Icariin promotes bone marrow mesenchymal stem cells osteogenic differentiation via the mTOR/autophagy pathway to improve ketogenic diet-associated osteoporosis.

BACKGROUND: Icariin, a traditional Chinese medicine, has demonstrated anti-osteoporotic properties in ovariectomized mice. However, its effectiveness in preventing bone loss induced by ketogenic diet (KD), which mimics osteoporosis in human, remains unexplored. This study aims to investigate icariin's impact on KD-induced bone loss in mice. METHODS: Thirty mice were divided into: sham, KD, and KD + icariin groups. Post a 12-week intervention, evaluation including bone microstructures, serum concentrations of tartrate-resistant acid phosphatase (TRAP) and bone-specific alkaline phosphatase (ALP), and femoral tissue expression levels of osteocalcin (OCN) and TRAP. The expression levels of mammalian target of rapamycin (mTOR), ALP, peroxisome proliferator-activated receptor gamma (PPAR-γ), phosphorylated mTOR (p-mTOR), and the autophagy adaptor protein (p62) were also analyzed. Alizarin granule deposition and cellular ALP levels were measured following the induction of bone marrow mesenchymal stem cells (BMSCs) into osteogenesis. RESULTS: The study found that KD significantly impaired BMSCs' osteogenic differentiation, leading to bone loss. Icariin notably increased bone mass, stimulated osteogenesis, and reduced cancellous bone loss. In the KD + icariin group, measures such as bone tissue density (TMD), bone volume fraction (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) were significantly higher than in the KD group. Additionally, bone trabecular separation (Tb.Sp) was markedly lower in the KD + icariin group. Moreover, icariin increased OCN and ALP levels while suppressing PPAR-γ, TRAP, p62, and p-mTOR. In cellular studies, icariin encouraged osteogenic development in BMSCs under KD conditions. CONCLUSIONS: Icariin effectively counteracts bone thinning and improves bone microstructure. Its mechanism likely involves stimulating BMSCs osteogenic differentiation and inhibiting bone resorption, potentially through mTOR downregulation. These findings suggest icariin's potential as an alternative treatment for KD-induced bone loss.

Breaking NGF-TrkA immunosuppression in melanoma sensitizes immunotherapy for durable memory T cell protection.

Melanoma cells, deriving from neuroectodermal melanocytes, may exploit the nervous system's immune privilege for growth. Here we show that nerve growth factor (NGF) has both melanoma cell intrinsic and extrinsic immunosuppressive functions. Autocrine NGF engages tropomyosin receptor kinase A (TrkA) on melanoma cells to desensitize interferon γ signaling, leading to T and natural killer cell exclusion. In effector T cells that upregulate surface TrkA expression upon T cell receptor activation, paracrine NGF dampens T cell receptor signaling and effector function. Inhibiting NGF, either through genetic modification or with the tropomyosin receptor kinase inhibitor larotrectinib, renders melanomas susceptible to immune checkpoint blockade therapy and fosters long-term immunity by activating memory T cells with low affinity. These results identify the NGF-TrkA axis as an important suppressor of anti-tumor immunity and suggest larotrectinib might be repurposed for immune sensitization. Moreover, by enlisting low-affinity T cells, anti-NGF reduces acquired resistance to immune checkpoint blockade and prevents melanoma recurrence.

[Corrigendum] Icaritin promotes the osteogenesis of bone marrow mesenchymal stem cells via the regulation of sclerostin expression.

Following the publication of the above article, the authors have contacted the Editorial Office to explain that they had assembled the cellular morphological images in Fig. 1A on p. 819 incorrectly; essentially, the cell morphology of 2 passages of hBMSCs (centre panel) should have been shown as the data panel for 3 passages of hBMSCs (right-hand panel), and likewise, the cell morphology of 3 passages of hBMSCs should have been shown as the data panel for 2 passages of hBMSCs. The revised version of Fig. 1 is shown below. The authors confirm that the errors associated with this figure did not have any significant impact on either the results or the conclusions reported in this study, and are grateful to the Editor of International Journal of Molecular Medicine for allowing them the opportunity to publish this Corrigendum. Furthermore, they apologize to the readership of the Journal for any inconvenience caused. [International Journal of Molecular Medicine 45: 816-824, 2020; DOI: 10.3892/ijmm.2020.4470].

Dynamics of a disinhibitory prefrontal microcircuit in controlling social competition.

Social competition plays a pivotal role in determining individuals' social status. While the dorsomedial prefrontal cortex (dmPFC) is essential in regulating social competition, it remains unclear how information is processed within its local networks. Here, by applying optogenetic and chemogenetic manipulations in a dominance tube test, we reveal that, in accordance with pyramidal (PYR) neuron activation, excitation of the vasoactive intestinal polypeptide (VIP) or inhibition of the parvalbumin (PV) interneurons induces winning. The winning behavior is associated with sequential calcium activities initiated by VIP and followed by PYR and PV neurons. Using miniature two-photon microscopic (MTPM) and optrode recordings in awake mice, we show that VIP stimulation directly leads to a two-phased activity pattern of both PYR and PV neurons-rapid suppression followed by activation. The delayed activation of PV implies an embedded feedback tuning. This disinhibitory VIP-PV-PYR motif forms the core of a dmPFC microcircuit to control social competition.

Ectopic Expression of miR-532-3p Suppresses Bone Metastasis of Prostate Cancer Cells via Inactivating NF-κB Signaling.

miR-532-3p is a widely documented microRNA (miRNA) involved in multifaceted processes of cancer tumorigenesis and metastasis. However, the clinical significance and biological functions of miR-532-3p in bone metastasis of prostate cancer (PCa) remain largely unknown. Herein, we report that miR-532-3p was downregulated in PCa tissues with bone metastasis, and downexpression of miR-532-3p was significantly associated with Gleason grade and serum prostate-specific antigen (PSA) levels and predicted poor bone metastasis-free survival in PCa patients. Upregulating miR-532-3p inhibited invasion and migration abilities of PCa cells in vitro, while silencing miR-532-3p yielded an opposite effect on invasion and migration abilities of PCa cells. Importantly, upregulating miR-532-3p repressed bone metastasis of PCa cells in vivo. Our results further demonstrated that overexpression of miR-532-3p inhibited activation of nuclear facto κB (NF-κB) signaling via simultaneously targeting tumor necrosis factor receptor-associated factor 1 (TRAF1), TRAF2, and TRAF4, which further promoted invasion, migration, and bone metastasis of PCa cells. Therefore, our findings reveal a novel mechanism contributing to the sustained activity of NF-κB signaling underlying the bone metastasis of PCa.

Construction of a human cell landscape at single-cell level.

Single-cell analysis is a valuable tool for dissecting cellular heterogeneity in complex systems1. However, a comprehensive single-cell atlas has not been achieved for humans. Here we use single-cell mRNA sequencing to determine the cell-type composition of all major human organs and construct a scheme for the human cell landscape (HCL). We have uncovered a single-cell hierarchy for many tissues that have not been well characterized. We established a 'single-cell HCL analysis' pipeline that helps to define human cell identity. Finally, we performed a single-cell comparative analysis of landscapes from human and mouse to identify conserved genetic networks. We found that stem and progenitor cells exhibit strong transcriptomic stochasticity, whereas differentiated cells are more distinct. Our results provide a useful resource for the study of human biology.

Icaritin promotes the osteogenesis of bone marrow mesenchymal stem cells via the regulation of sclerostin expression.

Icaritin, a metabolite of icariin, is a potent promoter of bone marrow­derived mesenchymal stem cells (BMSCs) osteogenesis, but the underlying mechanisms remain unclear. To examine the effects of icaritin on osteogenic differentiation, BMSCs were exposed to osteogenic induction medium with or without icaritin pretreatment in the present study. It was identified that icaritin (0.01­1 µM) exhibited no cytotoxicity on the proliferative abilities of the BMSCs. Icaritin at 1 µM increased alkaline phosphatase activity, mineral deposition and osteoblast­specific gene expression. Treatment with 1 µM Icaritin upregulated osteocalcin, RUNX family transcription factor 2, tissue­nonspecific alkaline phosphatase and ß­catenin, and suppressed sclerostin (SOST) gene expression in different stages of osteogenic differentiation. It was also demonstrated that SOST overexpression inhibited icaritin­induced osteogenesis. The western blot analysis data suggested that ICI 182780, which causes estrogen receptor α (ERα) degradation, reversed the icaritin­induced decrease in SOST expression, which was inconsistent with the results of immunofluorescence analysis. In conclusion, icaritin was demonstrated to promote the osteogenesis of hBMSCs by downregulating SOST expression, and icaritin­induced suppression of SOST was regulated in part via the Wnt/ß­catenin/ERα axis.

Use of TAI-FISH to visualize neural ensembles activated by multiple stimuli.

Researchers in behavioral neuroscience have long sought imaging techniques that can identify and distinguish neural ensembles that are activated by sequentially applied stimuli at single-cell resolution across the whole brain. Taking advantage of the different kinetics of immediate-early genes' mRNA and protein expression, we addressed this problem by developing tyramide-amplified immunohistochemistry-fluorescence in situ hybridization (TAI-FISH), a dual-epoch neural-activity-dependent labeling protocol. Here we describe the step-by-step procedures for TAI-FISH on brain sections from mice that were sequentially stimulated by morphine (appetitive first stimulus) and foot shock (aversive second stimulus). We exemplify our approach by FISH-labeling the neural ensembles that were activated by the second stimulus for the mRNA expression of c-fos, a well-established marker of neural activation. We labeled neuronal ensembles activated by the first stimulus using fluorescence immunohistochemistry (IHC) for the c-fos protein. To further improve the temporal separation of the c-fos mRNA and protein signals, we provide instructions on how to enhance the protein signals using tyramide signal amplification (TSA). Compared with other dual-epoch labeling techniques, TAI-FISH provides better temporal separation of the activated neural ensembles and is better suited to investigation of whole-brain responses. TAI-FISH has been used to investigate neural activation patterns in response to appetitive and aversive stimuli, and we expect it to be more broadly utilized for visualizing brain responses to other types of stimuli, such as sensory stimuli or psychiatric drugs. From first stimulation to image analysis, TAI-FISH takes ∼9 d to complete.

[Clinical Analysis of 4 AML Patients Aged Over 80 Years Treated with Chemotherapy Combined with Haploidentical Hematopoietic Stem Cell Infusion].

OBJECTIVE: To investigate the efficacy and safety of MA (mitoxantrone and cytarabine) regimen chemotherapy combined with granulocyte-colony stimulating factor (G-CSF)-mobilized family related HLA-haploidentical donor peripheral blood hematopoietic stem cell (G-PBHSC) infusion for the treatment of acute myeloid leukemia (AML) patients aged over 80 years. METHODS: Four elderly patients with AML were treated in Chinese Second Artillery General Hospital from August 2008 to September 2013. The proportion of male to female was 1 : 3 and the median age 83 (80-85) years. All patients received programmed infusions of G-PBHSC after MA regimen chemotherapy without graft-versus-host disease (GVHD) prophylaxis. After complete remission (CR), patients only received G-PBHSC infusion without chemotherapy. RESULTS: Three cases achieved CR and their disease free survival (DFS) time was 18, 8, 6 months, respectively. 1 case did not reach remission after 2 cycles chemotherapy. The median overall survival (OS) time was 10 (3-20) months. No GVHD was observed in any of the patients during treatment. Concludsion: The combination of chemotherapy and programmed haploidentical G-PBHSC infusion is an alternative approach for AML patients aged over 80 years.

[Clinical analysis of lymphoblastic lymphoma/leukemia treated with Hyper-CVAD/MA regimen chemotherapy combined with haploidentical hematopoietic stem cell infusion].

This study was aimed to investigate the efficacy of Hyper-CVAD/MA regimen chemotherapy combined with haploidentical hematopoietic stem cell infusion for the treatment of lymphoblastic lymphoma/leukemia (LBL/ALL). Seven patients with LBL/ALL were treated in Second Artillery General Hospital from August 2009 to September 2012. All patients received programmed infusions of granulocyte-colony stimulating factor (G-CSF)-mobilized family related HLA-haploidentical donor peripheral blood hematopoietic stem cell (G-PBHSC) after each of cycle of Hyper-CVAD/MA regimen chemotherapy without graft-versus-host disease (GVHD) prophylaxis. A total of four cycles of therapy were planned. The interval between each cycle of treatment was 8 to 12 weeks. By April 2014, the median follow-up time was 41 (20-57) months. The results showed that the 7 patients totally received 30 cycles of treatment, and all patients achieved complete remission (CR). The patients were generally well-tolerated to therapy, and the most significant toxicities of grade 3 to 4 neutropenia and thrombocytopenia developed in nearly all of the patients after each course of the Hyper-CVAD/MA regimen. No GVHD was observed in any of the patients during treatment. Up to now, 5 patients were still alive, 2 patients were died of relapse. It is concluded that the combination of chemotherapy and programmed haploidentical G-PBHSC infusion is a promising approach to the treatment of LBL/ALL.

[Clinical investigation of refractory lymphoma with HLA-mismatched stem-cell microtransplantation].

OBJECTIVE: To explore the efficacy and safety of HLA-mismatched stem-cell microtransplantation in patients with refractory lymphoma. METHODS: This study included 10 patients with relapsing or refractory lymphoma at Department of Hematology, Second Artillery General Hospital from October 2009 to February 2012. All patients received programmed infusions of G-CSF-mobilized HLA-mismatched donor peripheral blood stem cell (G-PBSC) after each cycle of Hyper-CVAD/MA conditioning without graft-versus-host disease (GVHD) prophylaxis. RESULTS: A total of 31 cycles of microtransplantation were performed. Among them, 6 patients achieved a complete remission (CR) and 2 got partial remission (PR). And the overall response was 8/10. The occurrence of grades III-IV neutrocytopenia and thrombocytopenia was almost 100% after Hyper-CVAD/MA conditioning, but the median recovery times of neutrophils and platelets were 9 days and 14 days respectively because of programmed infusions of G-PBSCs. And 18 bouts of G-PBSC infusion related fever were observed. No GVHD was observed in any of them during treatment. Up to March 2013, 6 patients survived while another 4 died. The 1- and 3-year overall survival and disease-free survival rates were the same (60%). CONCLUSION: The novel therapy of microtransplantation may improve outcome and avoid GVHD in patients with relapsing or refractory lymphoma.

[Prophylactic G-CSF mobilized donor lymphocytes infusion after non-myeloablative stem cell transplantation prevents relapse in patients with high-risk leukemia].

OBJECTIVE: To evaluate the anti-leukemia effects of prophylactic G-CSF mobilized donor lymphocytes infusion (pG-DLI) and its relationship with the incidence of graft-versus-host disease (GVHD) in high-risk leukemia patients with non-myeloablative stem cell transplantation (NST). METHODS: 12 patients with high-risk leukemia were analyzed, including Ph⁺ acute lymphocytic leukemia (n=1), acute leukemia (AL) with persistent non-complete remission (n=2), acute myeloid leukemia (AML) with central nervous system (CNS) relapse (n=3), hybrid AL (n=1), secondary AML evolving from myelodysplastic syndrome (MDS/AML) (n=2), chronic myeloid leukemia in accelerated phase (CML-AP) (n=1), CML in blastic phase (CML-BP) (n=2). All patients received non-myeloablative conditioning and pG-DLIs were administered 30-40 days post transplantation if no signs of GVHD were present. The percentage of donor cell chimera was analyzed by short tandem repeat-polymerase chain reaction (STR-PCR) just before and after pG-DLI. The incidence of leukemia relapse and GVHD were observed. RESULTS: 12 high-risk leukemia patients with a median age of 38 (range: 29-52) years received G-DLI at a median interval of 35 (32-40) days. The median numbers of infused mononuclear cells (MNCs), CD34⁺, and CD3⁺ cells/kg recipient body weight was 2.3×108/kg, 1.7×106/kg, and 0.6×108/kg, respectively. 10 of 12 patients had full donor chimera before pG-DLIs and conversion from mixed to full donor chimera occurred in the other 2 patients shortly after pG-DLI. Grade II acute GVHD (aGVHD) was observed in only 2 patients and chronic GVHD (cGVHD) developed in 6 patients, including involvement of skin (n=3), skin and intestine (n=2), liver (n=1). 1 patient died of cGVHD. With a median follow-up of 40 (24-64) months, 7 patients are alive in remission, with 3-year actuarial overall survival (OS) and disease-free survival (DFS) rates of the same 58.3%. CONCLUSION: Our findings indicate that pG-DLI after NST does not increase the risk of aGVHD, but could enhance the capacity graft-vs-leukemia and prevent relapse in high-risk leukemia patients.

Effects of recombinant human granulocyte colony-stimulating factor on central and peripheral T lymphocyte reconstitution after sublethal irradiation in mice.

Granulocyte colony-stimulating factor (G-CSF) is one of the most critical cytokines used for the treatment of acute radiation syndrome (ARS). In addition to the hematopoietic effects of G-CSF on the differentiation and proliferation of myeloid progenitor cells, G-CSF is also known to have immunomodulatory effects. The aim of the present study was to investigate whether G-CSF could accelerate central and peripheral T lymphocyte recovery after a sublethal dose of irradiation. Female BALB/c mice were subjected to 6 Gy of total body irradiation and then were treated with either 100 µg/kg G-CSF or an equal volume of PBS once daily for 14 days. Percentages of thymocyte subpopulations including CD4 - CD8 - , CD4 + CD8 + , CD4 + CD8- and CD4 - CD8+ T cells, peripheral CD3 + , CD4+ and CD8+ cells were analyzed by flow cytometry. Recent thymic emigrants (RTEs) were assessed by real-time polymerase chain reaction (PCR) using primers specific to the 257-bp T cell receptor rearrangement excision circles (sjTRECs). The proliferative capacity of splenic mononuclear cells upon exposure to ConA was measured by using the Cell Count Kit-8 (CCK-8). G-CSF treatment promoted thymocyte regeneration, accelerated the recovery of CD4 + CD8+ cells and increased the frequency of thymocyte sjTRECs. These effects were more prominent at early time points (Day 28) after irradiation. G-CSF also increased the rate of recovery of peripheral CD3 + , CD4+ and CD8+ cells and shortened the period of severe lymphopenia following irradiation. G-CSF also increased the splenic mononuclear cell mitotic responsiveness to ConA more than control-treated cells. Our results show that G-CSF accelerates T cell recovery through both thymic-dependent and thymic-independent pathways, which could be used to increase the rate of immune reconstitution after sublethal irradiation.

Ras signaling mechanisms underlying impaired GluR1-dependent plasticity associated with fragile X syndrome.

Fragile X syndrome, caused by the loss of FMR1 gene function and loss of fragile X mental retardation protein (FMRP), is the most commonly inherited form of mental retardation. The syndrome is characterized by associative learning deficits, reduced risk of cancer, dendritic spine dysmorphogenesis, and facial dysmorphism. However, the molecular mechanism that links loss of function of FMR1 to the learning disability remains unclear. Here, we report an examination of small GTPase Ras signaling and synaptic AMPA receptor (AMPA-R) trafficking in cultured slices and intact brains of wild-type and FMR1 knock-out mice. In FMR1 knock-out mice, synaptic delivery of GluR1-, but not GluR2L- and GluR4-containing AMPA-Rs is impaired, resulting in a selective loss of GluR1-dependent long-term synaptic potentiation (LTP). Although Ras activity is upregulated, its downstream MEK (extracellular signal-regulated kinase kinase)-ERK (extracellular signal-regulated kinase) signaling appears normal, and phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB; or Akt) signaling is compromised in FMR1 knock-out mice. Enhancing Ras-PI3K-PKB signaling restores synaptic delivery of GluR1-containing AMPA-Rs and normal LTP in FMR1 knock-out mice. These results suggest aberrant Ras signaling as a novel mechanism for fragile X syndrome and indicate manipulating Ras-PI3K-PKB signaling to be a potentially effective approach for treating patients with fragile X syndrome.