Effects of the combination of Epimedii Folium and Ligustri Lucidi Fructus on apoptosis and autophagy in SOP rats and osteoblasts via PI3K/AKT/mTOR pathway.

BACKGROUND: This study aimed to investigate the effects of the combination of Epimedii Folium (EF) and Ligustri Lucidi Fructus (LLF) on regulating apoptosis and autophagy in senile osteoporosis (SOP) rats. METHODS: Firstly, we identified the components in the decoction and drug-containing serum of EL (EF&LLF) by Ultra performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q-TOF-MS). Secondly, SOP rats were treated with EF, LLF, EL and caltrate to evaluate the advantages of EL. Finally, H2O2-, chloroquine-, and MHY1485-induced osteoblasts were treated with different doses of EL to reveal the molecular mechanism of EL. We detected bone microstructure, oxidative stress levels, ALP activity and the expressions of Bax, Bcl-2, caspase3, P53, Beclin-1, p-PI3K, PI3K, p-Akt, Akt, p-mTOR, mTOR, and LC3 in vivo and in vitro. RESULTS: 36 compounds in EL decoction and 23 in EL-containing serum were identified, including flavonoids, iridoid terpenoids, phenylethanoid glycosides, polyols and triterpenoids. EL could inhibit apoptosis activity and increase ALP activity. In SOP rats and chloroquine-inhibited osteoblasts, EL could improve bone tissue microstructure and osteoblasts functions by upregulating Bcl-2, Beclin1, and LC3-II/LC3-I, while downregulating p53 in all treatment groups. In H2O2-induced osteoblasts, EL could upregulate the protein and mRNA expressions of Bcl-2 while downregulate LC3-II/LC3-I, p53 and Beclin1. Besides, EL was able to down-regulate PI3K/AKT/mTOR pathway which activated in SOP rats and MHY1485-induced osteoblasts. CONCLUSIONS: These findings demonstrate that EL with bone protective effects on SOP rats by regulating autophagy and apoptosis via PI3K/Akt/mTOR signaling pathway, which might be an alternative medicine for the treatment of SOP.

Combined Epimedii Folium and Ligustri Lucidi Fructus with dexamethasone alleviate the proliferation of airway smooth muscle cells by regulating apoptosis/autophagy.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine (TCM) theory believes kidney deficiency is the root cause of chronic refractory asthma with pathological changes of airway remodeling. Our previous experiments confirmed that the combination of Epimedii Folium and Ligustri Lucidi Fructus (ELL) with the effect of nourishing Yin and Yang of the kidney could improve the pathological changes of airway remodeling in asthmatic rats, but the specific mechanism remains unclear. AIM OF THE STUDY: This research was designed to reveal the synergy of ELL and dexamethasone (Dex) in the proliferation, apoptosis, and autophagy of airway smooth muscle cells (ASMCs). MATERIALS AND METHODS: Primary cultures of ASMCs from rats were prepared and induced with histamine (Hist), Z-DEVD-FMK (ZDF), rapamycin (Rap), or 3-Methyladenine (3-MA) at generation 3-7 for 24 or 48 h. Subsequently, the cells were treated with Dex, ELL, and ELL&Dex for 24 or 48 h. The effect of various concentrations of inducers and drugs on cell viability was detected by Methyl Thiazolyl Tetrazolium (MTT) assay, cell proliferation was tested using immunocytochemistry (ICC) by detecting Ki67 protein, cell apoptosis was measured by Annexin V-FITC/PI assay and Hoechst nuclear staining, cell ultrastructure was observed by transmission electron microscopy (TEM), and immunofluorescence (IF), Western blot (WB) combined with quantitative real-time PCR (qPCR) were used for measuring autophagy and apoptosis-related genes including protein 53 (P53), cysteinyl aspartate-specific proteinase (Caspase)-3, microtubule-associated protein 1 light chain 3 (LC3), Beclin-1, mammalian target of rapamycin (mTOR) and p-mTOR. RESULTS: In ASMCs, Hist and ZDF promoted cell proliferation, significantly decreased Caspase-3 protein expression, and up-regulated Beclin-1 levels; Dex alone and in combination with ELL promoted Beclin-1, Caspase-3, and P53 expression, enhancing autophagy activity and apoptosis in Hist and ZDF-induced AMSCs. In contrast, Rap inhibited cell viability, increased Caspase-3, P53, Beclin-1, and LC3-II/I and decreased the levels of mTOR and p-mTOR with promoting apoptosis and autophagy; ELL or ELL&Dex reduced P53, Beclin-1, and LC3-II/I to down-regulate apoptosis and the excessive autophagic state of ASMCs induced by Rap. In the 3-MA model, cell viability and autophagy were reduced; ELL&Dex significantly upgraded the expression of Beclin-1, P53, and Caspase-3 and promoted apoptosis and autophagy of ASMCs. CONCLUSIONS: These results suggest that ELL combined with Dex may regulate the proliferation of ASMCs by promoting apoptosis and autophagy and be a potential medicine for the treatment of asthma.

Epimedii Folium and Ligustri Lucidi Fructus Promote Osteoblastogenesis and Inhibit Osteoclastogenesis against Osteoporosis via Acting on Osteoblast-Osteoclast Communication.

Osteoblast (OB) and osteoclast (OC) play important roles in bone formation and bone resorption, which can communicate with each other through cytokine paracrine. Previous studies have confirmed that Epimedii Folium (EF) and Ligustri Lucidi Fructus (LLF) used alone or in combination can treat osteoporosis (OP) through regulating bone remodeling, but the effects of EF and LLF on osteoblastogenesis, osteoclastogenesis, and OB-OC communication are unclear. In this study, we investigated the direct and indirect effects of EF and LLF on OBs and OCs via monoculture and coculture (transwell) models of OBs and OCs. We found that the combination of EF and LLF (EF&LLF) could promote osteoblastogenesis and inhibit osteoclastogenesis directly and indirectly. In order to study the mechanisms of EF&LLF on indirectly regulating osteoblastogenesis and osteoclastogenesis, we detected the expression of cytokines by which OBs and OCs could communicate with each other. We found that EF&LLF could downregulate the expression of RANKL and M-CSF and the protein ratio of RANKL/OPG of OBs and Atp6v0d2 expression of OCs and upregulate the expression of OPG and TGF-ß1 of OBs and the expression of TGF-ß1, BMP-2, and IGF-1 of OCs, indicating that EF&LLF could regulate cytokine expressions of OBs/OCs to affect OB-OC communication. In addition, EF&LLF had a better effect on regulating cytokines of OBs and OCs than EF or LLF in single use. This study suggested that EF&LLF exhibited the effects of promoting osteoblastogenesis and inhibiting osteoclastogenesis via acting on OB-OC communication and provided some scientific evidences for EF&LLF against OP.

Transcriptome analysis reveals the mechanisms of hepatic injury caused by long-term environmental exposure to atrazine in juvenile common carp (Cyprinus carpio L.).

Atrazine (ATZ) is the second most commonly used herbicide worldwide, resulting in the pollution of water bodies and affecting the economic benefits of aquaculture. ATZ is known to cause liver damage in the common carp, Cyprinus carpio L., one of the most widely cultivated fish in China, but the underlying mechanisms are poorly understood. In this study, juvenile common carp Cyprinus carpio L. were exposed to three different environmental levels (0.4, 0.8, and 1.2 µg/L) of ATZ for 12 weeks and changes in the liver transcriptomes between the high-dose group and the control group were analyzed. The data showed that different levels of ATZ exposure caused hepatotoxicity in juvenile carp, shown by biochemical parameters and histopathological changes. Comparative transcriptomics showed that high-dose ATZ exposure led to alterations in the expression of various lipid metabolism-related gene changes, including genes associated with metabolic pathways, fatty acid metabolism, and fatty acid elongation. Furthermore, a connection network analysis of the top 100 differentially expressed genes (DEGs) showed a variety of associations between high-dose ATZ-induced liver damage and the principal DEGs, indicating the complexity of hepatotoxicity induced by ATZ. In conclusion, the molecular mechanisms underlying ATZ-triggered hepatotoxicity in juvenile carp are primarily related to impaired lipid metabolism.

The balance between NANOG and SOX17 mediated by TET proteins regulates specification of human primordial germ cell fate.

BACKGROUND: Human primordial germ cells (hPGCs) initiate from the early post-implantation embryo at week 2-3 and undergo epigenetic reprogramming during development. However, the regulatory mechanism of DNA methylation during hPGC specification is still largely unknown due to the difficulties in analyzing early human embryos. Using an in vitro model of hPGC induction, we found a novel function of TET proteins and NANOG in the hPGC specification which was different from that discovered in mice. METHODS: Using the CRISPR-Cas9 system, we generated a set of TET1, TET2 and TET3 knockout H1 human embryonic stem cell (hESC) lines bearing a BLIMP1-2A-mKate2 reporter. We determined the global mRNA transcription and DNA methylation profiles of pluripotent cells and induced hPGC-like cells (hPGCLCs) by RNA-seq and whole-genome bisulfite sequencing (WGBS) to reveal the involved signaling pathways after TET proteins knockout. ChIP-qPCR was performed to verify the binding of TET and NANOG proteins in the SOX17 promoter. Real-time quantitative PCR, western blot and immunofluorescence were performed to measure gene expression at mRNA and protein levels. The efficiency of hPGC induction was evaluated by FACS. RESULTS: In humans, TET1, TET2 and TET3 triple-knockout (TKO) human embryonic stem cells (hESCs) impaired the NODAL signaling pathway and impeded hPGC specification in vitro, while the hyperactivated NODAL signaling pathway led to gastrulation failure when Tet proteins were inactivated in mouse. Specifically, TET proteins stimulated SOX17 through the NODAL signaling pathway and directly regulates NANOG expression at the onset of hPGCLCs induction. Notably, NANOG could bind to SOX17 promoter to regulate its expression in hPGCLCs specification. Furthermore, in TKO hESCs, DNMT3B-mediated hypermethylation of the NODAL signaling-related genes and NANOG/SOX17 promoters repressed their activation and inhibited hPGCLC induction. Knockout of DNMT3B in TKO hESCs partially restored NODAL signaling and NANOG/SOX17 expression, and rescued hPGCLC induction. CONCLUSION: Our results show that TETs-mediated oxidation of 5-methylcytosine modulates the NODAL signaling pathway and its downstream genes, NANOG and SOX17, by promoting demethylation in opposition to DNMT3B-mediated methylation, suggesting that the epigenetic balance of DNA methylation and demethylation in key genes plays a fundamental role in early hPGC specification.

Structure and Crystallization of High-Calcium, CMAS Glass Ceramics Synthesized with a High Content of Slag.

In this work, more than 70 wt % of ferromanganese slag (containing 40 wt % CaO) was used to synthesize high-calcium, CaO-MgO-Al2O3-SiO2 (CMAS) glass ceramics. The effect of SiO2/CaO on the structure, crystallization behavior and microstructure of high-calcium, CMAS, slag glass ceramics was studied by IR, NMR, DSC, XRD and SEM. The results showed that in the high-calcium, CMAS glass ceramics, the main existing forms of silicon-oxygen tetrahedra (Qn) were Q0 and Q1. With the increase in the SiO2/CaO, Qn changed from Q0 and Q1 (main units) to Q1 (main units) and Q2, and then to Q1 and Q2 (main units). The polymerization degree of Qn changed from low to high, making the glass more stable, which led to the increase in crystallization temperature and the decrease in crystallization kinetic constant (k) and frequency factor (υ). At the same time, the change in the Qn structure resulted in a gradual change to the main crystal, from akermanite to diopside-wollastonite.

Valproic acid promotes the in vitro differentiation of human pluripotent stem cells into spermatogonial stem cell-like cells.

BACKGROUND: Studying human germ cell development and male infertility is heavily relied on mouse models. In vitro differentiation of human pluripotent stem cells into spermatogonial stem cell-like cells (SSCLCs) can be used as a model to study human germ cells and infertility. The current study aimed to develop the SSCLC induction protocol and assess the effects of the developed protocol on SSCLC induction. METHODS: We examined the effects of valproic acid (VPA), vitamin C (VC) and the combination of VPA and VC on the SSCLC induction efficiency and determined the expression of spermatogonial genes of differentiated cells. Haploid cells and cells expressed meiotic genes were also detected. RNA-seq analysis was performed to compare the transcriptome between cells at 0 and 12 days of differentiation and differently expressed genes were confirmed by RT-qPCR. We further evaluated the alteration in histone marks (H3K9ac and H3K27me3) at 12 days of differentiation. Moreover, the SSCLC induction efficiency of two hiPSC lines of non-obstructive azoospermia (NOA) patients was assessed using different induction protocols. RESULTS: The combination of low concentrations of VPA and VC in the induction medium was most effective to induce SSCLCs expressing several spermatogonial genes from human pluripotent stem cells at 12 days of differentiation. The high concentration of VPA was more effective to induce cells expressing meiotic genes and haploid cells. RNA-seq analysis revealed that the induction of SSCLC involved the upregulated genes in Wnt signaling pathway, and cells at 12 days of differentiation showed increased H3K9ac and decreased H3K27me3. Additionally, two hiPSC lines of NOA patients showed low SSCLC induction efficiency and decreased expression of genes in Wnt signaling pathway. CONCLUSIONS: VPA robustly promoted the differentiation of human pluripotent stem cells into SSCLCs, which involved the upregulated genes in Wnt signaling pathway and epigenetic changes. hiPSCs from NOA patients showed decreased SSCLC induction efficiency and Wnt signaling pathway gene expression, suggesting that SSC depletion in azoospermia testes might be associated with inactivation of Wnt signaling pathway. Our developed SSCLC induction protocol provides a reliable tool and model to study human germ cell development and male infertility.

MicroRNAs secreted by human embryos could be potential biomarkers for clinical outcomes of assisted reproductive technology.

Introduction: MicroRNAs (miRNAs) are important regulators of many biological functions, including embryo implantation and development. Recently, it has been reported that miRNAs in biofluids are predictive for physiological and pathological processes. Objectives: In this study, we aim to investigate whether the miRNAs secreted by human embryos in culture medium can be used as embryonic biomarkers. Methods: The culture media were prospectively collected from embryos of patients at reproductive medicine center with informed consent. A high-throughput miRNA sequencing method was applied to detect the miRNA profiles in the human embryo culture media. After bioinformatics analysis and screening of differentially expressed miRNAs, quantitative real-time polymerase chain reaction (qRT-PCR) assay was subsequently performed to further confirm the sequencing results with mixed samples. Furthermore, we performed droplet digital PCR (ddPCR) to verify the target miRNAs at single sample level. Receiver operating characteristic (ROC) analyses were performed for differentially expressed miRNAs. Results: Compared with embryos with failed pregnancy, the embryos with successful pregnancy secreted different miRNA profiles into the culture media, which were predicted to be involved in multiple biological processes. Validated by droplet digital polymerase chain reaction (ddPCR), the expression of hsa-miR-26b-5p and hsa-miR-21-5p in the culture media of cleavage embryos with successful pregnancy was significantly lower than that of embryos with failed pregnancy. Moreover, the Receiver Operating Characteristic (ROC) curve analysis indicated that hsa-miR-26b-5p and hsa-miR-21-5p could serve as potential biomarkers for reproductive outcomes. Conclusion: Together, our findings highlight the important predictive potential of miRNAs secreted by human embryos in culture media, which is meaningful for non-invasive embryo selection in assisted reproductive technology.

Confined Gaussian-distributed electromagnetic field of tin(II) chloride-sensitized surface-enhanced Raman scattering (SERS) optical fiber probe: From localized surface plasmon resonance (LSPR) to waveguide propagation.

Surface-enhanced Raman scattering (SERS) induced by largely enhanced electromagnetic (EM) field provides a solid and promising avenue for ultrasensitive molecular detection. Here, a confined Gaussian-distributed EM field for SERS fiber probe with two influencing factors (localized surface plasmon resonance (LSPR) of silver and waveguide propagation of optical fiber) are proposed for the first time. SERS fiber probes with high sensitivity and good reproducibility were synthesized via a novel SnCl2 sensitization aided solvothermal method. The influencing factors and EM field distribution are investigated experimentally and theoretically. The LSPR-induced EM enhancement is observed. By introducing a sensitization procedure, silver particles show smaller sizes and narrower interparticle gaps, significantly influencing the LSPR and EM enhancement of the SERS fiber probe. Moreover, a unique waveguide-propagation-induced EM enhancement is brought up. Waveguide propagation modes of optical fibers influence the intensity and enhancement area of EM field. Further, the EM field distribution of SERS fiber probe is studied. It exhibits a concentrically-increased intensity gradient that is confined in core area with maximum enhancement at fiber core center. This confined Gaussian-distributed configuration of EM field on SERS fiber probe facet is induced by the LSPR of plasmons and waveguide propagation of optical fiber.

Impaired spermatogenesis in COVID-19 patients.

BACKGROUND: The current study aimed to determine the impact of SARS-CoV-2 infection on male fertility. METHODS: This is a single-center, hospital-based observational study that included autopsied testicular and epididymal specimens of deceased COVID-19 male patients (n=6) and recruited recovering COVID-19 inpatients (n=23) with an equal number of age-matched controls, respectively. We performed histopathological examinations on testicular and epididymal specimens, and also performed TUNEL assay and immunohistochemistry. Whereas, we investigated the semen specimen for sperm parameters and immune factors. FINDINGS: Autopsied testicular and epididymal specimens of COVID-19 showed the presence of interstitial edema, congestion, red blood cell exudation in testes, and epididymides. Thinning of seminiferous tubules was observed. The number of apoptotic cells within seminiferous tubules was significantly higher in COVID-19 compared to control cases. It also showed an increased concentration of CD3+ and CD68+ in the interstitial cells of testicular tissue and the presence of IgG within seminiferous tubules. Semen from COVID-19 inpatients showed that 39.1% (n=9) of them have oligozoospermia, and 60.9% (n=14) showed a significant increase in leucocytes in semen. Decreased sperm concentration, and increased seminal levels of IL-6, TNF-α, and MCP-1 compared to control males were observed. INTERPRETATION: Impairment of spermatogenesis was observed in COVID-19 patients, which could be partially explained as a result of an elevated immune response in testis. Additionally, autoimmune orchitis occurred in some COVID-19 patients. Further research on the reversibility of impairment and developing treatment are warranted. FUNDING: This study was supported by Ministry of Science and Technology of China Plan, Hubei Science and Technology Plan, National Key Research and Development Program of China, HUST COVID-19 Rapid Response Call, China and National Natural Science Foundation of China; these funding bodies are public institutions, and they had no role in study conception, design, interpretation of results, and manuscript preparation.

Abce1 orchestrates M-phase entry and cytoskeleton architecture in mouse oocyte.

ATP-binding cassette E1 (ABCE1) is a member of the ATP-binding cassette transporters and essential for diverse biological events regulating abroad range of biological functions including viral infection, cell proliferation, anti-apoptosis, translation initiation and ribosome biogenesis. Here, we discovered that Abce1 also plays indispensable roles in mouse oocyte meiotic progression. In the present study, we examined the expression, localization, and function of Abce1 during mouse oocyte meiotic maturation. Immunostaining and confocal microscopy identified that Abce1 localized as small dots in nucleus in germinal vesicle stage. After germinal vesicle breakdown, it dispersedly localized around the whole spindle apparatus. During the anaphase and telophase stages, Abce1 was just like a cap to localize around the two pole region of spindle but not the midbody and chromosome. Knockdown of Abce1 by specific siRNA injection delayed the resumption of meiosis (GVBD) and affected the extrusion of first polar body. Moreover, the process of spindle assembly and chromosome alignment were severely impaired. Abce1-RNAi led to the dissociation of γ-tubulin and p-MAPK from spindle poles, thus caused mounts of spindle morphology abnormities and chromosome alignment defects, leading to high incidence of aneuploidy. Our findings refresh the knowledge of Abce1 function by exploring its role in oocyte meiotic resumption, spindle assembly and chromosome alignment.