Buyang Huanwu decoction promotes angiogenesis after cerebral ischemia through modulating caveolin-1-mediated exosome MALAT1/YAP1/HIF-1α axis.

BACKGROUND: Angiogenesis is an effective method for promoting neurological function recovery after cerebral ischemia (CI). Buyang Huanwu decoction (BHD) is a traditional Chinese medicinal recipe that is frequently employed for CI treatment. Previous investigations have validated that it promotes angiogenesis following CI. Nevertheless, the precise mechanism by which it does this has yet to be completely understood. OBJECTIVE: This study aims to examine the underlying mechanism through which BHD facilitates angiogenesis following CI by regulating the exosomal MALAT1/YAP1/HIF-1α signaling axis, specifically via the involvement of caveolin-1 (Cav1), an endocytosis-associated protein. METHODS: A CI model was created using middle cerebral artery occlusion (MCAO). Following the administration of multiple doses of BHD, various parameters, including the neurobehavioral score, pathological damage, and angiogenesis, were assessed in each group of mice to identify the optimal dosage of BHD for treating CI. The molecular processes underlying the angiogenic implications of BHD following CI were investigated exhaustively by employing single-cell sequencing. Finally, the involvement of Cav1 was confirmed in Cav1 knockout mice and Cav1-silenced stably transfected strains to validate the mechanism by which BHD increases angiogenesis following CI. RESULTS: BHD could promote angiogenesis after CI. Single-cell sequencing results suggested that its potential mechanism of action might be connected with Cav1 and the exosomal MALAT1/YAP1/HIF-1α signaling axis. BHD could promote angiogenesis after CI by regulating the exosomal MALAT1/YAP1/HIF-1α axis through Cav1, as validated in vivo and in vitro experiments. Accordingly, Cav1 may be a key target of BHD in promoting angiogenesis after CI. CONCLUSION: This investigation represents the initial attempt to comprehensively ascertain the underlying mechanism of action of BHD in treating CI using single-cell sequencing, gene-knockout mice, and stable transfected cell lines, potentially associated with the modulation of the exosomal MALAT1/YAP1/HIF-1α axis by Cav1. Our findings offer novel empirical evidence for unraveling the regulatory pathways through which Cav1 participates in angiogenesis following CI and shed light on the potential mechanisms of BHD.

UTX inhibition suppresses proliferation and promotes apoptosis in patient-derived glioblastoma stem cells by modulating periostin expression.

Glioblastoma stem cells (GSCs) exert a crucial influence on glioblastoma (GBM) development, progression, resistance to therapy, and recurrence, making them an attractive target for drug discovery. UTX, a histone H3K27 demethylase, participates in regulating multiple cancer types. However, its functional role in GSCs remains insufficiently explored. This study aims to investigate the role and regulatory mechanism of UTX on GSCs. Analysis of TCGA data revealed heightened UTX expression in glioma, inversely correlating with overall survival. Inhibiting UTX suppressed GBM cell growth and induced apoptosis. Subsequently, we cultured primary GSCs from three patients, observing that UTX inhibition suppressed cell proliferation and induced apoptosis. RNA-seq was performed to analyze the gene expression changes after silencing UTX in GSCs. The results indicated that UTX-mediated genes were strongly correlated with GBM progression and regulatory tumor microenvironment. The transwell co-cultured experiment showed that silencing UTX in the transwell chamber GSCs inhibited the well plate cell proliferation. Protein-protein interaction analysis revealed that periostin (POSTN) played a role in the UTX-mediated transcriptional regulatory network. Replenishing POSTN reversed the effects of UTX inhibition on GSC proliferation and apoptosis. Our study demonstrated that UTX inhibition hindered POSTN expression by enhancing the H3K27me2/3 level, eventually resulting in inhibiting proliferation and promoting apoptosis of patient-derived GSCs. Our findings may provide a novel and effective strategy for the treatment of GBM.

CCN3/NOV Regulates Proliferation and Neuronal Differentiation in Mouse Hippocampal Neural Stem Cells via the Activation of the Notch/PTEN/AKT Pathway.

Neural stem cells (NSCs) persist in the subgranular zone (SGZ) throughout the lifespan and hold immense potential for the repair and regeneration of the central nervous system, including hippocampal-related diseases. Several studies have demonstrated that cellular communication network protein 3 (CCN3) regulates multiple types of stem cells. However, the role of CCN3 in NSCs remains unknown. In this study, we identified CCN3 expression in mouse hippocampal NSCs and observed that supplementing CCN3 improved cell viability in a concentration-dependent manner. Additionally, in vivo results showed that the injection of CCN3 in the dentate gyrus (DG) increased Ki-67- and SOX2-positive cells while decreasing neuron-specific class III beta-tubulin (Tuj1) and doublecortin (DCX)-positive cells. Consistently with the in vivo results, supplementing CCN3 in the medium increased the number of BrdU and Ki-67 cells and the proliferation index but decreased the number of Tuj1 and DCX cells. Conversely, both the in vivo and in vitro knockdown of the Ccn3 gene in NSCs had opposite effects. Further investigations revealed that CCN3 promoted cleaved Notch1 (NICD) expression, leading to the suppression of PTEN expression and eventual promotion of AKT activation. In contrast, Ccn3 knockdown inhibited the activation of the Notch/PTEN/AKT pathway. Finally, the effects of changes in CCN3 protein expression on NSC proliferation and differentiation were eliminated by FLI-06 (a Notch inhibitor) and VO-OH (a PTEN inhibitor). Our findings imply that while promoting proliferation, CCN3 inhibits the neuronal differentiation of mouse hippocampal NSCs and that the Notch/PTEN/AKT pathway may be a potential intracellular target of CCN3. Our findings may help develop strategies to enhance the intrinsic potential for brain regeneration after injuries, particularly stem cell treatment for hippocampal-related diseases.

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

Background and Objectives: 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. Methods and Results: 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. Conclusions: 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.

Humidified versus nonhumidified low-flow oxygen therapy in children with Pierre-Robin syndrome: A randomized controlled trial.

BACKGROUND: Humidification is an important process in clinical oxygen therapy. We aimed to evaluate the effects and safety of humidified versus nonhumidified low-flow oxygen therapy in children with Pierre-Robin syndrome. METHODS: This study was an open-label, single-centered randomized controlled trial (RCT) with a parallel group design. The study protocol has been registered in Chinese Clinical Trial Registry (ChiCTR1900021584). The children were randomized to the humidified versus nonhumidified groups. Average arterial oxygen partial pressure (PaO2) and carbon dioxide partial pressure (PaCO2), incidence of ventilator-associated pneumonia (VAP), nasal cavity dryness, nasal mucosal bleeding and bacterial contamination of the humidified bottle, the cost of nasal oxygen therapy and duration of intensive care unit (ICU) stay were analyzed. RESULTS: A total of 213 children with Pierre-Robin syndrome were included. There were no significant differences in the gender, age, weight, prematurity, duration of anesthesia and surgery duration of mandibular traction between humidified group and nonhumidified group (all P > .05). No significant differences in the average arterial PaO2 and PaCO2 level on the postoperative day 1, 2, and ICU discharge between humidified group and nonhumidified group were found (all P > .05). There were no significant differences in the incidence of nasal cavity dryness, nasal mucosal bleeding, bacterial contamination and VAP, the duration of ICU stay between humidified group and nonhumidified group (all P > .05). The cost of nasal oxygen therapy in the humidified group was significantly less than that of nonhumidified group (P = .013). CONCLUSIONS: Humidifying the oxygen with cold sterile water in the low-flow oxygen therapy in children may be not necessary. Future RCTs with lager sample size and rigorous design are warranted to further elucidate the effects and safety of humidified versus nonhumidified low-flow oxygen therapy.

ISL1 Promotes Human Glioblastoma-Derived Stem Cells' Self-Renewal by Activation of Sonic Hedgehog/GLI1 Function.

Glioblastoma (GBM), the most aggressive primary heterogeneous primary brain tumor, is a glioma subtype that originates from the glial cells of the central nervous system. Glioblastoma stem cells (GSCs), situated at the top of the hierarchy, initiate and maintain the tumor and are largely accountable for GBM resistance to the mainstay treatment and recurrence. The LIM homeobox transcription factor islet 1 (ISL1) induces tumorigenicity in various tumors; however, its function in GSCs has been less reported. We aimed to generate GSCs from surgical specimens of human GBM and investigate the effect of ISL1 knockdown on GSCs. We established patient-derived GSCs, determined cancer stem cell marker expression, and immunostained GSCs to assess cell viability and apoptosis. We demonstrated that ISL1 deletion decreased the GSC viability and proliferation, and upregulated apoptosis. Moreover, we performed enzyme-linked immunosorbent assay and western blotting and found that ISL1 knockdown affected the expression of sonic hedgehog (SHH) and its downstream regulator GLI1, and further validated these results by supplementing the cells with recombinant SHH. Our results suggested that ISL1 played a critical role in regulating GBM growth and that an ISL1/SHH/GLI1 pathway was required for the maintenance of GBM progression and malignancy. The regulation of GSC growth through ISL1 might be a mechanism of interest for future therapeutic studies.

Glypican 4 Regulates Aß Internalization in Neural Stem Cells Partly via Low-Density Lipoprotein Receptor-Related Protein 1.

Neural stem cell (NSC) damage has been reported in patients with Alzheimer's disease. Intracellular Aß plays a vital role in NSC damage. Heparan sulfate proteoglycans are potent mediators of Aß enrichment in the brain. We hypothesized the heparan sulfate proteoglycan glypican 4 (Gpc4) regulates Aß internalization by NSCs. We evaluated Gpc4 expression in NSCs from P0-P2 generations using immunofluorescence. Adenovirus and lentivirus were used to regulate Gpc4 expression in NSCs and APP/PS1 mice, respectively. Co-immunoprecipitation was used to determine the relationship between Gpc4, Aß, and low-density lipoprotein receptor-related protein 1 (LRP1). Intracellular Aß concentrations were detected using enzyme-linked immunosorbent assay and immunofluorescence. The role of Gpc4/LRP1 on toxic/physical Aß-induced effects was evaluated using the JC-1 kit, terminal deoxynucleotidyl transferase dUPT nick end labeling, and western blotting. Gpc4 was stably expressed in NSCs, neurons, and astrocytes. Gpc4 was upregulated by Aß in NSCs and regulated Aß internalization. Gpc4 attenuation reduced Aß uptake; Gpc4 overexpression increased Aß uptake. Gpc4 regulated Aß internalization through LRP1 and contributed to Aß internalization and toxic/physical concentrations of Aß-induced mitochondrial membrane potential and cell apoptosis, partly via LRP1. Therefore, Gpc4 is a key regulator of Aß enrichment in NSCs. Inhibiting Gpc4 rescued the Aß-induced toxic effect and attenuated the nontoxic Aß enrichment into intracellular toxic concentrations. Gpc4 contributed to Aß internalization and toxic/physical concentrations of Aß-induced mitochondrial membrane potential damage and cell apoptosis, partly via LRP1. These findings suggest a potential role of Gpc4 in treating Alzheimer's disease at an early stage, by targeting NSCs.

Optogenetic manipulation of cellular communication using engineered myosin motors.

Cells achieve highly efficient and accurate communication through cellular projections such as neurites and filopodia, yet there is a lack of genetically encoded tools that can selectively manipulate their composition and dynamics. Here, we present a versatile optogenetic toolbox of artificial multi-headed myosin motors that can move bidirectionally within long cellular extensions and allow for the selective transport of GFP-tagged cargo with light. Utilizing these engineered motors, we could transport bulky transmembrane receptors and organelles as well as actin remodellers to control the dynamics of both filopodia and neurites. Using an optimized in vivo imaging scheme, we further demonstrate that, upon limb amputation in axolotls, a complex array of filopodial extensions is formed. We selectively modulated these filopodial extensions and showed that they re-establish a Sonic Hedgehog signalling gradient during regeneration. Considering the ubiquitous existence of actin-based extensions, this toolbox shows the potential to manipulate cellular communication with unprecedented accuracy.

Activation of metabotropic glutamate receptor 4 regulates proliferation and neural differentiation in neural stem/progenitor cells of the rat subventricular zone and increases phosphatase and tensin homolog protein expression.

Neural stem/progenitor cells (NSPCs) persist in the mammalian subventricular zone throughout life, where they can be activated in response to physiological and pathophysiological stimuli. A recent study indicates metabotropic glutamate receptor 4 (mGluR4) is involved in regulating NSPCs behaviors. Therefore, defining mGluR4 function in NSPCs is necessary for determining novel strategies to enhance the intrinsic potential for brain regeneration after injuries. In this study, mGluR4 was functionally expressed in SVZ-derived NSPCs from male Sprague-Dawley rats, in which the cyclic adenosine monophosphate concentration was reduced after treatment with the mGluR4-specific agonist VU0155041. Additionally, lateral ventricle injection of VU0155041 significantly decreased 5-bromo-2'-deoxyuridine (BrdU)+ and Ki67+ cells, while increased Doublecortin (DCX)/BrdU double-positive cells in SVZ. In cultured NSPCs, mGluR4 activation decreased the ratio of BrdU+ cells, G2/M-phase cells, and inhibited Cyclin D1 expression, whereas it increased neuron-specific class III ß-tubulin (Tuj1) expression and the number of Tuj1, DCX, and PSA-NCAM-positive cells. However, pharmacological blocking mGluR4 with the antagonist MSOP or knockdown of mGluR4 abolished the effects of VU0155041 on NSPCs proliferation and neuronal differentiation. Further investigation demonstrated that VU0155041 treatment down-regulated AKT phosphorylation and up-regulated expression of the phosphatase and tensin homolog protein (PTEN) in NSPCs culture. Moreover VU0155041-induced proliferating inhibition and neuronal differentiating amplification in NSPCs were significantly hampered by VO-OHpic, a PTEN inhibitor. We conclude that activation of mGluR4 in SVZ-derived NSPCs suppresses proliferation and enhances their neuronal differentiation, and regulation of PTEN may be involved as a potential intracellular target of mGluR4 signal. Cover Image for this issue: https://doi.org/10.1111/jnc.15052.

Is routine nasogastric decompression after hepatic surgery necessary? A systematic review and meta-analysis.

OBJECTIVES: Currently the nasogastric tube (NGT) is routinely inserted in clinical after abdominal surgery for decompression in China, yet the practice varies between regions, the role of NGT for the patients after hepatic surgery remains unclear. Therefore, this present meta-analysis aimed to assess the efficacy and safety of NGT placement after hepatic surgery. DESIGN: A systematic review and meta-analysis DATA SOURCES: PUBMED, EMBASE, Science Direct, Cochrane Central Register of Controlled Trials, China National Knowledge Infrastructure (CNKI) and Wanfang Database (until Mar 30, 2019) were systematically searched. REVIEW METHODS: Randomized controlled studies (RCTs) comparing the efficacy and safety of NGT and no NGT treatment after hepatic surgery were included. Data were synthesized using a random-effects or fixed effect model according to the heterogeneity. Outcomes were presented as Mantel-Haenszel style odd ratios (ORs) or mean differences (MDs) with 95% confidence intervals (95% CIs). RESULTS: Seven studies with 1306 patients were eligible for inclusion. Compared with NGT treatment, the no NGT decompression could shorten the time to first defecation (MD -0.59; -0.79, -0.39), reduce the time to start diet (MD -0.46; -0.90, -0.03), and decrease the length of hospital stay (MD 0.48; -0.93, -0.03), but it could also increase the risk of NGT re-intubation (OR 6.8; 1.77, 26.72), no significant differences were detected on the first passage of flatus (MD -0.34; -0.86, 0.18), the incidence of nausea (OR 0.81; 0.40, 1.67), vomiting (OR 1.06; 0.19, 5.93), abdominal distention (OR 0.87; 0.60, 1.25). CONCLUSION: Given that very limited information for some endpoints in this present meta-analysis, the routinely insertion of NGT after hepatic surgery is not justified, the no NGT decompression seems to be more beneficial to the prognosis of patients after hepatic surgery, more related studies on this issue are needed.

Activation of type 4 metabotropic glutamate receptor promotes cell apoptosis and inhibits proliferation in bladder cancer.

Bladder cancer, the second most common genitourinary malignancy, severely endangers the human health. Rising evidence suggests that metabotropic glutamate receptors (mGluRs) are involve in tumor progression. In this study, we observed that metabotropic glutamate receptor 4 (mGluR4) was functionally expressed in normal and cancerous bladder cells and its expression was positively correlated with high bladder cancer grading. We further confirmed that the activation of mGluR4 by VU0155041, an mGluR4-specific agonist, decreased cyclic adenosine monophosphate (cAMP) concentration and cell viability, promoted apoptosis and inhibited proliferation in bladder cancer cells, whereas MSOP (group III mGluR antagonist) or mGluR4 knockdown eliminated the effects of mGluR4 activity. Western blotting revealed the decreased cyclin D1 expression, increased procaspase-8/9/3 cleavage, and unbalanced Bcl-2/Bax expression in bladder cancer cell lines after mGluR4 activation, and likewise MSOP and mGluR4 knockdown abrogated the actions of mGluR4 activity. In vivo study showed that mGluR4 activation significantly inhibited tumor growth of bladder cancer via suppressing proliferation and promoting apoptosis. Furthermore, upregulation of phosphatase and tensin homolog (PTEN) and inhibition of Akt phosphorylation were also observed after mGluR4 activation. Similar with VU0155041, the Akt-specific inhibitor markedly promoted apoptosis and inhibited proliferation. Nevertheless, the PTEN-specific inhibitor significantly abolished the mGluR4 activation-induced cell apoptosis and proliferative inhibition in bladder cancer cell lines. These results indicate that mGluR4 can regulate the switch between survival and death via the cAMP/PTEN/AKT signaling pathway in bladder cancer cells. Our findings suggest that mGluR4 has diagnostic and prognostic potential for bladder cancer, and the development of mGluR4 agonist may be a promising strategy for bladder cancer treatment.

Activity of Metabotropic Glutamate Receptor 4 Suppresses Proliferation and Promotes Apoptosis With Inhibition of Gli-1 in Human Glioblastoma Cells.

Glioblastoma multiforme (GBM) is the most lethal glioma variant in the adult brain and among the deadliest of human cancers. Increasing evidence has shown that metabotropic glutamate receptor subtype 4 (mGluR4) expression may play roles in regulating the growth of neural stem cells as well as several cancer cell lines. Here, we investigated the effects of mGluR4 on the growth and apoptosis of the LN229 GBM cell line. Involvement of Gli-1, one of the key transcription factors in the sonic Hedgehog (SHH) signaling pathway, was further explored. In this study, mGluR4 was activated using selective agonist VU0155041; and gene-targeted siRNAs were used to generate loss of function of mGluR4 and Gli-1 in LN229 cells. The results demonstrated that LN229 cells expressed mGluR4 and the agonist VU0155041 decreased cell viability in a dose- and time-dependent manner. Activation of mGluR4 inhibited cyclin D1 expression, activated pro-caspase-8/9/3, and disrupted the balance of Bcl-2/Bax expression, which indicated cell cycle arrest and apoptosis of LN229 cells, respectively. Furthermore, Gli-1 expression was reduced by mGluR4 activation in LN229 cells, and downregulation of Gli-1 expression by gene-targeted siRNA resulted in both inhibition of cell proliferation and promotion of apoptosis. Moreover, VU0155041 treatment substantially blocked SHH-induced cyclin D1 expression and cell proliferation, while increasing TUNEL-positive cells and the activation of apoptosis-related proteins. We concluded that activation of mGluR4 expressed in LN229 cells could inhibit GBM cell growth by decreasing cell proliferation and promoting apoptosis. Further suppression of intracellular Gli-1 expression might be involved in the action of mGluR4 on cancer cells. Our study suggested a novel role of mGluR4, which might serve as a potential drug target for control of GBM cell growth.

BMP­6 protects retinal pigment epithelial cells from oxidative stress­induced injury by inhibiting the MAPK signaling pathways.

Worldwide, neovascular age­related macular degeneration (nAMD) is one of the most common causes of blindness in the elderly. In particular, degeneration of retinal pigment epithelial (RPE) cells represents the main pathological process in the development of nAMD, and oxidative stress serves a major role. The present study aimed to investigate the association between bone morphogenetic protein 6 (BMP­6) and nAMD. BMP­6 concentration was significantly reduced in patients with wet nAMD compared with in the control group. Furthermore, the present study investigated the protective effects of BMP­6 on RPE cells following oxidative stress­induced injury. Cell Counting Kit­8 assay and terminal deoxynucleotidyl transferase dUTP nick­end labeling staining demonstrated that BMP­6 increased RPE cell viability, which was decreased following treatment with hydrogen peroxide (H2O2), and reduced H2O2­induced apoptosis. In addition, western blotting revealed that BMP­6 reversed the decrease in pro­caspase­3 levels and the dysregulation of the B­cell lymphoma 2 (Bcl­2)/Bcl­2­associated X protein (Bax) balance caused by H2O2. In addition, alterations in c­Jun N­terminal protein kinase (JNK) and p38 mitogen­activated protein kinase (MAPK) expression were examined, and pretreatment with BMP­6 was demonstrated to reduce H2O2­induced activation of JNK and p38 MAPK. Conversely, the effects of BMP­6 were attenuated by its inhibitor noggin. In conclusion, the present study demonstrated that BMP­6 may protect RPE cells from oxidative stress injury to a certain extent, which may be associated with alterations in the MAPK signaling pathway. However, the specific mechanism of action underlying this effect requires further investigation. Overall, the present study laid a foundation for exploring novel nAMD treatment methods.

The inhibiting effect of neural stem cells on proliferation and invasion of glioma cells.

The invasive and infiltrative nature of tumor cells leads to the poor prognosis of glioma. Currently, novel therapeutic means to eliminate the tumor cells without damaging the normal brain tissue are still strongly demanded. Significant attentions had been paid to stem cell-based therapy and neural stem cell (NSC) had been considered as one of the efficient delivery vehicles for targeting therapeutic genes. However, whether the NSCs could directly affect glioma cells remains to be seen. In this study, both rat and human glioma cells (C6 and U251) were co-cultured with normal rat embryonic NSCs directly or in-directly. We found the survival, proliferation, invasion and migration of glioma cells were significantly inhibited, while the differentiation was not affected in the in vitro co-culture system. In nude mice, although no significant difference was observed in the tumor growth, survival status and time of tumor-bearing mice were significantly promoted when U251 cells were subcutaneously injected with NSCs. In coincidence with the suppression of glioma cell growth in vitro, expression of mutant p53 and phosphorylation of AKT, ERK1/2 decreased while the level of caspase-3 increased significantly. Our results suggested that normal NSCs could possess direct anti-glioma properties via inhibiting the glioma cell viability, proliferation, invasion and migration. It could be a very promising candidate for glioma treatment.