Alistipes indistinctus-derived hippuric acid promotes intestinal urate excretion to alleviate hyperuricemia.

Hyperuricemia induces inflammatory arthritis and accelerates the progression of renal and cardiovascular diseases. Gut microbiota has been linked to the development of hyperuricemia through unclear mechanisms. Here, we show that the abundance and centrality of Alistipes indistinctus are depleted in subjects with hyperuricemia. Integrative metagenomic and metabolomic analysis identified hippuric acid as the key microbial effector that mediates the uric-acid-lowering effect of A. indistinctus. Mechanistically, A. indistinctus-derived hippuric acid enhances the binding of peroxisome-proliferator-activated receptor γ (PPARγ) to the promoter of ATP-binding cassette subfamily G member 2 (ABCG2), which in turn boosts intestinal urate excretion. To facilitate this enhanced excretion, hippuric acid also promotes ABCG2 localization to the brush border membranes in a PDZ-domain-containing 1 (PDZK1)-dependent manner. These findings indicate that A. indistinctus and hippuric acid promote intestinal urate excretion and offer insights into microbiota-host crosstalk in the maintenance of uric acid homeostasis.

[Oxidative Damage of Bone Marrow Stromal Cells Caused by Chemotherapy Drugs].

OBJECTIVE: To explore the oxidative damage of OP9 cells induced by daunorubicin (DNR) treatment. METHODS: The TMRM probe was used to detect mitochondrial membrane potential by flow cytometry; the reactive oxygen species (ROS) was determined by flow cytometry DCFDA probe; the real-time PCR was used to detect the molecular expression of antioxidant enzyme，glutathione peroxidase (GPX) in OP9 cells; the expression of γ-H2AX was determined by flow cytometry. RESULTS: Compared with normal OP9 cells, the positive rate of TMRM in DNR-treated OP9 cells decreased by 56.7% (P＜0.05); the positive rate of DCFDA in DNR-treated OP9 cells increased by 3.52 times (P＜0.01). Compared with normal OP9 cells, DNR-treated OP9 cells showed a decrease in the expression of GPX4 by 44.22% (P＜0.001); the expression of GPX7 decreased by 65.7% (P＜0.001); the expression of GPX8 decreased by 24.7% (P＜0.001); the positive rate of γ-H2AX in DNR-treated OP9 cells increased (P＜0.05). CONCLUSION: After DNR treatment, mitochondrial membrane potential of OP9 cells decreases; the level of reactive oxygen species increases; the expression of glutathione peroxidase (GPX) molecules decreases significantly; genomic instability increases obviously; the oxidative damage of cells increased.

[Effect of Chemotherapeutic Drug-Induced Damage of Bone Marrow Stroma Cells on Normal Hematopoietic Cells].

OBJECTIVE: To explore the effect of damage of bone marrow stroma cells induced by chemotherapeutic drug on the function of normal hematopoitic cells. METHODS: Senescence cells were detected by flow cytometry after SA-ß-gal staining; real-time PCR was used to detect the expression of a serial molecules in bone marrow stromal cell line OP9 cells; the expression of γ-H2AX was determined by flow cytometry after histone γ-H2AX staining; the colony forming ability of hematopoietic cells was tested by colony formation assay. RESULTS: The percentage of senescence cells in OP9 cells after DNR treatment was 2.24 times as much as that in untreated OP9 cells (P<0.05). Compared with normal OP9 cells, the expression levels of IL-6 and TNF-alpha in DNR-treated OP9 cells increased by 2.73 times (P<0.01) and 0.56 times (P<0.01), and the expression levels of N-cadherin, alpha smooth muscle actin (alpha-SMA), angiopoietin1 (Angpt1) and osteopontin (OPN) decreased by 69.54%（P<0.01），63.90%（P<0.01），87.41%（P<0.01）and 42.78%（P<0.01）respectively. After the co-culture with DNR-treated OP9 cells, the colony formation of normal hematopoietic cells decreased by 47.10% than that co-cultured with untreated OP9 cells (P< 0.05), meanwhile, the percentage of γ-H2AX+ cells in normal hematopoietic cells increased by 2.19 times (P<0.05). CONCLUSION: After treatment with DNR, the senescence cell number of OP9 cells sgnificantly increases; the expression of TNF-α and IL-6 is up-regulated, while the expression of α-SMA, Angpt-1 and OPN is down-regulated as compared with normal OP9 cells. In addition, after co-culture of DNR-treated OP9 cells with normal hematopoietic cells, the colony formation ability of hematopoietic cells decreases and the genome instability of hematopoietic cells increases as compared with normal hematopoietic cells.

[Exploration of Mechanism for Meisoindigo-Inducing K562 Cell Apoptosis by Using Quantitative Proteomic Analysis].

OBJECTIVE: To screen the differentially expressed proteins at the early stage of K562 cells treated with meisoindigo by using tandem mass tags (TMT)-based proteomics technology, and to explore the mechanism for meisoindigo-inducing apoptosis. METHODS: The half inhibitory concentration (IC50) of mesoindigo on K562 cells was determined by CCK8. The flow cytometry was used to assay the apoptosis of K562 cells treated by meisoindigo or DMSO. Total proteins were extracted from the cells treated with 0.2% DMSO (control) or 20 µmol/L meisoindigo (Test) for 2 hours. Then, the TMT-labeling HPLC-MS/MS was used to identify and quantify the peptides and their abundance, all the tests were repeated for 3 times. The Mascot software was used to identify the proteins; the GO annotations, enrichment and cluster analysis were used to analyze the differentially expressed proteins. RESULTS: Meisoindigo-induced K562 cell apoptosis in a dose-dependent manner (r=0.98), 5 544 proteins were identified, 4792 of which were quantified. The protein with expression difference>1.5-folds in Test group accoanted for 8, out of which the expression of 4 proteins were up-regulated and 4 were down-regulated. The differentially expressed proteins mainly associated with reactive oxygen species (ROS). CONCLUSION: Several proteins including DDIT4 were found to have dramatic changes in the early stage of K562 cells treated with meisoindigo by using quantitative proteomics technology. The ROS metabolic process may play important roles in meisoindigo-inducing apoptosis of K562 cells.

[Relationship between High Expression of c-FLIP and Drug Resistance of Leukemia Cells].

OBJECTIVE: To explore the effect of c-FLIP expression on drug resistance of Kasumi-1 leukemia cells and its mechanisms. METHODS: Tet-on inducible system was used to construct the conditional expression vector of c-FLIP by cloning the c-FLIP gene into lentivirus vector pLVX-Tight-Puro, then the Kasumi-1 cells were transfected with lentivirus pLVX-Tight-Puro-c-FLIP. The expression of c-FLIP was induced by doxycycline(Dox) for different time and doses, and verified by qRT-PCR and Western blot. On the basis of the overexpression of c-FLIP, the Kasumi-1-c-FLIP cells were treated with CH11 and PB in order to induce apoptosis, and the Giemsa staining was used to show the apoptotic cell morphology. RESULTS: qRT-PCR and Western blot showed the overexpression of c-FLIP, the CH11 and PB can induce Kasumi-1 cell apoptosis, while the c-FLIP overexpression weakened this effects. Western blot showed that the c-FLIP blocked the caspase-8 activation. CONCLUSION: The overexpression of c-FLIP inhibits the apoptosis caused by CH11 and PB, and leads to drug resistance in leukemia cells.

[Expression and Asymmetric Division of Numb in Leukemia Cell K562 Line].

OBJECTIVE: To investigate the role of asymmetric division in leukemia cells through detection of expression and asymmetric division of Numb in differentiated and undifferentiated K562 cells. METHODS: Firstly, Hemin was used to induce K562 cell differentiation, and the expression of Numb was detected by the real-time quantitative RT-PCR and flow cytometry. After K562 cells were synchronized by nocodazole, the Numb protein was labeled by immunohistochemical staining, followed by the determination of the terminally differentiated cells through confocal microscopy. The fluorescence intensity was calculated by Image J software, and the cell division pattern was analyzed on the basis of the fluorescence intensities of Numb in 2 divided daughter cells. RESULTS: Compared with the undifferentiated K562 cells, the level of Numb mRNA expression increased 2.3 times (P<0.001). The ratio of Numb positive cells was(67.37±5.01)% in differentiated K562 cells, while that was (43.97±5.72)% in undifferentiated K562 cells (P<0.01). Compared with undifferentiated K562 cells, the ratio of cells with asymmetric division in differentiated K562 cells increased 18.3%, the percentage of cells with symmetry self-renewal reduced 49.7%(P<0.001) and that with symmetry differentiation increased 32%(P<0.001). CONCLUSION: In differentiated K562 cells, expression of Numb and proportion of cells with asymmetric division were higher than that in undifferentiated cells. With the differentiation of leukemia cells, the proportion of cells with asymmetrical division increases, and the proportion of cells with symmetrical self-renewal decreases. The stemness of leukemia cells is maintained mainly through the symmetrical self-renewal.