Number Of BrdU-positive Cells Research Articles

Epigenetic modifier G9a is involved in regulation of mouse tongue development

ObjectivesThe tongue comprises multiple tissues of different embryonic origins, including pharyngeal arch, somite, and cranial neural crest (CNC). However, its developmental regulatory mechanisms, especially those involving epigenetic modifiers, remain poorly understood. This study examined the roles of the epigenetic modifier G9a in murine tongue development. MethodsWe deleted G9a using Sox 9 (SRY-related HMG-box gene 9)-Cre recombinase, which acts in tongue progenitor cells, including CNC-derived cells, to generate G9a conditional knockout (cKO) mice. Histochemical and immunohistochemical analyses were conducted on sections prepared from tongue tissues of control and cKO mice. ResultsCre-dependent LacZ reporter mice, generated by crossing Rosa-LacZ mice with sox9-Cre mice, revealed Cre recombinase activity in the mucosal epithelium and tongue connective tissue of the embryonic tongue. Tongue volume was significantly reduced on embryonic day 17.5 (E17.5) and postnatal day 0 (P0) in cKO mice. Histological sections showed that the lingual mucosal epithelium was thinner in cKO mice. Reduced G9a levels were accompanied by decreased levels of a G9a substrate, dimethylated lysine 9 in histone H3, in the embryonic tongue. BrdU injection at E16.5 revealed reduced numbers of BrdU-positive cells in the mucosal epithelium and underlying connective tissue at E17.5 in cKO mice, indicating suppression of cell proliferation in both tissues. Investigation of keratin 5 and 8 protein localization showed significantly suppressed expression in the lingual mucosal epithelium in cKO mice. ConclusionsG9a is required for proper proliferation and differentiation of sox9-expressing tongue progenitor cells and is thereby involved in tongue development.

Glucocorticoid Receptor Down-Regulation Affects Neural Stem Cell Proliferation and Hippocampal Neurogenesis.

Dysregulation of the hypothalamic-pituitary-adrenal axis and abnormalities in the glucocorticoid receptor (GR) have been linked to major depressive disorder. Given the critical role of GR in stress response regulation, we investigated the impact of GR changes on neural stem cells (NSCs) proliferation and hippocampal neurogenesis. Stress response was induced using dexamethasone (DEX), a GR agonist, which led to reduced proliferation of neural stem cells and neural progenitor cells, as well as decreased expression of GR. Additionally, a reduction of serum concentration within the culture media resulted in suppressed cell proliferation, accompanied by decreased GR expression. The association between GR expression and cell proliferation was further confirmed through GR siRNA knockdown and overexpression experiments. Furthermore, in vivo studies utilizing young male C57BL/6 mice demonstrated that corticosterone (CORT) (35μg/ml) administered through drinking water for four weeks induced depression-like behavior, as indicated by increased immobility times in forced swimming and tail suspension tests. CORT exposure led to reduced GR and nestin expression levels, along with diminished numbers of BrdU-positive cells in the hippocampi, indicating impaired hippocampal neurogenesis. Taken together, our findings provide the first evidence that stress-induced downregulation of GR negatively affects neurogenesis by inhibiting NSCs proliferation.

PDGFR-beta signaling mediates endogenous neurogenesis after postischemic neural stem/progenitor cell transplantation in mice

ABSTRACT Objective Although platelet-derived growth factor receptor (PDGFR)-β mediates the self-renewal and multipotency of neural stem/progenitor cells (NSPCs) in vitro and in vivo, its mechanisms of activating endogenous NSPCs following ischemic stroke still remain unproven. Methods The exogenous NSPCs were transplanted into the ischemic striatum of PDGFR-β conditionally neuroepithelial knockout (KO) mice at 24 h after transient middle cerebral artery occlusion (tMCAO). 5-Bromo-2’-deoxyuridine (BrdU) was intraperitoneally injected to label the newly formed endogenous NSPCs. Infarction volume was measured, and behavioral tests were performed. In the subventricular zone (SVZ), proliferation of endogenous NSPCs was tested, and synapse formation and expression of nutritional factors were measured. Results Compared with control mice, KO mice showed larger infarction volume, delayed neurological recovery, reduced numbers of BrdU positive cells, decreased expression of neurogenic factors (including neurofilament, synaptophysin, and brain-derived neurotrophic factor), and decreased synaptic regeneration in SVZ after tMCAO. Moreover, exogenous NSPC transplantation significantly alleviated neurologic dysfunction, promoted neurogenesis, increased expression of neurologic factors, and diminished synaptic deformation in SVZ of FL mice after tMCAO but had no beneficial effect in KO mice. Conclusion PDGFR-β signaling may promote activation of endogenous NSPCs after postischemic NSPC transplantation, and thus represents a novel potential regeneration-based therapeutic target.

Atractylenolide promotes trophoblast cell proliferation and migration in recurrent spontaneous abortion via ERK pathway

Purpose: To investigate the effect of atractylenolide on recurrent spontaneous abortion (RSA).Methods: The HTR-8/SVneo was established as an in vitro cell model of RSA. Cell viability and proliferation were determined using CCK8 and BrdU staining, while cell migration and invasion were determined by cell scratch and transwell assays.Results: Atractylenolide significantly increased cell viability, and enhanced the number of BrdU-positive cells of HTR-8/SVneo (p < 0.01). Atractylenolide also significantly promoted cell migration and invasion (p < 0.01), and increased protein expression of MMP-9, MMP-2, and N-cadherin, but reduced Ecadherin. Atractylenolide also increased the phosphorylation of ERK (p < 0.01).Conclusion: Atractylenolide enhances cell proliferation and migration of HTR-8/SVneo through activation of ERK signaling. Further studies using animal models are recommended to determine the protective role of atractylenolide against RSA, in vivo.

N-palmitoylethanolamine modulates hippocampal neuroplasticity in rats with stress-induced depressive behavior phenotype

Bioactive lipid mediator N-palmitoylethanolamide (PEA) is an endocannabinoid-like molecule. Based on our previous data, this study aimed to further investigate the antidepressant property of PEA via the peroxisome proliferator-activated receptor alpha (PPARα) pathway, focusing on the intervention of PEA on hippocampal neuroplasticity. Behavioral tests were performed in rats induced by unpredictable chronic mild stress (uCMS) in the last week of the experiment, and then the brain tissue samples were retained for subsequent immunohistochemical detection and Western blot analysis. In vitro, the apoptosis of HT22 cells induced by CORT and apoptosis-related proteins were detected by Hoechst staining and Western blot, respectively. The results showed that PEA ameliorated the depression-like phenotype in rats induced by uCMS, prevented the uCMS-induced reduction in the number of BrdU-positive cells, and increased BrdU/NeuN co-localization in the hippocampus, and upregulated the levels of synapse associated protein NCAM, MAP2, SYN and PSD95 in the hippocampus. Hoechst staining results showed that PEA significantly increased the CORT-induced reduction in the number of hippocampal neurons. Western blot analysis showed that PEA decreased the expression of caspase-3 and c-caspase-3, and increased the ratio of Bcl-2/Bax in CORT-induced HT22 cells. MK886, a PPARα antagonist, partially or completely reversed these effects. In conclusion, the therapeutic potential of PEA for depressive mood disorders may be through targeting the hippocampal neuroplasticity, including increasing adult neurogenesis and synaptic plasticity, as well as down-regulated neuronal apoptosis, to remodel hippocampal circuitries upon functional integration and PPARα pathway may be involved in this process.

Photobiomodulation Promotes Hippocampal Neurogenesis and Improves Cognitive Function and Anti-Inflammatory Injury in Rats With Chronic Cerebral Hypoperfusion

To investigate the effect of photobiomodulation (PBM) on hippocampal neurogenesis, cognitive function, and inflammatory injury in rats with chronic cerebral hypoperfusion. Bilateral ovariectomy (OVX) was performed on female Sprague-Dawley (SD) rats. One week later, the rats were randomly assigned to three groups, Sham surgery (or Sham) group, bilateral common carotid artery occlusion (BCCAO) group, and PBM intervention (or BCCAO+PBM) group. There were 8 rats in each group. In the BCCAO group, chronic cerebral hyporeperfusion was induced by permanent ligation of bilateral common carotid arteries and no PBM was given. Rats in the Sham group underwent the same surgical procedure except for the occlusion of the two carotids arteries and no PBM was given. In addition to the BCCAO surgery, rats in the BCCAO+PBM group received 808 nm laser therapy (5 min each time at a laser dose of 20 mW/cm 2) of the frontal cortex every other day for 1 month. Between 86 and 90 days after BCCAO, Morris water maze (MWM) was used to observe the spatial learning and memory function of the rats. The rats were sacrificed on day 90 and immunofluorescence staining and Western blot were performed thereafter. Immunofluorescence staining was used to determine the expression of 5-bromodeoxyuracil nucleoside (BrdU), a cell proliferation marker, glial fibrillary acidic protein (GFAP), an astrocyte marker, doublecortin (DCX), a specific marker of newborn neuron precursor cells, NeuN, a marker of mature neurons, and Iba1, a microglia marker, in the hippocampal dentate gyrus (DG) region. Western blot was performed to analyze the protein expressions of inflammasome components, NLRP3, ASC, cleaved caspase-1, and Iba1 in the hippocampus. In the latency trial of MWM test, BCCAO+PBM rats spent shorter periods of time finding the underwater platform than the BCCAO rats did. In the probe trial, after the platform that was original placed in a quadrant was removed, the BCCAO+PBM rats spent longer periods of time exploring the quadrant than the BCCAO animals did ( P<0.05). Compared with BCCAO rats, BCCAO+PBM rats showed significant decrease in the immunofluorescence intensities of GFAP and Iba1 ( P<0.01). PBM intervention significantly increased the number of BrdU-positive cells in the hippocampal DG region compared with those of Sham and BCCAO groups ( P<0.05). Furthermore, the number of NeuN positive cells showed no significant difference among the three groups, while in BCCAO+PBM group, the number of DCX-positive cells was significantly increased ( P<0.001) and the number of DCX +/NeuN + co-located cells was significantly increased compared to that of the BCCAO group ( P<0.001). Compared with those of the BCCAO group, Western blot results showed that the protein expression levels of Iba1, NLRP3, and cleaved caspase-1 in the BCCAO+PBM group were significantly decreased ( P<0.05), while the ASC protein expression level showed no significant difference. PBM can effectively improve the spatial learning and memory function in rats with chronic cerebral hypoperfusion, inhibit the activation of glial cells, reduce inflammatory damage mediated by NLRP3 inflammasome, and promote the regeneration of endogenous neural stem cells in the hippocampal DG region of rats.

Parathyroid Hormone-Related Protein Promotes the Proliferation of Patient-Derived Glioblastoma Stem Cells via Activating cAMP/PKA Signaling Pathway.

Glioblastoma (GBM) is an aggressive primary brain tumor characterized by its heterogeneity and high recurrence and lethality rates. Glioblastoma stem cells (GSCs) play a crucial role in therapy resistance and tumor recurrence. Therefore, targeting GSCs is a key objective in developing effective treatments for GBM. The role of Parathyroid hormone-related peptide (PTHrP) in GBM and its impact on GSCs remains unclear. This study aimed to investigate the effect of PTHrP on GSCs and its potential as a therapeutic target for GBM. Using the Cancer Genome Atlas (TCGA) database, we found higher expression of PTHrP in GBM, which correlated inversely with survival. GSCs were established from three human GBM samples obtained after surgical resection. Exposure to recombinant human PTHrP protein (rPTHrP) at different concentrations significantly enhanced GSCs viability. Knockdown of PTHrP using target-specific siRNA (siPTHrP) inhibited tumorsphere formation and reduced the number of BrdU-positive cells. In an orthotopic xenograft mouse model, suppression of PTHrP expression led to significant inhibition of tumor growth. The addition of rPTHrP in the growth medium counteracted the antiproliferative effect of siPTHrP. Further investigation revealed that PTHrP increased cAMP concentration and activated the PKA signaling pathway. Treatment with forskolin, an adenylyl cyclase activator, nullified the antiproliferative effect of siPTHrP. Our findings demonstrate that PTHrP promotes the proliferation of patient-derived GSCs by activating the cAMP/PKA signaling pathway. These results uncover a novel role for PTHrP and suggest its potential as a therapeutic target for GBM treatment.

Limonin, a Component of Immature Citrus Fruits, Activates Anagen Signaling in Dermal Papilla Cells.

Hair loss remains a significant problem that is difficult to treat; therefore, there is a need to identify safe natural materials that can help patients with hair loss. We evaluated the hair anagen activation effects of limonin, which is abundant in immature citrus fruits. Limonin increased the proliferation of rat dermal papilla cells (rDPC) by changing the levels of cyclin D1 and p27, and increasing the number of BrdU-positive cells. Limonin increased autophagy by decreasing phosphorylated mammalian target of rapamycin levels and increasing the phospho-Raptor, ATG7 and LC3B. Limonin also activated the Wnt/β-catenin pathway by increasing phospho-β-catenin levels. XAV939, a Wnt/β-catenin inhibitor, inhibited these limonin-induced changes, including induced autophagy, BrdU-positive cells, and cell proliferation. Limonin increased the phosphorylated AKT levels in both two-dimensional cultured rDPC and three-dimensional spheroids. Treatment with the PI3K inhibitor wortmannin inhibited limonin-induced proliferation, and disrupted other limonin-mediated changes, including decreased p27, increased BrdU-positive cells, induced autophagy, and increased ATG7 and LC3B levels. Wortmannin also inhibited limonin-induced cyclin D1 and LC3 expression in spheroids. Collectively, these results indicate that limonin can enhance anagen signaling by activating autophagy via targeting the Wnt/β-catenin and/or PI3K/AKT pathways in rDPC, highlighting a candidate nutrient for hair loss treatment.

Effects of β-amyloid (1-42) Administration on the Main Neurogenic Niches of the Adult Brain: Amyloid-Induced Neurodegeneration Influences Neurogenesis

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder and warrants further study as well as timely treatment. Additionally, the mechanisms of the brain's intrinsic defense against chronic injury are not yet fully understood. Herein, we examined the response of the main neurogenic niches to amyloid exposure and the associated changes in structure and synaptic activity. Flow cytometry of Nestin-, Vimentin-, Nestin/Vimentin-, NeuN-, GFAP-, NeuN/GFAP-, NSE-, BrdU-, Wnt-, BrdU/Wnt-, VEGF-, Sox14-, VEGF/Sox14-, Sox10-, Sox2-, Sox10/Sox2-, Bax-, and Bcl-xL-positive cells was performed in the subventricular zone (SVZ), hippocampus, and cerebral cortex of rat brains on 90th day after intracerebroventricular (i.c.v.) single injection of a fraction of β-amyloid (Aβ) (1-42). The relative structural changes in these areas and disruptions to synaptic activity in the entorhinal cortex-hippocampus circuit were also evaluated. Our flow analyses revealed a reduction in the numbers of Nestin-, Vimentin-, and Nestin/Vimentin-positive cells in neurogenic niches and the olfactory bulb. These changes were accompanied by an increased number of BrdU-positive cells in the hippocampus and SVZ. The latter changes were strongly correlated with changes in the numbers of VEGF- and VEGF/Sox14-positive cells. The morphological changes were characterized by significant neural loss, a characteristic shift in entorhinal cortex-hippocampus circuit activity, and decreased spontaneous alternation in a behavioral test. We conclude that although an injection of Aβ (1-42) induced stem cell proliferation and triggered neurogenesis at a certain stage, this process was incomplete and led to neural stem cell immaturity. We propose the idea of enhancing adult neurogenesis as a promising strategy for preventing dementia at healthy elderly people andpeople at high risk for developing AD, or treating patients diagnosed with AD.

Targeted delivery of harmine to xenografted human pancreatic islets promotes robust cell proliferation

Type 1 diabetes (T1D) occurs as a consequence of the autoimmune destruction of insulin-producing pancreatic beta (β) cells and commonly presents with insulin deficiency and unregulated glycemic control. Despite improvements in the medical management of T1D, life-threatening complications are still common. Beta-cell replication to replace lost cells may be achieved by using small-molecule mitogenic drugs, like harmine. However, the safe and effective delivery of such drugs to beta cells remains a challenge. This work aims to deploy an antibody conjugated nanocarrier platform to achieve cell-specific delivery of candidate therapeutic and imaging agents to pancreatic endocrine cells. We approached this goal by generating core–shell type micellar nanocarriers composed of the tri-block copolymer, Pluronic®F127 (PEO100–PPO65–PEO100). We decorated these nanocarriers with a pancreatic endocrine cell-selective monoclonal antibody (HPi1), with preference for beta cells, to achieve active targeting. The PPO-based hydrophobic core allows encapsulation of various hydrophobic cargoes, whereas the PEO-based hydrophilic shell curbs the protein adhesion, hence prolonging the nanocarriers' systemic circulation time. The nancarriers were loaded with quantum dots (QDots) that allowed nanocarrier detection both in-vitro and in-vivo. In-vitro studies revealed that HPi1 conjugated nanocarriers could target endocrine cells in dispersed islet cell preparations with a high degree of specificity, with beta cells exhibiting a fluorescent quantum dot signal that was approximately five orders of magnitude greater than the signal associated with alpha cells. In vivo endocrine cell targeting studies demonstrated that the HPi1 conjugated nanocarriers could significantly accumulate at the islet xenograft site. For drug delivery studies, the nanocarriers were loaded with harmine. We demonstrated that HPi1 conjugated nanocarriers successfully targeted and delivered harmine to human endocrine cells in a human islet xenograft model. In this model, targeted harmine delivery yielded an ~ 41-fold increase in the number of BrdU positive cells in the human islet xenograft than that observed in untreated control mice. By contrast, non-targeted harmine yielded an ~ 9-fold increase in BrdU positive cells. We conclude that the nanocarrier platform enabled cell-selective targeting of xenografted human pancreatic endocrine cells and the selective delivery of the hydrophobic drug harmine to those cells. Further, the dramatic increase in proliferation with targeted harmine, a likely consequence of achieving higher local drug concentrations, supports the concept that targeted drug delivery may promote more potent biological responses when using harmine and/or other drugs than non-targeting approaches. These results suggest that this targeted drug delivery platform may apply in drug screening, beta cell regenerative therapies, and/or diagnostic imaging in patients with type 1 diabetes.

Acute radiofrequency electromagnetic radiation exposure impairs neurogenesis and causes neuronal DNA damage in the young rat brain

A mobile phone is now a commonly used device for digital media and communication among all age groups. Young adolescents use it for longer durations, which exposes them to radiofrequency electromagnetic radiation (RF-EMR). This exposure can lead to neuropsychiatric changes. The underlying cellular mechanism behind these changes requires detailed investigation. In the present study, we investigated the effect of RF-EMR emitted from mobile phones on young adolescent rat brains. Wistar rats (5 weeks, male) were exposed to RF-EMR signal (2115 MHz) at a head average specific absorption rate (SAR) of 1.51 W/kg continuously for 8 h. Higher level of lipid peroxidation, carbon-centered lipid radicals, and single-strand DNA damage was observed in the brain of rat exposed to RF-EMR. The number of BrdU-positive cells in the dentate gyrus (DG) decreased in RF-EMR-exposed rats, indicating reduced neurogenesis. RF-EMR exposure also induced degenerative changes and neuronal loss in DG neurons but had no effect on the CA3 and CA1 neurons of the hippocampus and cerebral cortex. The activity of Pro-caspase3 did not increase upon exposure in any of the brain regions, pointing out that degeneration observed in the DG region is not dependent on caspase activation. Results indicate that short-term acute exposure to RF-EMR induced the generation of carbon-centered lipid radicals and nuclear DNA damage, both of which likely played a role in the impaired neurogenesis and neuronal degeneration seen in the young brain's hippocampus region. The understanding of RF-EMR-induced alteration in the brain at the cellular level will help develop appropriate interventions for reducing its adverse impact.

Interleukin-11 (IL11) inhibits myogenic differentiation of C2C12 cells through activation of extracellular signal-regulated kinase (ERK)

Cancer-associated cachexia (CAC) is a multifactorial wasting syndrome characterized by loss of skeletal muscle. Interleukin-11 (IL11), one of the IL6 family cytokines, is highly expressed in various types of cancer including cancers frequently associated with cachexia. However, the impact of IL11 on muscle metabolism remains to be determined. Since one of the mechanisms of muscle wasting in cachexia is defective muscle regeneration due to impaired myogenic differentiation, we examined the effect of IL11 on the differentiation of C2C12 mouse myoblasts. Treatment of C2C12 cells with recombinant mouse IL11 resulted in decreased myotube formation. In addition, IL11 treatment reduced the protein and mRNA levels of myosin heavy chain (MHC), a marker of myogenic differentiation. Moreover, the levels of myogenic regulatory factors including myogenin and Mrf4 were significantly reduced by IL11 treatment. IL11 treatment increased the number of BrdU-positive cells and the level of phosphorylated retinoblastoma (Rb) protein, while the levels of p21Waf1 and p27Kip1 were reduced by IL11 treatment in differentiating C2C12 cells, suggesting that IL11 interferes with cell cycle exit during the early stages of myogenic differentiation. Consistent with this, IL11 treatment at the late stage of differentiation did not affect myotube formation and MHC expression. IL11 treatment resulted in an activation of ERK, STAT3, and AKT in differentiating C2C12 cells. However, only ERK inhibitors including PD98059 and U0126 were able to ameliorate the suppressive effect of IL11 on the expression of MHC and myogenin. Additionally, pretreatment with PD98059 and U0126 resulted in improved myotube formation and reduced BrdU staining in IL11-treated cells. Together, our results suggest that IL11 inhibits myogenic differentiation through delayed cell cycle exit in an ERK-dependent manner. To our knowledge, this study is the first to demonstrate an inhibitory role of IL11 in myogenic differentiation and identifies the previously unrecognized role of IL11 as a possible mediator of CAC.

Aloin promotes oral squamous cell carcinoma cell apoptosis and autophagy through Akt/mTOR pathway

Aloin, a natural product, has demonstrated anticancer activity in a variety of cancers, but its role in oral squamous cell carcinoma (OSCC) has not been reported. In this study, we aimed to investigate the effects of Aloin on the biological functions of OSCC cells. Additionally, we evaluated the classical Akt/mTOR signal transduction pathway. CAL-27 OSCC cells were treated with Aloin for in vitro experiments. 5-bromo-2’-deoxyuridine (BrdU) and colony formation assays were applied to evaluate cell proliferation potential. Wound scratch and Transwell assays detected cell migration and invasion, respectively. Flow cytometry detected apoptosis. Autophagy was assessed by p62, Beclin-1, light chain 3 (LC3)-I and LC3-II protein expressions through Western blot test. Akt/mTOR signal pathway-related protein levels were also detected by Western blot test. Here, compared with the control group, Aloin treatment memorably reduced the number of BrdU-positive cells and colonies, and undermined the ability of cell migration and invasion. Moreover, Aloin promoted apoptosis and autophagy. Western blot test proved that the protein expression of p62 was down-regulated, while Beclin-1 and LC3-I/LC3-II protein expressions were up-regulated. Further investigations revealed that Aloin inhibited p-Akt and p-mTOR, suggesting that Aloin could block the activation of AKT/mTOR pathway. Our findings demonstrated that Aloin could inhibit the activation of the AKT/mTOR pathway to induce autophagic cell death, which could provide a novel vision for OSCC treatment.

Dexmedetomidine represses TGF-β1-induced extracellular matrix production and proliferation of airway smooth muscle cells by inhibiting MAPK signaling pathway.

Airway remodeling is implicated in the pathogenesis of asthma, and abnormal proliferation of airway smooth muscle cells (ASMCs) contribute to airway remodeling. Inflammatory mediator, transforming growth factor-β1 (TGF-β1), stimulates the proliferation of ASMCs, and is associated with airway remodeling in asthma. Dexmedetomidine (DEX) has been widely used in the adjuvant therapy of acute asthma. The potential effects of DEX on extracellular matrix (ECM) production and proliferation of ASMCs were investigated in this study. Human ASMCs were incubated with TGF-β1 for 48 hours, and then treated with different concentrations of DEX for another 24 hours. Cell proliferation was detected by MTT and BrdU (5'-bromo-2'-deoxyuridine) staining. Flow cytometry was used to assess cell apoptosis, and western blot was applied to identify the underlying mechanism. TGF-β1 induced increase in cell viability and bromodeoxyuridine (BrdU) positive cells in ASMCs while repressed cell apoptosis. Second, TGF-β1-induced ASMCs were then treated with different concentrations of DEX. Cell viability of TGF-β1-induced ASMCs was decreased by incubation of DEX. The number of BrdU positive cells in TGF-β1-induced ASMCs was reduced by incubation of DEX. Moreover, incubation of DEX promoted cell apoptosis of TGF-β1-induced ASMCs. Third, incubation of DEX attenuated TGF-β1-induced increase in fibronectin, collagen I, MMP9, and versican in ASMCs. Lastly, the up-regulation of phosphorylated extracellular receptor kinase (p-ERK), phosphorylated Jun N-terminal Kinase (p-JNK), and p-p38 in TGF-β1-induced ASMCs was reversed by incubation of DEX. DEX suppressed TGF-β1-induced ECM production and proliferation of ASMCs through inactivation of p38 mitogen-activated protein kinase (MAPK) pathway, providing a potential strategy for prevention of asthma.

Bone Marrow Mesenchymal Stem Cells (BMSCs)-Derived Exosomes Promotes Proliferation and Differentiation of Retinal Neuron-Like Cells to Repair Corneal Epithelial Damage

Dry eye disease (DED) is a common ocular surface disease. Bone marrow mesenchymal stem cells (BMSCs) can differentiate into various cells, and BMSC-derived exosomes (BMSC-exo) is essential to maintaining BMSCs stemness. This study aimed to elucidate the mechanism underlying BMSCexo in DED. Sixty rats with corneal epithelial injury were treated with BMSCs or BMSC-exo and untreated (each group, n = 20) followed by analysis of the effect of BMSCs and BMSC-exo by evaluating the corneal epithelium damage via measuring the Basso-Beattie-Bresnahan (BBB) score on 1st, 3rd, 7th, 14th, 28th day after treatments. TUNEL staining assessed cell apoptosis, NF200 expression and the number of BrdU-positive cells. There was no significant difference in BBB scores among three groups on the 1st and 3rd day after treatment (p &gt; 0.05) with significant difference on the 7th, 14th, and 28th day (p &lt;0.05); compared with control group, BMSCs group and combination group had significantly higher BBB score (p &lt; 0.05). The amount of apoptotic cells rose on 3rd and then gradually decreased since 7th day. Moreover, BMSCs and BMSC-exo decreased the apoptotic index and increased absorbance of NF200 and BrdU-positive rate (p &lt; 0.05). BMSC-exo alleviates corneal epithelial damage in DED and facilitates wound healing possibly through reducing cell apoptosis and increasing retinal neuron-like cell proliferation protein.

Neuroprotective and Proneurogenic Effects of Glucosamine in an Internal Carotid Artery Occlusion Model of Ischemia.

Neuroprotective, antineuroinflammatory, and proneurogenic effects of glucosamine, a naturally occurring amino sugar, have been reported in various animal models of brain injury including cerebral ischemia and hypoxic brain damage. Given that clinical translation of therapeutic candidates identified in animal models of ischemic stroke has remained unsatisfactory in general, possibly due to inadequacy of existing models, we sought to study the effects of glucosamine in a recently developed, clinical condition mimicking mouse model of internal cerebral artery occlusion. In this model of mild to moderate striatal damage, glucosamine ameliorated behavioral dysfunction, rescued ischemia-induced striatal damage, and suppressed ischemia-induced upregulation of proinflammatory genes in striatal tissue. Further, in ex vivo neurosphere assay involving neural stem cells/neural progenitor cells from subventricular zone, glucosamine increased the number of large neurospheres, along with enhancing mRNA levels of the proliferation markers Nestin, NeuroD1, and Sox2. Lastly, coronal brain sections containing the striatal region with subventricular zone showed increased number of BrdU positive cells and DCX positive cells, a marker for newly differentiating and immature neurons, in glucosamine-treated ischemic mice. Cumulatively, the results confirming neuroprotective, antineuroinflammatory, and proneurogenic effects of glucosamine enhance drug repurposing potential of glucosamine in cerebral ischemia.

CHML targeted by miR-199a-3p promotes non-small cell lung cancer cell growth via binding to Rab5A

Choroideremia-like (CHML) has been demonstrated to be related to the development of urothelial carcinoma, multiple myeloma, and hepatocellular carcinoma. Whereas, the association between CHML and lung cancer remains dimness. CHML expression was analyzed in NSCLC patients from TCGA dataset and evaluated in our collected NSCLC tissues and NSCLC cell lines. The effects of CHML on the proliferation and apoptosis of NSCLC were investigated in A549 and H1299 cells that downregulation of CHML as well as in H1299-induced xenograft mouse model. An upstream miRNA of CHML was further analyzed. Moreover, bioinformatics analysis and co-immunoprecipitation assay were carried out to explore the mechanism of CHML in NSCLC. We found CHML expression was upregulated in NSCLC patients and cell lines compared with their controls. Knockdown of CHML suppressed the viability and BrdU-positive cell number, and elevated the proportion of Tunel-positive cells and levels of Bax/Bcl-2 and cleaved-caspase-3 in NSCLC cells. In mouse models, downregulation of CHML decreased tumor volume and weight, attenuated Ki-67 staining, whereas elevated numbers of Tunel-positive cells, and upregulated levels of Bax/Bcl-2 and cleaved-caspase-3. CHML was demonstrated to be a target of miR-199a-3p. miR-199a-3p inhibitor significantly promoted the proliferation, and attenuated the apoptosis of H1299 cells, which were abrogated by CHML silencing. CHML promoted the proliferation of NSCLC cells via directly binding to Rab5A. Taken together, this study revealed that CHML was an oncogene in NSCLC and it could promote the proliferation and inhibit apoptosis of NSCLC cells through binding to Rab5A. CHML was targeted by miR-199a-3p in this cancer.

The effect induced by alternated mechanical loading on Notch-1 in mandibular condylar cartilage of growing rabbits.

AimsThe aim of our study is to investigate the effect induced by alternated mechanical loading on Notch-1 in mandibular condylar cartilage (MCC) of growing rabbits.MethodsA total of 64 ten-day-old rabbits were randomly divided into two groups according to dietary hardness: normal diet group (pellet) and soft diet group (powder). In each group, the rabbits were further divided into four subgroups by feeding time: two weeks, four weeks, six weeks, and eight weeks. Animals would be injected 5-bromo-2′-deoxyuridine (BrdU) every day for one week before sacrificing. Histomorphometric analysis of MCC thickness was performed through haematoxylin and eosin (HE) staining. Immunochemical analysis was done to test BrdU and Notch-1. The quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were used to measure expression of Notch-1, Jagged-1, and Delta-like 1 (Dll-1).ResultsThe thickness of MCC in the soft diet group was thinner than the one in normal diet group. Notch-1 was restricted in fibrous layer, proliferative layer, and hypertrophic layer. The expression of Notch-1 increased from two weeks to six weeks and then fell down. Notch-1 in normal diet group was higher than that in soft diet group in anterior part of MCC. The statistical differences of Notch-1 were shown at two, four, and six weeks (p < 0.05). The result of western blot and quantitative real-time PCR (qRT-PCR) showed the expression of Dll-1 and Jagged-1 rose from two to four weeks and started to decrease at four weeks. BrdU distributed in all layers of cartilage and subchondral bone. The number of BrdU-positive cells, which were less in soft diet group, was decreasing along with the experiment period. The significant difference was found at four, six, and eight weeks in anterior and posterior parts (p < 0.05).ConclusionThe structure and proliferation of MCC in rabbits were sensitive to dietary loading changes. The proper mechanical loading was essential for transduction of Notch signalling pathway and development of mandibular condylar cartilage.Cite this article: Bone Joint Res 2021;10(7):437–444.

Neuroprotective response and efficacy of intravenous administration of mesenchymal stem cells in traumatic brain injury mice.

Cellular transplantation of stem cells can be a beneficial treatment approach for neurodegenerative diseases such as traumatic brain injury (TBI). In this study, we investigated the proliferation and differentiation potential of infused mesenchymal stem cells (MSCs) after localisation at the injury site. We evaluated the appropriate homing of infused MSCs through immunohistochemistry, followed by Y-chromosome-specific polymerase chain reaction and fluorescent in situ hybridisation analyses. The proliferation and differentiation of infused MSCs were analysed using exogenous cell tracer 5'-bromo-2'-deoxyuridine (BrdU) labelling and neuronal specific markers, respectively. Structural and functional recovery in TBI mice were examined by performing magnetic resonance imaging and different behavioural assessments, respectively. Results demonstrated a significantly high number of BrdU-positive cells in the lesion region in the MSC-infused group compared with control and TBI groups. Infused MSCs were well differentiated into neural-like cells and expressed significantly more neural markers (neuronal nuclear antigen [NeuN], microtubule-associated protein 2 [MAP2] and glial fibrillary acid protein [GFAP]). Improved tissue abnormalities as well as functional behaviours were observed in MSC-infused TBI mice, implying the substantial proliferation and differentiation of infused MSCs. Our findings support the neuroprotective response and efficacy of MSCs after transplantation in TBI mice, and MSCs may serve as potential therapeutic candidates in regenerative medicine.

Effect of Sonic hedgehog on the proliferation in mouse tongue epithelial cells.

To investigate Sonic hedgehog (Shh) effects on proliferation and apoptosis of tongue epithelial cells in embryonic and ageing mice. Embryonic day 13.5 (E13.5), E14.5, E16.5 and postnatal day 0.5 (PN0.5) K14-Cre;Shhfl/fl mice, and E14.5, E16.5, PN0.5, PN90.5 and postnatal 1.5years (PN1.5Y) wild-type (Wt) mice were employed. Scanning electron microscopy, haematoxylin-eosin and immunohistochemistry were performed. Gel beads containing exogenous Shh protein were embedded in the tongue of PN90.5 and PN1.5Y Wt mice. Three days later, 5-bromodeoxyuridine (BrdU), proliferating cell nuclear antigen (PCNA) immunohistochemical and TUNEL staining were performed. The number of fungiform papillae was decreased with age. The numbers of BrdU- and PCNA-positive cells were the highest at PN0.5 and the lowest at PN1.5Y. Compared with Wt mice, K14-Cre;Shhfl/fl mice had decreased PCNA-positive cells in the epithelium, a smaller tongue volume, and fewer papillae at PN0.5. At E14.5, the number of BrdU-positive cells was decreased in K14-Cre;Shh fl/fl mice. At PN1.5Y, the number of apoptotic cells in tongue tissue exposed to Shh protein was less than that in the BSA group and the numbers of BrdU- and PCNA-positive proliferating cells were increased. Shh maintains cell proliferation and reduces apoptosis during tongue development and ageing.