In this study, RAW264.7 cells were employed to investigate the effects of honey-processed Astragalus on their energy metabolism and polarization, and explore the scientific connotation of the enhanced efficacy of honey-processed Astragalus on invigorating spleen-stomach and replenishing Qi. The medicated sera were prepared by intragastric administration of rats with water extracts of crude and honey-processed Astragalus, and the composition changes of medicated sera of crude and honey-processed Astragalus were analyzed by using LC-MS technology. The cell survival rates were detected and the concentrations of medicated sera were screened through CCK-8 assay. The differences of cell phagocytic rates, ATP energy metabolism, and NO secretion between crude and honey-processed Astragalus were evaluated by using neutral red phagocytosis assay, ATP detection kit, and NO detection kit. The effects of crude and honey-processed Astragalus on TNF-α secretion of RAW264.7 cells were detected by employing ELISA kit. The effects of crude and honey-processed Astragalus on polarization of RAW264.7 cells were evaluated by utilizing flow cytometry. The differential metabolites related to glycolysis in cell lysates and culture media were screened by using LC-MS technology. The experiment was approved by the experimental animal ethics committee from Shanxi University of Chinese Medicine (No. AWE202407352). The results showed that the contents of betaine, amino acids, and ononin in the prepared medicated sera of rats treated by intragastric administration with water extracts of honey-processed Astragalus increased compared to those in crude one. The results of CCK-8 experiment showed that there were no cytotoxic effects on RAW264.7 cells in the medicated sera of crude and honey-processd Astragalus at different concentration. The phagocytic index and ATP yield both increased to varying degrees after administration of the medicated sera to cells. The secretion of NO in normal and inflammatory cells increased and decreased respectively, and the effect of honey-processed Astragalus was better than that of crude one. The results of ELISA kit showed that the medicated sera of both crude and honey-processed Astragalus could promote the secretion of TNF-α in a concentration-dependent manner, and the promoting effect of honey-processed Astragalus was stronger than that of crude one. The results of flow cytometry showed that the medicated sera of both crude and honey-processed Astragalus could promote M1-type polarization and inhibit M2-type polarization of RAW264.7 cells, and the effect of honey-processed Astragalus was better than that of crude one. Compared to crude Astragalus, the metabolites related to glycolysis in the cell lysates and culture media of the medicated sera of honey-processed Astragalus were generally on the rise, indicating that the effect on promoting glycolysis of honey-processed Astragalus was better than that of crude one. In summary, honey-processed Astragalus can promote the polarization and energy metabolism of RAW264.7 cells, and participate in positive immune regulation, which is correlated with its enhanced effect of invigorating spleen-stomach and replenishing Qi.

OBJECTIVE: To investigate the effects and underlying mechanism of ionizing radiation on the adipogenic of mesenchymal stem cells (MSCs). METHODS: Mouse MSCs were cultured in vitro and treated with 2 Gy and 6 Gy radiation with ⁶⁰Co, and the radiation dose rate was 0.98 Gy/min. Bulk RNA-seq was performed on control and irradiated MSCs. The changes of adipogenic differentiation and oxidative stress pathways of MSC were revealed by bioinformatics analysis. Oil Red O staining was used to detect the adipogenic differentiation ability of MSCs in vitro, and real-time fluorescence quantitative PCR (qPCR) was used to detect the expression differences of key regulatory factors Cebpa, Lpl and Pparg after radiation treatment. At the same time, qPCR and Western blot were used to detect the effect of inhibition of Nrf2, a key factor of antioxidant stress pathway, on the expression of key regulatory factors of adipogenesis. Moreover, the species conservation of the irradiation response of human bone marrow MSCs and mouse MSC was determined by qPCR. RESULTS: Bulk RNA-seq suggested that ionizing radiation promotes adipogenic differentiation of MSCs and up-regulation of oxidative stress-related genes and pathways. The results of Oil Red O staining and qPCR showed that ionizing radiation promoted the adipogenesis of MSCs, with high expression of Cebpa, Lpl and Pparg, as well as oxidative stress-related gene Nrf2. Nrf2 pathway inhibitors could further enhance the adipogenesis of MSCs in bone marrow after radiation. Notably, the similar regulation of oxidative pathways and enhanced adipogenesis post irradiation were observed in human bone marrow MSCs. In addition, irradiation exposure led to up-regulated mRNA expression of interleukin-6 and down-regulated mRNA expression of colony stimulating factor 2 in human bone marrow MSCs. CONCLUSION Ionizing radiation promotes adipogenesis of MSCs in mice, and oxidative stress pathway participates in this effect, blocking Nrf2 further promotes the adipogenesis of MSCs. Additionally, irradiation activates oxidative pathways and promotes adipogenic differentiation of human bone marrow MSCs.
Mesenchymal Stem Cells/cytology* ; Oxidative Stress/radiation effects* ; Animals ; Adipogenesis/radiation effects* ; Mice ; Radiation, Ionizing ; Cell Differentiation/radiation effects* ; Humans ; NF-E2-Related Factor 2/metabolism* ; PPAR gamma ; Cells, Cultured

OBJECTIVE: To establish an in vitro cell model simulating acute graft-versus-host disease (aGVHD) bone marrow microenvironment injury with the advantage of mouse serum of aGVHD model and explore the effect of serum of aGVHD mouse on the adipogenic differentiation ability of mesenchymal stem cells (MSCs). METHODS: The 6-8-week-old C57BL/6N female mice and BALB/c female mice were used as the donor and recipient mice of the aGVHD model, respectively. Bone marrow transplantation (BMT) mouse model (n=20) was established by being injected with bone marrow cells (1×10⁷ per mouse) from donor mice within 4-6 hours after receiving a lethal dose (8.0 Gy, 72.76 cGy/min) of γ ray general irradiation. A mouse model of aGVHD (n=20) was established by infusing a total of 0.4 ml of a mixture of donor mouse-derived bone marrow cells (1×10⁷ per mouse) and spleen lymphocytes (2×10⁶ per mouse). The blood was removed from the eyeballs and the mouse serum was aspirated on the 7^th day after modeling. Bone marrow-derived MSCs were isolated from 1-week-old C57BL/6N male mice and incubated with 2%, 5% and 10% BMT mouse serum and aGVHD mouse serum in the medium, respectively. The effect of serum in the two groups on the in vitro adipogenic differentiation ability of mouse MSCs was detected by Oil Red O staining. The expression levels of related proteins PPARγ and CEBPα were detected by Western blot. The expression differences of key adipogenic transcription factors including PPARγ, CEBPα, FABP4 and LPL were determined by real-time quantitative PCR (RT-qPCR). RESULTS: An in vitro cell model simulating the damage of bone marrow microenvironment in mice with aGVHD was successfully established. Oil Red O staining showed that the number of orange-red fatty droplets was significantly reduced and the adipogenic differentiation ability of MSC was impaired at aGVHD serum concentration of 10% compared with BMT serum. Western blot experiments showed that adipogenesis-related proteins PPARγ and CEBPα expressed in MSCs were down-regulated. Further RT-qPCR assay showed that the production of PPARγ, CEBPα, FABP4 and LPL, the key transcription factors for adipogenic differentiation of MSC, were significantly reduced. CONCLUSION The adipogenic differentiation capacity of MSCs is inhibited by aGVHD mouse serum.
Animals ; Mesenchymal Stem Cells/cytology* ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Adipogenesis ; Female ; Cell Differentiation ; Graft vs Host Disease/blood* ; Bone Marrow Cells/cytology* ; PPAR gamma/metabolism* ; Disease Models, Animal ; CCAAT-Enhancer-Binding Protein-alpha/metabolism*

OBJECTIVE: To detect and analyze the expression and clinical significance of long non-coding RNA tyrosine kinase non-catalytic region adaptor protein 1-antisense RNA1 (NCK1-AS1) in patients with acute myeloid leukemia (AML). METHODS: 89 AML patients and 23 healthy controls were included from the People's Hospital Affiliated to Jiangsu University. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression levels of NCK1-AS1 and NCK1 in bone marrow samples. The relationship between the expression of NCK1-AS1 and the clinical characteristics of patients were analyzed, as well as the correlation between NCK1-AS1 and NCK1. RESULTS: The expression level of NCK1-AS1 in all AML, non-M3 AML and cytogenetically normal AML (CN-AML) patients was significantly higher than that in the control group (P < 0.01, P < 0.05, P < 0.01, respectively). In non-M3 AML, patients with high NCK1-AS1 expression had a significantly lower hemoglobin level than those with low NCK1-AS1 expression (P =0.036), furthermore, NCK1-AS1 ^high patients had shorter overall survival than NCK1-AS1^low patients (P =0.0378). Multivariate analysis showed that NCK1-AS1 expression was an independent adverse factor in patients with non-M3 AML ( HR =2.392, 95% CI :1.089-5.255, P =0.030). In addition, NCK1 expression was also significantly upregulated in all AML, non-M3 AML and CN-AML patients compared with controls (P < 0.01, P < 0.01, P < 0.001, respectively). There was a certain correlation between NCK1-AS1 and NCK1 expression (r =0.37, P =0.0058). CONCLUSION High expression of NCK1-AS1 in AML indicates poor prognosis of AML patients.
Humans ; Leukemia, Myeloid, Acute/genetics* ; RNA, Long Noncoding/genetics* ; Oncogene Proteins/genetics* ; Adaptor Proteins, Signal Transducing/genetics* ; Prognosis ; Male ; Female ; Middle Aged ; Adult ; Case-Control Studies ; Clinical Relevance

The growth of three plague phages from Qinghai Plateau in two Yersinia pestis strains(plague vaccine strains EV76 and 614F)and four non-Yersinia pestis strains(Yersinia pseudotuberculosis PTB3,PTB5,Escherichia coli V517,and Yersinia enterocolitica 52302-2)were detected through a micromethod based on the OmniLogTM microbial identification system and by the drop method,to provide a scientific basis for future ecological studies and classification based on the host range.For plague vaccine strains EV76 and 614F,successful phage infection and subsequent phage growth were observed in the host bacte-rium.Diminished bacterial growth and respiration and a concomitant decrease in color were observed with the OmniLogTM mi-crobial identification system at 33 ℃ for 48 h.Yersinia pseudotuberculosis PTB5 was sensitive to Yersinia pestis phage 476,but Yersinia pseudotuberculosis PST5 was insensitive to phage 087 and 072204.Three strains of non-Yersinia pestis(Yersinia pseudotuberculosis PTB3,Escherichia coli V517,and Yersinia enterocolitica 52302-2)were insensitive to Yersinia pestis pha-ges 087,072204,and 476 showed similar growth curves.The growth of phages 476 and 087,as determined with the drop method,in two Yersinia pestis strains(plague vaccine strains EV76 and 614F)and four non-Yersinia pestis strains(Yersinia pseudotuberculosis PTB3,Escherichia coli V517,and Yersin-ia enterocolitica 52302-2)showed the same results at 37 ℃,on the basis of comparisons with the OmniLogTM microbial i-dentification system;in contrast,phages 072204 did not show plaques on solid medium at 37 ℃ with plague vaccine strains EV76 and 614F.Determination based on the OmniLogTM detection system can be used as an alternative to the traditional determination of the host range,thus providing favorable application val-ue for determining the interaction between the phage and host bacteria.

Three 2,3-diketoquinoxaline alkaloids were isolated from Heterosmilax yunnanensis Gagnep. Their structures were determined through 1D and 2D NMR, HR-ESI-MS, UV, and IR as 1-[5′-(3″-hydroxy-3″-methyl) glutaryl] ribityl-2,3-diketo-1,2,3,4-tetrahydro-6,7-dimethylquinoxaline (1), 1-[2′-(3″-hydroxy-3″-methyl) glutaryl]ribityl-2,3-diketo-1,2,3,4-tetrahydro-6,7-dimethylquinoxaline (2), and 1-ribityl-2,3-diketo-1,2,3,4-tetrahydro-6,7-dimethylquinoxaline (3). Compounds 1 and 2 are novel compounds, and 3 was isolated from H. yunnanensis for the first time. The hepatoprotective activity of these three compounds was evaluated, with compound 3 showing promising hepatoprotective activity.

Objective:To investigate the effect of selenium on cyclophosphamide(CTX)-induced spermatogenic impairment(SI)in mice and its underlying mechanism.Methods:We equally randomized 36 male KM mice into 3 SI model and 3 control groups,the first 3 treated by intraperitoneal injection of CTX at 100 mg/kg(the SI model control group),CTX plus SI model control group,selenium deficient model group(-Se SI),selenium supplemented model group(+Se SI),while latter 3 by intraperitoneal injection of normal saline(the normal control),selenium deficiency control group(-Se control),selenium addition control group(+Se control),respectively,all once a week for 6 successive weeks.Then we observed the histopathological changes in the testes of all the mice by HE staining,obtained the sperm count in the epididymides,determined the expressions of glutathione peroxidase 4(GPx4)and SLC7A11 proteins by Western blot and ferroptosis-related genes by RT-qPCR,and examined the changes in the expres-sions of ferroptosis-related proteins and genes in the GC2-spd cells treated with ferroptosis inhibitors and inducers in combination with different concentrations of inorganic sodium selenite(SeS)and organic selenomethionine(SeM).Results:Compared with the nor-mal controls,the SI model mice showed significantly decreased testicular and prostatic organ coefficients,reduced spermatogenic lay-ers,increased voids,decreased serum ferritin concentration(P<0.05),and elevated transferrin concentration(P<0.05).The or-gan coefficients were significantly higher in the+Se SI and+Se control than in the-Se SI and-Se control groups(P<0.05,P<0.01),with evident pathological improvement of the testis tissue in the+Se controls.The expressions of the GPx4 and solute carrier family 7 members 11(SLC7A11)genes in the testis were dramatically down-regulated in the SI model controls(P<0.01),but up-reg-ulated in the+Se SI and+Se control compared with those in the-Se SI and-Se control group(P<0.01 and P<0.05),but there were no statistically significant differences between their protein expressions.The results of in vitro GC2 spd cell experiments indicated that the GPx4 gene and GPx4 protein levels in the-Se group were significantly lower than those in the normal control group(P<0.05),while the SLC7A11 gene level decreased(P<0.01).Different doses of SeS and SeM significantly increased the GPx4 protein expression compared to the average Se group.Low doses of SeM promoted a significant increase in GPx4 gene levels,while high doses of SeS increased the expression levels of SLC7A11 gene and SLC7A11 protein(P<0.05,P<0.01).The Se group showed a signifi-cant decrease in the levels of acsl4 and ptgs2 genes compared to the normal control group.SeM promoted the expression of acsl4,while SeS promoted the expression of ptgs2 and fth1(P<0.01,P<0.05).The intervention results of GC2 spd showed that the Erastin group had a decrease in ptgs2 compared to the normal control group,while the SeS+Erastin and SeM+Erastin groups had an increase in ptgs2 gene expression compared to the Erastin group.However,the ptgs2 expression of Fer-1 was lower than that of the normal con-trol group,and the ptgs2 gene level of SeS+Fer-1 and SeM+Fer-1 groups was lower than that of Fer-1 group(P<0.05);The gene quantity of GPx4 in the SeM+Erastin and SeM+Fer-1 groups increased compared to the Erastin and Fer-1 groups(P<0.01,P<0.05);SeM+Erastin and SeS+Erastin showed a decrease in SLC7A11 compared to the Erastin group,as well as SeM+Fer-1 and SeS+Fer-1 groups compared to the Fer-1 group,accompanied by an increase in acsl4 and fth1(P<0.01).Conclusion:Selenium deficiency causes the reduction of the SLC7A11 and GPx4 gene levels,disorder of ferroptosis-related genes and down-regulation of the GPx4 protein expression in the mouse testis and spermatocytes.Selenium can promote the expression of GPx4,up-regulate the level of SLC7A11,and improve spermatogenesis in the testis of the mouse with SI.There are differences between organic SeM and inorganic SeS in regulating the ferroptosis pathway-related genes.

Humans ; Prevalence ; Mitral Valve ; Mitral Valve Prolapse ; Adventitia

Objective To investigate the release of enterogenic and hepatogenic high mobility group protein B1(HMGB1)through exosomes and its regulatory pathway.Methods We used wild-type(WT)and ASC-/-mice for this study.We randomly selected five mice per group from each strain and fed them either a normal diet(ND)or a high-fat diet(HFD)for eight weeks.The control group consisted of WT mice fed with the normal diet;the HFD group were WT mice with the HFD;the microflora disturbance(MD)group were ASC-/-mice fed with the normal diet;the high-lipid microflora disturbance(HLMD)group were ASC-/-mice with HFD.We used confocal microscopy to detect the co-localization of liver and intestinal exosome markers with HMGB1.We then measured the expression level of HMGB1 content in exosomes by Western blotting and PCR.The AML12 cells were treated with palmitic acid(PA)and lipopolysaccharide(LPS)for 24 h to build an in vitro model.We also detected HMGB1/CD63 levels using Western blotting.To understand the regulatory mechanism of exosome release,we employed siRNA intervention.Results The secretion of exosomes increased significantly in HFD group compared with control group[(3.5±0.2)ng/ml vs.(1.1±0.3)ng/ml,P<0.05],HLMD group compared with those in MD group[(3.2±0.2)ng/ml vs.(1.9±0.4)ng/ml,P<0.05].Using immunofluorescence detection,we observed increased co-localization of exosome markers(ALP or VPS16)with HMGB1 in HFD group compared with control group.We also observed this in AML12 cells treated with PA and LPS compared with blank control.The PCR data showed that HMGB1 in hepatocyte exosomes was higher in HFD group compared with control group(41.5±10.2 vs.1.3±0.3,P<0.05),HLMD group was significantly higher than that in MD group(48.6±7.2 vs.1.5±0.5,P<0.05).TLR4 expression was higher in HFD group compared with control group(13.8±6.2 vs.2.8±0.9,P<0.05),HLMD group compared with MD group(22.6±4.1 vs.2.5±1.5,P<0.05).In intestinal mucosal cells,the co-location of HMGB1 and exosome marker CD63 was significantly higher in HFD group compared with control group(0.6±0.2 vs.0.4±0.1,P<0.05),and HLMD group compared with MD group(0.9±0.2 vs.0.5±0.1,P<0.05).In vitro,the HMGB1 of exosomes was increased in endotoxin group(5.1±0.8)and high lipid endotoxin group(5.5±0.7)compared with control group(3.8±0.6,P<0.05).On the other hand,the HMGB1 of exosomes in the cell siRNA intervention group was not increased compared with control group(3.7±0.6 vs.3.8±0.6,P>0.05).Conclusion HMGB1 is released by exosomes in hepatocytes and intestinal cells,and regulated by Toll-like receptor 4(TLR4)under a high-fat diet and intestinal flora disorder,which may be one of the contributing factors in promoting the development of steatohepatitis.

OBJECTIVE: To evaluate the characteristics of severe periodontitis with various number of tooth loss during 4-year natural progression, and to analyze the factors related to higher rate of tooth loss. METHODS: A total of 217 patients aged 15 to 44 years with severe periodontitis were included, who participated in a 4-year natural progression research. Data obtained from questionnaire survey, clinical examination and radiographic measurement. Tooth loss during 4-year natural progression was evaluated. The baseline periodontal disease related and caries related factors were calculated, including number of teeth with bone loss > 50%, number of missing molars, number of teeth with widened periodontal ligament space (WPDL), number of teeth with periapical lesions and etc. Characteristics of populations with various number of tooth loss and the related factors that affected higher rate of tooth loss were analyzed. RESULTS: In 4 years of natural progression, 103 teeth were lost, and annual tooth loss per person was 0.12±0.38. Nine patients lost 3 or more teeth. Thirty-four patients lost 1 or 2 teeth, and 174 patients were absent of tooth loss. Molars were mostly frequent to lose, and canines presented a minimum loss. The number of teeth with WPDL, with periapical lesions, with intrabony defects, with probing depth (PD)≥7 mm, with PD≥5 mm, with clinical attachment loss≥5 mm, with bone loss > 50% and with bone loss > 65% were positively correlated to number of tooth loss. Results from orderly multivariate Logistic regression showd that the number of teeth with bone loss > 50% OR=1.550), baseline number of molars lost (OR=1.774), number of teeth with WPDL (1 to 2: OR=1.415; ≥3: OR=13.105), number of teeth with periapical lesions (1 to 2: OR=4.393; ≥3: OR=9.526) and number of teeth with caries/residual roots (OR=3.028) were significant risk factors related to higher likelihood of tooth loss and multiple tooth loss. CONCLUSION In 4 years of natural progression, the number of teeth with bone loss > 50%, baseline number of missing molars, number of teeth with WPDL, baseline number of teeth with periapical lesions and number of teeth with caries/residual roots were significantly related to higher risk of tooth loss and multiple tooth loss among Chinese young and middle-aged patients with severe periodontitis in rural areas.
Humans ; Tooth Loss/etiology* ; Periodontitis/complications* ; Tooth ; Periodontal Diseases ; Molar