Angelica Sinensis Polysaccharide Suppresses the Aging of Hematopoietic Stem Cells Through Sirt1/FoxO1 Signaling.

BACKGROUND: This study investigated the anti-aging effects of Angelica sinensis polysaccharide (ASP) on mouse hematopoietic stem cells (HSCs) and related mechanisms. METHODS: Seventy-two C57BL/6J mice were randomly divided into the following three groups (n = 24 per group): control group; aging group, in which mice were irradiated with X-ray uniformly to establish the aging model of HSCs; and ASP group, in which mice were given 200 mg/kg ASP during irradiation. HSCs were collected by immunomagnetic beads, transmission electron microscopy was used to examine the morphological changes in HSCs, SA-ß-Gal staining was used to detect the aging cells, immunofluorescence staining was used to detect the reactive oxygen species (ROS), and western blotting was used to evaluate the expression levels of sirtuin 1 (Sirt1) and forkhead box O1 (FoxO1). RESULTS: HSCs in the control group had an intact cytoplasmic structure and many mitochondria. In the aging group, HSCs had many vacuoles in the cytoplasm, and few and irregular mitochondria. In the ASP group, HSCs had a normal cytoplasmic structure and more mitochondria compared with the aging group. The aging group had a significantly higher positive rate of HSCs SA-ß-Gal staining and ROS production than the control group (p < 0.05), but had lower expression of Sirt1 and FoxO1 (p < 0.05). These patho(physio)logical changes were ameliorated by ASP treatment (all p < 0.05). CONCLUSIONS: ASP inhibits irradiation-induced oxidative stress and aging of HSCs at least in part by regulating the Sirt1/FoxO1 pathway, thereby delaying aging of HSCs in mice.

Gastrodin promotes CNS myelination via a lncRNA Gm7237/miR-142a/MRF pathway.

Treatment of central nervous system (CNS) demyelination is greatly hindered by lack of the knowledge regarding to underlying molecular mechanisms as well as therapeutic agents. Here, we report a novel small molecule agent, gastrodin (GAS), which can significantly promote CNS myelination in in vivo mice models. By using high-throughput sequencing analysis, we discover a key long non-coding RNA Gm7237 that can enhance CNS myelination and is up-regulated by GAS. Through using bioinformatic analysis and experimental validations, we further unravel that microRNA-142a (miR-142a) and its target myelin gene regulatory factor (MRF) is under the direct regulation by Gm7237. Finally, we demonstrate that Gm7237/miR-142a/MRF axis is the key pathway involved in CNS myelination mediated by GAS. Overall, our results provide not only a novel agent for therapeutic treatment of CNS demyelination but also a molecular basis responsible for GAS-promoted CNS myelination.

A critical period of progesterone withdrawal precedes endometrial breakdown and shedding in mouse menstrual-like model.

STUDY QUESTION: Is there a critical period of progesterone (P4) withdrawal in a mouse menstrual-like model, and at what time after P4 withdrawal endometrial breakdown become irreversible? STUDY ANSWER: Our results showed that a 12-16 h critical period of P4 withdrawal exists in the mouse menstrual-like model. WHAT IS KNOWN ALREADY: P4 withdrawal is the trigger for endometrial breakdown and shedding during menstruation. To date, the molecular mechanisms responsible for endometrial breakdown have not been fully elucidated. In an ovariectomized macaque model, P4 replacement could reduce or block menses during a period of 36-48 h after P4 withdrawal, but after this, P4 supplementation did not reduce or block menses. Thus, in the macaque, a critical period of P4 withdrawal lasting 36-48 h exists before menses. STUDY DESIGN, SIZE, DURATION: We created a mouse menstrual-like model and restored P4 at four time points. The total number of mice was 120 and the duration of treatment was 26 days. PARTICIPANTS, SETTING, METHODS: A mouse menstrual model was characterized by endometrial morphology and plasma P4 levels. P4 was then replaced at 8, 12, 16 and 20 h after the removal of P4 implants. Vaginal smears, endometrial morphology, plasma P4 levels and expression patterns of matrix metalloproteinases (MMP-2, MMP-3, MMP-9, MMP-10, MMP-11 and MMP-13) were investigated. MAIN RESULTS AND THE ROLE OF CHANGE: Replacement of P4 at 8 and 12 h blocked menstrual-like bleeding and endometrial shedding; however, replacement at 16 and 20 h did not suppress bleeding or shedding. Furthermore, P4 replacement at 12 h inhibited the expression of all latent or active MMPs; however, replacement at 16 h inhibited only pMMP-13. LIMITATIONS, REASONS FOR CAUTION: Although determination of the critical period in vivo using a mouse model was successfully demonstrated, the mechanisms of P4 regulation need to be further explored. WIDER IMPLICATIONS OF THE FINDINGS: The experimental opportunities provided by the mouse model will facilitate understanding the role of P4 in the regulation of menstruation and help to identify new targets for the clinical intervention of menstrual disorders.

[Angelica sinensis polysaccharides delay aging of hematopoietic stem cells through inhibitting oxidative damge].

OBJECTIVE: The effect of angelica sinensis polysaccharides (ASP) on the production of reactive oxygen specie (ROS), the capability of total anti-oxidant (T-AOC), and the expression of p16 in mRNA level in mice hematopoietic stem cells (HSCs) were observed to explore the underlying mechanism that ASP delay aging of HSCs in vivo. METHOD: C57BL/6J mice were randomly divided into normal group, aging group, and the above groups treated with ASP. Mice were uniformly explored in X-ray (3.0 Gy/8 F) to erect model of aging. Normal and aging ASP intervention groups mice were treated with ASP by intragastric administration, while normal and aging groups were treated with equal-volume NS during X-ray irradiation. Mice HSCs were isolated by magnetic cell sorting and cultured in vitro. Senescence-associated beta-galactosidase (SA-beta-Gal) staining was used to detect aging HSCs. Cell cycles analysis and CFU-Mix cultivation were used to evaluate the capability of self-renewing and colony forming in HSCs. The production of ROS in HSCs was evaluated by flow cytometry analysis and immunofluorescence assess, respectively. T-AOC was detected by chemical colorimetric method. The expression of p16 was determined by real-time quantitative PCR (qRT-PCR). RESULT: Exogenous X-ray irradiation induced HSCs aging was compared with normal group without irradiation. Biological feature of HSCs in aging group with X-ray irradiation as follows: The percentage of SA-beta-Gal positive cells, the ratio of G1 stages and the production of ROS were significantly increased , the expression of p16 in mRNA level was also upregulated. The capacility of colony forming and T-AOC in HSCs were decreased. ASP could significantly decrease the percentage of SA-beta-Gal positive cells, the ratio of G1 stages and the production of ROS in HSCs, and downregulate the expression of p16 in mRNA level in HSCs contrast to aging group without ASP treatment. In addition, ASP could remarkably increase T-AOC and the capacility of colony forming in HSCs compared with aging group without ASP treatment. CONCLUSION: X-ray (3.0 Gy/8 F) could induce mice HSCs aging. ASP could delay senescence HSCs aging which maybe partly ascribed to the inhibition of oxidative damage and the downregulation of p16 mRNA expression.

CXCR4, regulated by HIF1A, promotes endometrial breakdown via CD45+ leukocyte recruitment in a mouse model of menstruation.

Menstruation is a specific physiological phenomenon in female humans that is regulated by complex molecular mechanisms. However, the molecular network involved in menstruation remains incompletely understood. Previous studies have suggested that C-X-C chemokine receptor 4 (CXCR4) is involved; however, how CXCR4 participates in endometrial breakdown remains unclear, as do its regulatory mechanisms. This study aimed to clarify the role of CXCR4 in endometrial breakdown and its regulation by hypoxia-inducible factor-1 alpha (HIF1A). We first confirmed that CXCR4 and HIF1A protein levels were significantly increased during the menstrual phase compared with the late secretory phase using immunohistochemistry. In our mouse model of menstruation, real-time PCR, western blotting, and immunohistochemistry showed that CXCR4 mRNA and protein expression levels gradually increased from 0 to 24 h after progesterone withdrawal during endometrial breakdown. HIF1A mRNA and HIF1A nuclear protein levels significantly increased and peaked at 12 h after progesterone withdrawal. Endometrial breakdown was significantly suppressed by the CXCR4 inhibitor AMD3100 and the HIF1A inhibitor 2-methoxyestradiol in our mouse model, and HIF1A inhibition also suppressed CXCR4 mRNA and protein expression. In vitro studies using human decidual stromal cells showed that CXCR4 and HIF1A mRNA expression levels were increased by progesterone withdrawal and that HIF1A knockdown significantly suppressed the elevation in CXCR4 mRNA expression. CD45+ leukocyte recruitment during endometrial breakdown was suppressed by both AMD3100 and 2-methoxyestradiol in our mouse model. Taken together, our preliminary findings suggest that endometrial CXCR4 expression is regulated by HIF1A during menstruation and may promote endometrial breakdown, potentially via leukocyte recruitment.

The elevated level of IL-1α in the bone marrow of aged mice leads to MSC senescence partly by down-regulating Bmi-1.

Osteoporosis is becoming increasingly prevalent with individual aging. Recent studies found that bone marrow mesenchymal stem cells (MSCs) undergo senescence along with the progression of age-related osteoporosis, leading to a decreased rate of new bone formation and fracture repair. The underlying mechanism of MSC senescence in the aged bone marrow has not been clarified yet. Here we found that MSCs from aged mice (12-month-old, O-MSCs) exhibited apparent senescent phenotypes compared with those from young controls (2-month-old, Y-MSCs), including lower proliferation rate, impaired self-renewal capacity, increased p16Ink4a expression and shifted differentiation balance to favor adipocytes over osteoblasts. Bmi-1, one of the main factors that regulate stem cell self-renewal, is dramatically decreased in O-MSCs. Knocking-down of Bmi-1 in Y-MSCs lead to cellular senescence, while over-expression of it rejuvenated O-MSCs. We further showed that the level of IL-1α is much higher in the bone marrow fluid of aged mice, which significantly inhibited Bmi-1 expression in MSCs. Our present study indicated that IL-1α, a key component of the senescence-associated secretory phenotype (SASP), is elevated in the aged bone marrow microenvironment, leading to decreased Bmi-1 expression in MSCs and consequently, MSC senescence.

Establishment of a Mouse Asthenospermia Model through Triggering D-Galactose Mediated Oxidative Stress Injury.

BACKGROUND: Asthenospermia is defined as the forward motility of sperm less than 32%. AIM/OBJECTIVE: This study aimed to establish a mouse model of asthenospermia through triggering D-galactose mediated oxidative stress. METHODS: A total of 40 Kunming male mice were randomly divided into control group, low-dose group (administrating D-galactose at 60 mg/kg), high-dose group (administrating D-galactose at 120 mg/kg), and high-dose+feed addition group (administrating D-galactose at 120 mg/kg together with oral D-galactose). The testicular weight, testicular organ coefficient, sperm viability, sperm concentration, and survival rate of the tail of epididymis were measured. Oxidative damage of D-- galactose to the reproductive system of mice was evaluated by measuring superoxide dismutase (SOD) and malondialdehyde (MDA) in the testicular homogenate of mice. RESULTS: The sperm motility, motility rate, concentration, and survival rate of low-dose, high-dose and high-dose+feed addition group were decreased, compared to that in the control group. However, there background:was a significant difference between high-dose group/high dose+feed group and the control group (p<0.05): the forward motile sperm motility rate and total motility rate are accorded with critical criteria of asthenospermia. As compared with the control group, the activity of SOD of model group mice significantly decreased, and MDA concentration significantly increased (p<0.05), except for low-dose versus control group for SOD activity. This suggests that testicular tissues suffered from oxidative damage. CONCLUSION: This study successfully established a mouse asthenospermia model through D-galactose mediated oxidative stress injury. The establishment of asthenospermia model in this study would provide new promising insight and act as a potential approach for studying asthenospermia in vivo levels.

De Novo assembly, characterization and development of EST-SSRs from Bletilla striata transcriptomes profiled throughout the whole growing period.

Bletilla striata is an endangered orchid that has been used for millennia as a medicinal herb, in cosmetics and as a horticultural plant. To construct the first nucleotide database for this species and to develop abundant EST-SSR markers for facilitating further studies, various tissues and organs of plants in the main developmental stages were harvested for mRNA isolation and subsequent RNA sequencing. A total of 106,054,784 clean reads were generated by using Illumina paired-end sequencing technology. The reads were assembled into 127,261 unigenes by the Trinity package; the unigenes had an average length of 612 bp and an N50 of 957 bp. Of these unigenes, 67,494 (51.86%) were annotated in a series of databases. Of these annotated unigenes, 41,818 and 24,615 were assigned to gene ontology categories and clusters of orthologous groups, respectively. Additionally, 20,764 (15.96%) unigenes were mapped onto 275 pathways using the KEGG database. In addition, 25,935 high-quality EST-SSR primer pairs were developed from the 15,433 unigenes by MISA mining. To validate the accuracy of the newly designed markers, 87 of 100 randomly selected primers were effectively amplified; 63 of those yielded PCR products of the expected size, and 25 yielded products with significant amounts of polymorphism among the 4 landraces. Furthermore, the transferability test of the 25 polymorphic markers was performed in 6 individuals of two closely related genus Phalaenopsis and dendrobium. Which results showed a total of 5 markers can successfully amplified among these populations. This research provides a comprehensive nucleotide database and lays a solid foundation for functional gene mining and genomic research in B. striata. The developed EST-SSR primers could facilitate phylogenetic studies and breeding.

Membrane chromatographic method for the rapid purification of vitellogenin from fish plasma.

A membrane chromatographic method was developed for the rapid purification of vitellogenin (Vtg) from the plasma of 17beta-estradiol induced loach (Misgurnus angaillicaud atus) and carp (Cyprinus carpio). The time required for the proposed procedure is less then 10 min at a flow-rate of 5 ml/min of the mobile phase, and 0.5 ml of fish plasma could be separated in one cycle. Multistep gradient elution was more suitable for the separation than linear gradient elution. Under optimized conditions, a single Vtg peak can be obtained and its identity was confirmed by SDS-PAGE and gel-permeation chromatography assessment. This method is rapid and easy to operate compared to conventional HPLC and FPLC columns for Vtg separation.

ROS are critical for endometrial breakdown via NF-κB-COX-2 signaling in a female mouse menstrual-like model.

Progesterone withdrawal triggers endometrial breakdown and shedding during menstruation. Menstruation results from inflammatory responses; however, the role of reactive oxygen species (ROS) in menstruation remains unclear. In this study, we explored the role of ROS in endometrial breakdown and shedding. We found that ROS levels were significantly increased before endometrial breakdown in a mouse menstrual-like model. Vaginal smear inspection, morphology of uterine horns, and endometrial histology examination showed that a broad range of ROS scavengers significantly inhibited endometrial breakdown in this model. Furthermore, Western blot and immunohistochemical analysis showed that the intracellular translocation of p50 and p65 from the cytoplasm into the nucleus was blocked by ROS scavengers and real-time PCR showed that cyclooxygenase-2 (COX-2) mRNA expression was decreased by ROS scavengers. Similar changes also occurred in human stromal cells in vitro. Furthermore, Western blotting and real-time PCR showed that one ROS, hydrogen peroxide (H2O2), promoted translocation of p50 and p65 from the cytoplasm to the nucleus and increased COX-2 mRNA expression along with progesterone maintenance. The nuclear factor κB inhibitor MG132 reduced the occurrence of these changes in human stromal cells in vitro. Viewed as a whole, our results provide evidence that certain ROS are important for endometrial breakdown and shedding in a mouse menstrual-like model and function at least partially via nuclear factor-κB/COX-2 signaling. Similar changes observed in human stromal cells could also implicate ROS as important mediators of human menstruation.