肠道微生物 z-bo 2020-11-19 415

　　Background: Aging is a degenerative process closely related to inflammation. The typical aging of chronic low-grade inflammation, called "inflammatory aging", involves the activation of the inflammatory network and the release of aging-related factors, including key pro-inflammatory mediators, such as nuclear factor kappa B (NF-κB). Inflammatory aging may also be related to age-related oxidative/genotoxic stress and changes in gut microbial composition. In addition to gastrointestinal diseases, metabolic disorders, and antibiotic use, aging has also been shown to affect the composition of the intestinal microbiota; the content of intestinal bifidobacteria decreases with age, while Clostridium perfringens and Lactobacillus The content of Enterococcus and Enterococcus increases with age. It is prominent in the context of inflammation, because the composition of the gut microbiota has been shown to be closely related to intestinal inflammatory diseases. The mechanism of intestinal microbial components inducing low-grade inflammation at the molecular level is still unclear. Cell cycle regulators play an important role in the aging process of cultured cells. Among these molecules, P16 has recently been selected as a suitable marker for aging in the body. P16 induces senescence by inhibiting the activity of cyclin-dependent kinases CDK4 and CDK6, otherwise it will phosphorylate and inactivate retinoblastoma tumor suppressor factor. As age increases, the expression of p16 in mouse bone marrow stem cells and progenitor cells increases, inhibiting stem cell proliferation and tissue regeneration. The sterile α-motif domain and HD domain contain protein 1 (SAMHD1), a cellular deoxynucleoside triphosphate hydrolase involved in cell replication, which depletes cellular deoxynucleosides that can be used for retroviral DNA Triphosphate pool to prevent virus replication. SAMHD1 is highly expressed in non-dividing cells such as macrophages and dendritic cells, and regulates cell proliferation through cyclin A2/CDK1. SAMHD1 can regulate the cell cycle through the degradation of cellular DNTP. Little is known about the functional role of SAMHD1 in cells. In this study, we first studied the composition of inflammatory aging markers such as P16, NF-κB and SAMHD1 and the level of LPS production in young and old mice, as well as the role of aging. In addition, we studied the relationship between aging and inflammation induced by the gut microbial LPS.

　　Method: Animals: male C57BL/6J mice (4 months old or 18 months old) and TLR4-deficient C57BL/10SCNJ mice (4 months old). Each group consisted of eight mice. All mice were reared at 20-22°C and 50%±10% humidity. The mice were anesthetized, and blood samples were collected for biochemical analysis. The colon was quickly removed, opened longitudinally, and feces were gently removed with phosphate buffered saline (PBS) and used for ELISA and immunoblotting.

　　DNA extraction, pyrosequencing and data analysis: Use a commercial DNA isolation kit to extract genomic DNA from four sets of stool samples according to the instructions. The genomic DNA was amplified using barcode primers targeting the V1 to V3 region of the bacterial 16S rRNA gene. Sequencing and basic analysis were performed according to the described method. The sequence of each sample is sorted by a unique barcode and low-quality readings are eliminated. Based on the 16S rRNA sequence data, the EZOCTRONE E database is used for sequence reading. Sequence number analysis, observe diversity abundance (OTU), estimate OTU abundance (ACE and JAU1). The Mythr program was used to calculate the coverage of pyrosequencing, and the cutoff value of 97% similarity of the 16S rRNA gene sequence was considered. The distance between the microbial communities from each sample is expressed as an unweighted paired group method and the arithmetic mean (UPGMA) clustering tree describes the dissimilarity between multiple samples.

　　Limulus amebocyte lysis test: The plasma and fecal endotoxin content is determined by diazonium-conjugated limulus amoeba lysate (LAL). The plasma is simply diluted 1:10 in pyrogen-free water, inactivated at 70°C for 10 minutes, and then incubated with LAL at 37°C for 30 minutes. The addition of the reagent resulted in the formation of a light-absorbing magenta derivative at 545 nm. For the fecal endotoxin test, 20 mg feces from the cecum were placed in a pyrogen-free tube of 50 ml PBS and sonicated on ice for 1 hour. After centrifugation at 400 g for 15 minutes, the upper 30 ml was collected, filtered through a 0.45 μm filter, and then filtered through a 0.22 μm filter, and inactivated at 70°C for 10 minutes. Then use the LAL detection kit to determine the LPS content of the filtered ultrasound product.

　　ELISA and immunoblotting: ELISA kits were used to determine the concentrations of tumor necrosis factor α (TNFα), interleukin (IL)-1β and IL-6 in plasma and colon. The levels of p16, Bcl-2, ATG7, LC3, phosphorylated mammalian target of rapamycin, mTOR, phosphorylated p65, p65, SAMHD1, cyclin E, CDK2 and β-actin in the colon are as described above Method determination. Use ECL detection kit for immunoassay.

　　"Isolation and culture of peritoneal macrophages: As described above, peritoneal macrophages were isolated from male C57BL/6J mice or TLR4-deficient mice. Mice were injected intraperitoneally (ip) with 2 ml of 4% thioglycolate solution. The mice were sacrificed 4 days after the injection, and the abdominal cavity was washed with 10 ml of RPMI1640 medium. The peritoneal lavage fluid was centrifuged at 200 g for 10 min, and the cells were incubated with RPMI 1640 medium. After incubating at 37°C for 1 h, the cells were washed three times, and non-adherent cells were removed by suction. At 37°C, peritoneal macrophages were cultured in RPMI1640 medium containing 10% fetal bovine serum. To detect the inflammatory effect of mouse fecal lysate, peritoneal macrophages were incubated with 100 μl of fecal lysate for immunoblotting.

　　Result: Young and old mouse gut microbiota: We started our research by studying the gut microbiota of mice of different ages. Caecal samples were collected from mice maintained on a normal (low-fat) diet for 8 weeks, and changes in the gut microbiota were studied by pyrosequencing. The number of sequence analysis, OTU, estimated OTU richness (ACE and JAU1), and pyrosequencing coverage are calculated using a cluster database with high identity. Decreased bacterial abundance and diversity were observed in cecum samples collected from young mice, and these differences were not statistically significant. Taxonomy-based analysis shows that aging significantly modulates the dominant gut microbiota. Compared with young mice, the levels of bifidobacteria in old mice were reduced, mice showed anti-inflammatory effects, and the levels of lactobacilli and enterococci increased. In this study, aging increased the prevalence of Firmicutes and Actinomyces, and reduced the prevalence of bacilli, leading to an overall increase in the ratio of Firmicutes to Bacteroides in the intestinal flora.

　　Young and old mice plasma and fecal endotoxin levels: CD14 and other inflammatory markers CD14 and C-reactive protein in the NF-κB signaling pathway bound by TLR4 increase with age. The previously unconsidered source of inflammation is the translocation of the gut microbiota and its products, leading to inflammation, such as inflammatory bowel disease. In order to understand the effect of LPS on the aging of mice, the LPS levels of young and old mice were measured. Compared with young mice, the weight/body weight ratio of epididymal fat pad in old mice increased significantly, but there was no significant difference in weight gain. We assessed endotoxin levels in different treatment groups to investigate whether changes in gut microbial composition with age are related to systemic endotoxemia. The fecal endotoxin level of old mice is higher than that of young mice. Compared with young mice, we also found that the systemic endotoxin level of old mice was significantly increased.

　　The expression levels of P16, cell cycle regulators and SAMHD1 in young and aging mice: The expression of P16 is higher, while the expression levels of cyclin E and CDK2 in old mice are lower than that in young mice. We also observed that the activation of NF-κB and mTOR in aging mice was stronger than that in young mice. Interestingly, with the increase of p16 expression, the colonic expression of SAMHD1 increased in aging mice relative to young mice.

　　"LFL increases macrophage SAMHD1 expression and inflammatory response: LFS in aging mice induces the expression of P16 and SAMHD1 in peritoneal macrophages of WT mice in peritoneal macrophages treated with different concentrations of LPS significantly higher than that of young mice.

　　Conclusion: These results indicate that aging can accelerate inflammatory aging by increasing the LPS level of the intestinal microbiota, by inducing the expression of P16 and SAMHD1 and NF-κB activation in mice. In this study, we demonstrated that the expression of SAMHD1 increased under inflammatory aging or inflammatory aging conditions, suggesting that SAMHD1 can be used as a new marker of inflammatory aging in mice.