Predictive potentials of glycosylation-related genes in glioma prognosis and their correlation with immune infiltration.

Glycosylation is currently considered to be an important hallmark of cancer. However, the characterization of glycosylation-related gene sets has not been comprehensively analyzed in glioma, and the relationship between glycosylation-related genes and glioma prognosis has not been elucidated. Here, we firstly found that the glycosylation-related differentially expressed genes in glioma patients were engaged in biological functions related to glioma progression revealed by enrichment analysis. Then seven glycosylation genes (BGN, C1GALT1C1L, GALNT13, SDC1, SERPINA1, SPTBN5 and TUBA1C) associated with glioma prognosis were screened out by consensus clustering, principal component analysis, Lasso regression, and univariate and multivariate Cox regression analysis using the TCGA-GTEx database. A glycosylation-related prognostic signature was developed and validated using CGGA database data with significantly accurate prediction on glioma prognosis, which showed better capacity to predict the prognosis of glioma patients than clinicopathological factors do. GSEA enrichment analysis based on the risk score further revealed that patients in the high-risk group were involved in immune-related pathways such as cytokine signaling, inflammatory responses, and immune regulation, as well as glycan synthesis and metabolic function. Immuno-correlation analysis revealed that a variety of immune cell infiltrations, such as Macrophage, activated dendritic cell, Regulatory T cell (Treg), and Natural killer cell, were increased in the high-risk group. Moreover, functional experiments were performed to evaluate the roles of risk genes in the cell viability and cell number of glioma U87 and U251 cells, which demonstrated that silencing BGN, SDC1, SERPINA1, TUBA1C, C1GALT1C1L and SPTBN5 could inhibit the growth and viability of glioma cells. These findings strengthened the prognostic potentials of our predictive signature in glioma. In conclusion, this prognostic model composed of 7 glycosylation-related genes distinguishes well the high-risk glioma patients, which might potentially serve as caner biomarkers for disease diagnosis and treatment.

Identification of necroptosis-related long non-coding RNAs prognostic signature and the crucial lncRNA in bladder cancer.

BACKGROUND: Research on the relationships between long non-coding RNAs (lncRNAs) and cancer is attractive and has progressed very rapidly. Necroptosis-related biomarkers can potentially be used for predicting the prognosis of cancer patients. This study aimed to establish a necroptosis-related lncRNA (NPlncRNA) signature to predict the prognosis of patients with bladder cancer (BCa). METHODS: First, NPlncRNAs were identified using Pearson correlation analysis and machine learning algorithms, including SVM-RFE, least absolute shrinkage and selection operator (LASSO) regression, and random forest. The prognostic NPlncRNA signature was constructed using univariate and multivariate Cox regression analyses and the diagnostic efficacy and clinically predictive efficiency were evaluated and validated. The biological functions of the signature were analysed using gene set enrichment analysis (GSEA) and functional enrichment analysis. We further integrated the RNA-seq dataset (GSE133624) with our outcomes to reveal the crucial NPlncRNA that was functionally verified by assessing cell viability, proliferation, and apoptosis in BCa cells. RESULTS: The prognostic NPlncRNAs signature was composed of PTOV1-AS2, AC083862.2, MAFG-DT, AC074117.1, AL049840.3, and AC078778.1, and a risk score based on this signature was proven to be an independent prognostic factor for the BCa patients, indicated by poor overall survival (OS) of patients in the high-risk group. Additionally, the NPlncRNAs signature had a higher diagnostic validity than that of other clinicopathological variables, with a greater area under the receptor operating characteristic and concordance index curves. A nomogram established by integrating clinical variables and risk score confirmed that the signature can accurately predict the OS of patients and has high clinical practicability. Functional enrichment analysis and GSEA revealed that some cancer-related and necroptosis-related pathways were enriched in high-risk groups. The crucial NPlncRNA MAFG-DT was associated with poor prognosis and was highly expressed in BCa cells. MAFG-DT silencing notably inhibited proliferation and enhanced apoptosis of BCa cells. CONCLUSIONS: A novel prognostic NPlncRNAs signature was identified in BCa in this study, which provides potential therapeutic targets among which MAFG-DT plays critical roles in the tumorigenesis of BCa.

Dihydroartemisinin Inhibits Laser-Induced Choroidal Neovascularization in a Mouse Model of Neovascular AMD.

Purpose: Choroidal neovascularization (CNV) is the main pathogenic process and a leading cause of severe vision loss in neovascular age-related macular degeneration (AMD). We investigated the antiangiogenic efficacy of dihydroartemisinin (DHA) in an experimental laser-induced CNV mouse model. Methods: After fluorescein angiography confirmed that CNV was induced by laser photocoagulation in C57BL/6J mice, DHA or vehicle was given by intragastric administration once a day. On day 6 and day 12, fluorescein angiography, optic coherence tomography, and flat-mounting analysis were performed to grade CNV leakage, measure CNV thickness and evaluate CNV areas, respectively. Immunofluorescence staining and Western blot analysis were performed to evaluate the expression of NF-κB, VEGF, and VEGFR2. To confirm the safety of intragastric DHA application, changes in retinal morphology and neural cell apoptosis were tested by histopathological examination and TUNEL assay, and retinal function was determined by electroretinogram (ERG). Results: Intragastric administration of DHA significantly suppressed CNV leakage and CNV formation in both thickness and area. Immunofluorescence showed that DHA suppressed VEGFR2 and NF-κB p65 expression in laser-induced lesions. Compared to the normal group, the protein expression of VEGF, VGFER2, NF-κB p65, and NF-κB1 p50 increased significantly in the vehicle group after laser photocoagulation, while it was profoundly inhibited by DHA treatment. In addition, histopathological examination, TUNEL analysis, and ERG test showed no obvious evidence of retinal toxicity caused by DHA. Conclusion: Systemic administration of DHA can effectively inhibit laser-induced CNV formation in mice, which might be due to the suppression of the classic NF-κB signaling pathway and downregulation of VEGFR2 and VEGF expression. The current results suggest that DHA could be a natural potential alternative therapeutic strategy for neovascular AMD.

Corrigendum: Combined Scutellarin and C18H17NO6 Imperils the Survival of Glioma: Partly Associated With the Repression of PSEN1/PI3K-AKT Signaling Axis.

[This corrects the article DOI: 10.3389/fonc.2021.663262.].

Dual-target one-step nested PCR for sensitive detection of SARS-CoV-2 nucleic acids.

The Corona Virus Disease reported in 2019 (COVID-19) poses a significant threat to human and public health. Its early and accurate detection can reduce the spread and recurrence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Real-time reverse transcription fluorescent quantitative polymerase chain reaction (RT-qPCR) is the "gold standard" for detecting the nucleic acid of SARS-CoV-2. This study developed and tested a dual-target (ORF1ab and N gene) one-step nested RT-qPCR (DTO-N-PCR) to detect SARS-CoV-2. Ten-fold serial dilutions of mixed synthetic DNA from SARS-CoV-2 ORF1ab and N gene were used as templates to test the sensitivity of DTO-N-PCR. Its specificity was subsequently tested using throat swab specimens from 10 COVID-19 patients and 35 healthy participants. DTO-N-PCR was more sensitive and specific than conventional RT-qPCR. It has unique features, including a dual-target (ORF1ab and N gene), rapid one-step operation of reverse transcription and PCR, four pairs of inner and outer primers, and specific probes. These features aid in its rapid, accurate, and efficient detection of SARS-CoV-2 RNA.

Scutellarin ameliorates neonatal hypoxic-ischemic encephalopathy associated with GAP43-dependent signaling pathway.

BACKGROUND: Neonatal hypoxic-ischemic encephalopathy (HIE) refers to the perinatal asphyxia caused by the cerebral hypoxic-ischemic injury. The current study was aimed at investigating the therapeutic efficacy of Scutellarin (Scu) administration on neurological impairments induced by hypoxic-ischemic injury and exploring the underlying mechanisms. METHODS: Primary cortical neurons were cultured and subjected to oxygen-glucose deprivation (OGD), and then treated with Scu administration. The growth status of neurons was observed by immunofluorescence staining of TUJ1 and TUNEL. Besides, the mRNA level of growth-associated protein 43 (GAP43) in OGD neurons with Scu treatment was detected by quantitative real-time polymerase chain reaction (qRT-PCR). To further verify the role of GAP43 in Scu treatment, GAP43 siRNA and knockout were applied in vitro and in vivo. Moreover, behavioral evaluations were performed to elucidate the function of GAP43 in the Scu-ameliorated long-term neurological impairments caused by HI insult. The underlying biological mechanism of Scu treatment was further elucidated via network pharmacological analysis. Finally, the interactive genes with GAP43 were identified by Gene MANIA and further validated by qRT-PCR. RESULTS: Our data demonstrated that Scu treatment increased the number of neurons and axon growth, and suppressed cell apoptosis in vitro. And the expression of GAP43 was downregulated after OGD, but reversed by Scu administration. Besides, GAP43 silencing aggravated the Scu-ameliorated neuronal death and axonal damage. Meanwhile, GAP43 knockout enlarged brain infarct area and deteriorated the cognitive and motor dysfunctions of HI rats. Further, network pharmacological analysis revealed the drug targets of Scu participated in such biological processes as neuronal death and regulation of neuronal death, and apoptosis-related pathways. GAP43 exhibited close relationship with PTN, JAK2 and STAT3, and GAP43 silencing upregulated the levels of PTN, JAK2 and STAT3. CONCLUSIONS: Collectively, our findings revealed Scu treatment attenuated long-term neurological impairments after HI by suppressing neuronal death and enhancing neurite elongation through GAP43-dependent pathway. The crucial role of Scutellarin in neuroprotection provided a novel possible therapeutic agent for the treatment of neonatal HIE.

Combined Scutellarin and C18H17NO6 Imperils the Survival of Glioma: Partly Associated With the Repression of PSEN1/PI3K-AKT Signaling Axis.

Glioma, the most common intracranial tumor, harbors great harm. Since the treatment for it has reached the bottleneck stage, the development of new drugs becomes a trend. Therefore, we focus on the effect of scutellarin (SCU) and its combination with C18H17NO6 (abbreviated as combination) on glioma and its possible mechanism in this study. Firstly, SCU and C18H17NO6 both suppressed the proliferation of U251 and LN229 cells in a dose-dependent manner, and C18H17NO6 augmented the inhibition effect of SCU on U251 and LN229 cells in vitro. Moreover, there was an interactive effect between them. Secondly, SCU and C18H17NO6 decreased U251 cells in G2 phase and LN229 cells in G2 and S phases but increased U251 cells in S phase, respectively. Meanwhile, the combination could further reduce U251 cells in G2 phase and LN229 cells in G2 and S phases. Thirdly, SCU and C18H17NO6 both induced the apoptosis of U251 and LN229. The combination further increased the apoptosis rate of both cells compared with the two drugs alone. Furthermore, SCU and C18H17NO6 both inhibited the lateral and vertical migration of both cells, which was further repressed by the combination. More importantly, the effect of SCU and the combination was better than positive control-temozolomide, and the toxicity was low. Additionally, SCU and C18H17NO6 could suppress the growth of glioma in vivo, and the effect of the combination was better. Finally, SCU and the combination upregulated the presenilin 1 (PSEN1) level but inactivated the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) signaling in vitro and in vivo. Accordingly, we concluded that scutellarin and its combination with C18H17NO6 suppressed the proliferation/growth and migration and induced the apoptosis of glioma, in which the mechanism might be associated with the repression of PSEN1/PI3K-AKT signaling axis.

Interleukin 10 Plays an Important Role in Neonatal Rats with Hypoxic-Ischemia Associated with B-Cell Lymphoma 2 and Endoplasmic Reticulum Protein 29.

Interleukin 10 (IL-10) is a synthetic inhibitor of human cytokines with immunomodulatory and anti-inflammatory effects. This study was designed to investigate the expression variation of IL-10 in the multiple sites including cortex, hippocampus, and lung tissues of neonatal hypoxic-ischemic (HI) rats and explore the crucial role of IL-10 in alleviating HI brain damage. In this study, neonatal Sprague-Dawley rats were subjected to the right common carotid artery ligation, followed by 2 h of hypoxia. The expression of IL-10 in the cortex, hippocampus, and lung tissues was measured with immunohistochemistry, real-time quantitative polymerase chain reaction (RT-qPCR), and western blot (WB). Immunofluorescence double staining was performed to observe the localization of IL-10 in neurons and astrocytes. Moreover, not-targeting and targeting IL-10 siRNA lentivirus vectors were injected into the rats of the negative control (NC) and IL-10 group, respectively, and the mRNA levels of B-cell lymphoma 2 (Bcl-2) and endoplasmic reticulum protein 29 (ERp29) were detected by RT-qPCR following IL-10 silence. The results demonstrated that the IL-10 expression was markedly increased after HI and IL-10 were colocalized with neurons and astrocytes which were badly injured by HI insult. In addition, Bcl-2 and ERp29 were remarkably decreased following IL-10 mRNA interference compared with the NC group. Our findings revealed that IL-10 exerted its antiapoptotic and neuroprotective effects by regulating the expression of Bcl-2 and ERp29, indicating that IL-10 may be a promising molecule target for HIE treatment.

Increased sensitivity to TNF-α promotes keloid fibroblast hyperproliferation by activating the NF-κB, JNK and p38 MAPK pathways.

Hyperproliferation of fibroblasts is the main cause of keloid formation. However, the pathogenesis of keloids has yet to be fully elucidated. Tumor necrosis factor (TNF)-α may play an important role in the formation and proliferation of keloids, as it is implicated in the pathogenesis of various fibrous disorders. In the present study, the expression level of TNF-α and its receptors, soluble TNF receptor (sTNFR)1 and sTNFR2, in the peripheral blood and skin tissues was detected by ELISA, reverse transcription-quantitative PCR or immunohistochemistry. There was no statistically significant difference in the expression of TNF-α and sTNFR2 in the peripheral blood and skin tissues between patients with keloids and healthy participants (P>0.05), while the sTNFR1 mRNA level in fibroblasts cultured in vitro and its protein level in keloid skin samples were significantly higher compared with those in normal skin (P<0.05). Subsequently, TNF-α recombinant protein was used to treat keloid-derived and normal skin fibroblasts, and it was observed that TNF-α promoted the proliferation of keloid fibroblasts (KFs), but had little effect on normal skin fibroblasts. Furthermore, it was observed that TNF-α stimulation led to the activation of the nuclear factor (NF)-κB, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) pathways in KFs. In conclusion, KFs exhibited increased expression of sTNFR1, which may contribute to the increased sensitivity to TNF-α, resulting in low concentrations of TNF-α activating the NF-κB, JNK and p38 MAPK pathways, thereby promoting the sustained and excessive proliferation of KFs.

Dihydroartemisinin and its anticancer activity against endometrial carcinoma and cervical cancer: involvement of apoptosis, autophagy and transferrin receptor.

INTRODUCTION: Dihydroartemisinin (DHA) is a first-line antimalarial drug with relatively low toxicity. DHA has been speculated to possess a broad-spectrum antitumour effect. However, the potential value of DHA for the treatment of endometrial carcinoma or cervical cancer is unclear. METHODS: We used human endometrial cancer cells and cervical cancer cells to assess whether DHA alone or when combined with cisplatin would induce cell death. We aimed to elucidate the role of autophagy in DHA-induced cytotoxicity in both endometrial and cervical cancer cells, and explore the impact of DHA treatment on cell proliferation, apoptosis and autophagy. RESULTS: DHA alone or in combination with cisplatin induced cell death in a dose- and time-dependent manner. Caspase-3 mRNA and cleaved caspase-3 protein levels were markedly elevated following DHA treatment either in the presence or absence of cisplatin, suggesting a role of apoptosis in DHA-induced cell death. DHA treatment activated the autophagic pathway, as evidenced by increased monodansylcadaverine-positive staining, elevated microtubule-associated protein 1 light chain 3 (LC3)-II/LC3-I ratio, and enhanced p62/sequestosome 1 degradation. Inhibition of autophagy by 3-methyladenine further enhanced the cytotoxicity of DHA towards tumour cells. mRNA levels of transferrin receptor (TfR) were suppressed upon DHA treatment and knockdown of TfR by RNA interference caused further DHA induction of cancer cell death. CONCLUSION: Our results suggest a clinical value for DHA in the treatment of endometrial carcinoma and cervical cancer. Our data revealed possible anticancer mechanisms of DHA that involve regulating apoptosis, autophagy pathway and levels of TfR.

DPYSL2 is a novel regulator for neural stem cell differentiation in rats: revealed by Panax notoginseng saponin administration.

BACKGROUND: The limited neuronal differentiation of the endogenous or grafted neural stem cells (NSCs) after brain injury hampers the clinic usage of NSCs. Panax notoginseng saponins (PNS) were extensively used for their clinical value, such as in controlling blood pressure, blood glucose, and inhibiting neuronal apoptosis and enhancing neuronal protection, but whether or not it exerts an effect in promoting neuronal differentiation of the endogenous NSCs is completely unclear and the potential underlying mechanism requires further exploration. METHODS: Firstly, we determined whether PNS could successfully induce NSCs to differentiate to neurons under the serum condition. Mass spectrometry and quantitative polymerase chain reaction (Q-PCR) were then performed to screen the differentially expressed proteins (genes) between the PNS + serum and serum control group, upon which dihydropyrimidinase-like 2 (DPYSL2), a possible candidate, was then selected for the subsequent research. To further investigate the actual role of DPYSL2 in the NSC differentiation, DPYSL2-expressing lentivirus was employed to obtain DPYSL2 overexpression in NSCs. DPYSL2-knockout rats were constructed to study its effects on hippocampal neural stem cells. Immunofluorescent staining was performed to identify the differentiation direction of NSCs after 7 days from DPYSL2 transfection, as well as those from DPYSL2-knockout rats. RESULTS: Seven differentially expressed protein spots were detected by PD Quest, and DPYSL2 was found as one of the key factors of NSC differentiation in a PNS-treated condition. The results of immunostaining further showed that mainly Tuj1 and GFAP-positive cells increased in the DPYSL2-overexpressed group, while both were depressed in the hippocampal NSCs in the DPYSL2-knockout rat. CONCLUSIONS: The present study revealed that the differentiation direction of NSCs could be enhanced through PNS administration, and the DPYSL2 is a key regulator in promoting NSC differentiation. These results not only emphasized the effect of PNS but also indicated DPYSL2 could be a novel target to enhance the NSC differentiation in future clinical trials.

Offspring of rats with cerebral hypoxia-ischemia manifest cognitive dysfunction in learning and memory abilities.

Neonatal hypoxic-ischemic encephalopathy is a serious neurological disease, often resulting in long-term neurodevelopmental disorders among surviving children. However, whether these neurodevelopmental issues can be passed to offspring remains unclear. The right common carotid artery of 7-day-old parental-generation rats was subjected to permanent ligation using a vessel electrocoagulator. Neonatal hypoxic-ischemic rat models were established by subjecting the rats to 8% O2-92% N2 for 2 hours. The results showed that 24 hours after hypoxia and ischemia, pathological damage, cerebral atrophy, liquefaction, and impairment were found, and Zea-Longa scores were significantly increased. The parental-generation rats were propagated at 3 months old, and offspring were obtained. No changes in the overall brain structures of these offspring rats were identified by magnetic resonance imaging. However, the escape latency was longer and the number of platform crossings was reduced among these offspring compared with normal rats. These results indicated that the offspring of hypoxic-ischemic encephalopathy model rats displayed cognitive impairments in learning and memory. This study was approved by the Animal Care & Welfare Committee of Kunming Medical University, China in 2018 (approval No. kmmu2019072).

Establishment of Neurobehavioral Assessment System in Tree Shrew SCT Model.

Tree shrews, possessing higher developed motor function than rats, were more suitable to study neurological behavior after spinal cord injury (SCI). Here, we established a feasible behavioral assessment method to detect the degree of ethology recovery in tree shrew subjected to spinal cord transection (SCT). Tree shrews were divided into normal group, sham group, and SCT group. The tree shrew in sham group was subjected to laminectomy without SCI, while the tree shrews in the SCT group were subjected to a complete SCT in thoracic 10 (T10). A novel neurobehavior assessment scale was established, in which, the behavior index including slow advancement, fast advancement, standing, shaking head, voluntary jump, lateral movement, and tail status, was determined, respectively. Meanwhile, magnetic resonance imaging (MRI) was applied to observe the structure of the spinal cord, and diffusion tensor imaging (DTI)-based white matter mapping was used to show the fibers of the spinal cord. As a result, a marked decrease in locomotor function and consciousness was seen in tree shrews with SCT, and the detection of MRI showed the collapsing of nerve fibers after SCT is completely cut and there is corresponding to the behavior change. Together, the present study provided a novel and feasible method that can be used to assess the neurobehavior in SCT model from tree shrews, which may be useful to the SCI translational study in future preclinic trial.

The Dual Role of AQP4 in Cytotoxic and Vasogenic Edema Following Spinal Cord Contusion and Its Possible Association With Energy Metabolism via COX5A.

Spinal cord edema, mainly including vasogenic and cytotoxic edema, influences neurological outcome after spinal cord contusion (SCC). Aquaporin 4 (AQP4) is the most ubiquitous water channel in the central nervous system (CNS), which is a rate-limiting factor in vasogenic edema expressing in brain injury, and it contributes to the formation of cytotoxic edema locating in astrocytes. However, little is known about the regulatory mechanism of AQP4 within vasogenic and cytotoxic edema in SCC, and whether the regulation mechanism of AQP4 is related to Cytochrome coxidase (COX5A) affecting energy metabolism. Therefore, the SCC model is established by Allen's method, and the degree of edema and neuronal area is measured. The motor function of rats is evaluated by the Basso, Beattie, and Bresnahan (BBB) scoring system. Meanwhile, AQP4 and COX5A are detected by real-time quantitative PCR (qRT-PCR) and western blot (WB). The localization of targeted protein is exhibited by immunohistochemical staining (IHC) and immunofluorescence (IF). Additionally, the methodology of AQP4 lentivirus-mediated RNA interference (AQP4-RNAi) is used to reveal the effect on edema of SCC and the regulating molecular mechanism. Firstly, we observe that the tissue water content increases after SCC and decreases after the peak value of tissue water content at 3 days (P < 0.05) with abundant expression of AQP4 protein locating around vascular endothelial cells (VECs), which suggests that the increasing AQP4 promotes water reabsorption and improves vasogenic edema in the early stage of SCC. However, the neuronal area is larger than in the sham group in the 7 days (P < 0.05) with the total water content of spinal cord decrease. Meanwhile, AQP4 migrates from VECs to neuronal cytomembrane, which indicates that AQP4 plays a crucial role in aggravating the formation and development of cytotoxic edema in the middle stages of SCC. Secondly, AQP4-RNAi is used to elucidate the mechanism of AQP4 to edema of SCC. The neuronal area shrinks and the area of cytotoxic edema reduces after AQP4 downregulation. The BBB scores are significantly higher than in the vector group after AQP4-RNAi at 5, 7, and 14 (P < 0.05). There is a relationship between AQP4 and COX5A shown by bioinformatics analysis. After AQP4 inhibition, the expression of COX5A is significantly upregulated in the swelling astrocytes. Therefore, the inhibition of AQP4 expression reduces cytotoxic edema in SCC and improves motor function, which may be associated with upregulation of COX5A via affecting energy metabolism. Moreover, it is not clear how the inhibition of AQP4 directly causes the upregulation of COX5A.

Suppression of PDGF induces neuronal apoptosis after neonatal cerebral hypoxia and ischemia by inhibiting P-PI3K and P-AKT signaling pathways.

Neonatal hypoxic-ischemic encephalopathy (HIE) always results in severe neurologic dysfunction, nevertheless effective treatments are limited and the underlying mechanism also remains unclear. In this study, we firstly established the neonatal HIE model in the postnatal day 7 SD rats, Zea-Longa score and TTC staining were employed to assess the neurological behavior and infarct volume of the brain after cerebral hypoxia-ischemia (HI). Afterwards, protein chip was adopted to detect the differential proteins in the right cortex, hippocampus and lung, ultimately, PDGF was noticed. Then, immunohistochemistry, immunofluorescence double staining of NeuN/PDGF, and western blot were used to validate the expression level of PDGF in the cortex and hippocampus at 6 hours (h), 12 h and 24 h after HI. To determine the role of PDGF, the primary cortical neurons were prepared and performed PDGF shRNA administration. The results showed that HIE induced a severe behavioral dysfunction and brain infarction in neonatal rats, and the expression of PDGF in right cortex and hippocampus was remarkably increased after HI. Whereas, suppressing PDGF resulted in a significant loss of neurons and inhibition of neurite growth. Moreover, the protein level of P-PI3K and P-AKT signaling pathways were largely decreased following PDGF-shRNA application in the cortical neurons. In conclusion, PDGF suppression aggravated neuronal dysfunction, and the underlying mechanism is associated with inhibiting the phosphorylation of P-PI3K and P-AKT. Together, PDGF regulating PI3K and AKT may be an important panel in HIE events and therefore may provide possible strategy for the treatment of HIE in future clinic trail.

C18H17NO6 and Its Combination with Scutellarin Suppress the Proliferation and Induce the Apoptosis of Human Glioma Cells via Upregulation of Fas-Associated Factor 1 Expression.

BACKGROUND: Glioma is the most common malignant brain tumor and the patients are prone to poor prognosis. Due to limited treatments, new drug exploration has become a general trend. Therefore, the objective of this study is to investigate the effect of the new drugs C18H17NO6 and its combination with Scutellarin on glioma cells and the underlying mechanism. METHOD: U251 and LN229 cells were administrated with C18H17NO6 and its combination with Scutellarin. The proliferation ability of glioma cells was determined by cell counting kit-8, plate clone formation assay, and EdU incorporation assay. The cell cycle and apoptosis detection were detected by flow cytometry. Moreover, TUNEL assay was also used for cell apoptosis analysis. Then, the transfer ability of cells was achieved through wound healing assay. Furthermore, polymerase chain reaction (PCR) test and western bolt analysis were used to detect the mRNA expression and protein expression, respectively. Lastly, immunofluorescence was for the purity identification of astrocyte. RESULT: The results showed that, with the increasing dose of C18H17NO6, the cell inhibition rate, the cells in G1 phase, and the apoptosis rate were gradually increased, but the clone number, proliferation rate, and the cells in G2 and S phases were gradually decreased in comparison with control group. However, with the increase of C18H17NO6, the transferred rate of U251 and LN229 was not significantly augmented, expect that on U251 in C18H17NO6 5 µM group. In addition, Scutellarin 200 µM has little effect on proliferation, with the inhibition rate 10-20% and proliferation rate except U251 in Scutellarin 200 µM group similar to that in control group. Moreover, compared to control group, Scutellarin 300 µM increased the U251 cells in G2 and S phases and the apoptosis rate of LN229 but decreased the LN229 cells in G2 and S phases. Besides, in Scutellarin 200 µM group, the transfer ability of LN229 was inhibited, but not in U251. Furthermore, if C18H17NO6 was combined with Scutellarin 200/300µM, the proliferation and transferred ability were suppressed and the apoptosis was elevated in LN229 cell in comparison with C18H17NO6 alone. Dramatically, the combined effect on U251 was the exact opposite. Importantly, there was little toxicity on astrocyte under the dose of C18H17NO6 and Scutellarin in the study. In molecular level, the mRNA and protein expression of Fas-associated factor 1 (FAF1) expression in U251 and LN229 were upregulated by C18H17NO6 and its combination with Scutellarin, especially the protein expression. CONCLUSION: C18H17NO6 could efficiently suppress cell proliferation and induce cell apoptosis in glioma cells, and its combination with Scutellarin had a promoting effect, in which the underlying mechanism referred to the upregulation of Fas-associated factor 1.

Expression and localization of histamine H1, H2, and H3 receptors in rat olfactory epithelium.

OBJECTIVE: Histamine is an important chemical mediator in the development of allergic rhinitis and plays a key role in eliciting the nasal symptoms of the disorder. Histamine may also affect smell as a neurotransmitter. However, whether histamine receptors are present in the mammalian olfactory epithelium has not yet been examined. The aim of this study was to investigate the expression and distribution of histamine H1, H2, and H3 receptors in rat olfactory epithelium. METHODS: Real-time quantitative PCR and immunohistochemical staining were performed to examine the mRNA level and protein expression and localization of histamine receptors (H1, H2, and H3) in rat olfactory epithelium. RESULTS: We demonstrated that mRNAs encoding histamine H1, H2, and H3 receptors were detected in rat olfactory epithelium. Immunohistochemistry also showed strong positive staining for these receptors. Co-localization of histamine H1, H2, and H3 receptors with olfactory mature protein revealed that these three histamine receptors were mainly localized in olfactory receptor neurons. CONCLUSIONS: These findings indicate that histamine H1, H2, and H3 receptors are present in rat olfactory epithelium and may play a physiological role in olfactory transmission.

Neural Stem Cell Transplantation Is Associated with Inhibition of Apoptosis, Bcl-xL Upregulation, and Recovery of Neurological Function in a Rat Model of Traumatic Brain Injury.

Traumatic brain injury (TBI) is a common disease that usually causes severe neurological damage, and current treatment is far from satisfactory. The neuroprotective effects of neural stem cell (NSC) transplantation in the injured nervous system have largely been known, but the underlying mechanisms remain unclear, and their limited sources impede their clinical application. Here, we established a rat model of TBI by dropping a weight onto the cortical motor area of the brain and explored the effect of engrafted NSCs (passage 3, derived from the hippocampus of embryonic 12- to 14-d green fluorescent protein transgenic mice) on TBI rats. Moreover, RT-PCR and Western blotting were employed to investigate the possible mechanism associated with NSC grafts. We found rats with TBI exhibited a severe motor and equilibrium dysfunction, while NSC transplantation could partly improve the motor function and significantly reduce cell apoptosis and increase B-cell lymphoma-extra large (Bcl-xL) expression at 7 d postoperation. However, other genes including Bax, B-cell lymphoma 2, Fas ligand, and caspase3 did not exhibit significant differences in expression. Moreover, to test whether Bcl-xL could be used as a therapeutic target, herpes simplex virus (HSV) 1 carrying Bcl-xL recombinant was constructed and injected into the pericontusional cortices. Bcl-xL overexpression not only resulted in a significant improvement in neurological function but also inhibits cell apoptosis, as compared with the TBI rats, and exhibits the same effects as the administration of NSC. The present study therefore indicated that NSC transplantation could promote the recovery of TBI rats in a manner similar to that of Bcl-xL overexpression. Therefore, Bcl-xL overexpression, to some extent, could be considered as a useful strategy to replace NSC grafting in the treatment of TBI in future clinical practices.

Transplantation of Hematopoietic Stem Cells Promotes Functional Improvement Associated with NT-3-MEK-1 Activation in Spinal Cord-Transected Rats.

Transected spinal cord injury (SCT) is a devastating clinical disease that strongly affects a patient's daily life and remains a great challenge for clinicians. Stem-cell therapy has been proposed as a potential therapeutic modality for SCT. To investigate the effects of hematopoietic stem cells (HSCs) on the recovery of structure and function in SCT rats and to explore the mechanisms associated with recovery, 57 adult Sprague-Dawley rats were randomly divided into sham (n = 15), SCT (n = 24), and HSC transplantation groups (n = 15). HSCs (passage 3) labeled by Hoechst 33342, were transplanted intraspinally into the rostral, scar and caudal sites of the transected lesion at 14 days post-operation. Both in vitro and in vivo, HSCs exhibited a capacity for cell proliferation and differentiation. Following HSC transplantation, the animals' Basso, Beattie, and Bresnahan (BBB). locomotion scale scores increased significantly between weeks 4 and 24 post-SCT, which corresponded to an increased number of 5-hydroxytryptamine (5-HT) fibers and oligodendrocytes. The amount of astrogliosis indicated by immunohistochemical staining, was markedly decreased. Moreover, the decreased expression of neurotrophin- 3 (NT-3) and mitogen-activated protein kinase kinase-1 (MEK-1) after SCT was effectively restored by HSC transplantation. The data from the current study indicate that intraspinally administered HSCs in the chronic phase of SCT results in an improvement in neurological function. Further, the results indicate that intraspinally administered HSCs benefit the underlying mechanisms involved in the enhancement of 5-HT-positive fibers and oligogenesis, the suppression of excessive astrogliosis and the upregulation of NT3-regulated MEK-1 activation in the spinal cord. These crucial findings reveal not only the mechanism of cell therapy, but may also contribute to a novel therapeutic target for the treatment of spinal cord injury (SCI).