription factor EB and lysosome were measured,it was found that PSG increased the expression of TFEB in nucleus and enhanced the function of lysosomes.5.PSG reduced ROS levels in senescent A375 cells and alleviated mitochondrial dysfunction.The ROS level of senescent A375 cells after PSG treatment was detected by DHE staining,and the results showed that PSG significantly reduced the ROS level of senescent cells.Mito Tracker Red and JC-1staining were used to detect mitochondrial membrane potential of aging A375 cells,and PSG improved mitochondrial membrane potential abnormalities,thus alleviating mitochondrial dysfunction.6.PSG reduced ROS levels in senescent A375 cells by activating autophagy.DHE staining was used to detect the effects of the use of autophagy inhibitor Baf A1and autophagy activator Rapa on the level of ROS.The results showed that Rapa could reduce the level of ROS,while Baf A1 could increase the level of ROS,and the increase could be reversed by the treatment of PSG.It indicated that the activation of autophagy induced by PSG leaded to the decrease of ROS level.7.Remission of mitochondrial dysfunction of senescent A375 cells was associated with the reduction of ROS induced by PSG.Senescent A375 cells were treated with NAC,and the intracellular ROS level and mitochondrial membrane potential were detected by DHE,Mito Tracker Red and JC-1 staining.The results showed that NAC effectively eliminated intracellular ROS and alleviated the breakdown of mitochondrial membrane potential induced by H₂O₂.8.PSG alleviated A375 cells senescence by activating autophagy.Experiment results showed that PSG could activate autophagy in senescent A375 cells,and autophagy activation could reduce the ROS leve,which was closely related to mitochondrial dysfunction.This suggested that PSG reduced H₂O₂-induced rise in cell ROS levels and improved mitochondrial dysfunction by activating autophagy,thus alleviating cellular senescence.