| Nuclear Factor Erythroid-2 related factor 2(Nrf2),containing basic area leucine zipper,which has closed association with redox regulation,proteolysis,DNA repair,apoptosis prevention,iron and heme metabolism,as well as drugs and xenobiotic metabolism.Nrf2 dysfunction is a characteristic feature of aging and aging-related diseases.During aging,the expression of Nrf2 usually decreases,and cells are more sensitive to oxidative stress,endoplasmic reticulum stress and protein aggregation,which triggers the aging process.Studies have shown that aging is closely related to the gut microbiota.In aging organisms,the diversity and structure of gut microbiota change greatly,the abundance of beneficial bacteria decreases,and the facultative anaerobes increases.However,it is unclear whether there is a correlation or a causal link between the deficiency of Nrf2 during aging and the changes of the gut microbiota.Therefore,this project selects Nrf2 knockout mice as the research object,using D-galactose to accelerate the aging process of mice.Our experiment established a wild type with saline treatment group(WT-NC),D-galactose treatment wild type group(WT-D-gal),saline treatment Nrf2 knockout group(KO-NC),and D-gal treated Nrf2 knockout group(KO-D-gal).We analyzed the regulation mechanism of Nrf2 on the microecology during aging process through detecting mouse antioxidant capacity,aging phenotype and oxidative indicators and combining with colonic transcriptome and gut microbiota.Our results show:1.Compared with wild-type(WT),the food intake and weight have increased in Nrf2 knockout mice.However,after treating with D-gal,the mice in the KO group showed obvious listlessness,hair loss,reduced food intake,decreased weight gain and apparent weakness.In addition,the results of the antioxidant capacity of the liver and serum of the mice in each group showed that the effect of Nrf2 knockout and D-gal reduced the antioxidant capacity of the mice significantly.2.The results of HE staining about mouse colon showed that the colon structure of Nrf2 knockout mice was destroyed,the crypt structure was atrophied,and the goblet cells were reduced.In the mice treated with Dgal,the results showed that the colon structure of the KO group was severely damaged.In addition to the atrophy of the crypt structure and the decrease of goblet cells,mucosal damage and submucosal edema also appeared.Besides,in Nrf2 knocked out mice,the content of ROS in colon was increased,and after D-gal treatment,it was significantly higher in KO group than that of the WT group.Finally,the results of β-galactosidase staining of colon showed that after Nrf2 knockout and D-gal treatment,the number of senescent cells in colon of the KO group was significantly increased.3.Colonic transcriptome sequencing found that after knocking out Nrf2,the expression of Nqo1,Gstp1,Gstp2,Gstm1 and other antioxidant enzymes decreased significantly,the expression of phase II detoxification enzymes such as Cyp2l2 and Cyp4f14 increased significantly,as well as the expression of Casp4,Pcsk9,Kras,Ubaly.When Nrf2 knockout mice are treated with D-gal,the secretion and expression of Il-6,Mcp-2,Mmp3,Cxcl10,Igfbp3,Igfbp4,Igfbp5,Igfbp6 and some other senescence-related secretory phenotypes(SASP)increased significantly compared with the untreated group.In addition,the secretion of MUC-2,which is the main mucin component of the colon,is increased.The expression of Adamdec1 and Mmp7-α-defensin axis,which are positively correlated with intestinal inflammation,is significantly up-regulated.Atf3 and Duoxa2,which are negatively correlated with colonic homeostasis,are significantly upregulated as well.4.The 16 S r DNA sequencing results of fresh mouse feces found that Nrf2 deficiency changed the structure and diversity of gut microbiota significantly.After knocking out Nrf2,D-gal treatment makes the relative abundance of pathogenic bacteria such us Streptococcus,Staphylococcus,Acinetobater,Pantoea,Helicobacter,Enterococcus and Shigella increased significantly,while beneficial bacteria decreased,including Ruminococcus,Odoribacter,Oscillospira,Coprococcus and AF12.In summary,the research of this subject has found that the deficiency of Nrf2 significantly reduces the organism antioxidant capacity.Under the oxidative pressure of D-gal,it accelerates the aging process of the colon and the body,confirming that Nrf2 is an important anti oxidative senescence factor again.We further found that Nrf2 deficiency reduces the structural integrity of colon tissues,up-regulates intestinal inflammation signals,and significantly reduces the diversity of gut microbiota,suggesting that Nrf2 plays a primary role in maintaining gut microbiota during aging.Our research will provide reference for the research of aging mechanism based on Nrf2 and intestinal microecology as well as the development of anti-aging drugs. |
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