Combination of fMRI and PET reveals the beneficial effect of three-phase enriched environment on post-stroke memory deficits by enhancing plasticity of brain connectivity between hippocampus and peri-hippocampal cortex.

AIM: The three-phase enriched environment (EE) intervention paradigm has been shown to improve learning and memory function after cerebral ischemia, but the neuronal mechanisms are still unclear. This study aimed to investigate the hippocampal-cortical connectivity and the metabolic interactions between neurons and astrocytes to elucidate the underlying mechanisms of EE-induced memory improvement after stroke. METHODS: Rats were subjected to permanent middle cerebral artery occlusion (pMCAO) or sham surgery and housed in standard environment or EE for 30 days. Memory function was examined by Morris water maze (MWM) test. Magnetic resonance imaging (MRI) was conducted to detect the structural and functional changes. [18 F]-fluorodeoxyglucose (FDG) positron emission tomography (PET) was conducted to detect brain energy metabolism. PET-based brain connectivity and network analysis was performed to study the changes of hippocampal-cortical connectivity. Astrocyte-neuron metabolic coupling, including gap junction protein connexin 43 (Cx43), glucose transporters (GLUTs), and monocarboxylate transporters (MCTs), was detected by histological studies. RESULTS: Our results showed EE promoted memory function improvement, protected structure integrity, and benefited energy metabolism after stroke. More importantly, EE intervention significantly increased functional connectivity between the hippocampus and peri-hippocampal cortical regions, and specifically regulated the level of Cx43, GLUTs and MCTs in the hippocampus and cortex. CONCLUSIONS: Our results revealed the three-phase enriched environment paradigm enhanced hippocampal-cortical connectivity plasticity and ameliorated post-stroke memory deficits. These findings might provide some new clues for the development of EE and thus facilitate the clinical transformation of EE.

Buyang Huanwu Decoction promotes neurovascular remodeling by modulating astrocyte and microglia polarization in ischemic stroke rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Buyang Huanwu Decoction (BYHWD), one of the most commonly utilized traditional Chinese medicine prescription for treatment of cerebral ischemic stroke. However, the understanding of BYHWD on neurovascular repair following cerebral ischemia is so far limited. AIM OF THE STUDY: This research investigated the influence of BYHWD on neurovascular remodeling by magnetic resonance imaging (MRI) technology and revealed the potential neurovascular repair mechanism underlying post-treatment with BYHWD after ischemic stroke. MATERIALS AND METHODS: Male Sprague-Dawley rats were utilized as an ischemic stroke model by permanent occlusion of the middle cerebral artery (MCAO). BYHWD was intragastrically administrated once daily for 30 days straight. Multimodal MRI was performed to detect brain tissue injuries, axonal microstructural damages, cerebral blood flow and intracranial vessels on the 30th day after BYHWD treatment. Proangiogenic factors, axonal/synaptic plasticity-related factors, energy transporters and adenosine monophosphate-activated protein kinase (AMPK) signal pathway were evaluated using western blot. Double immunofluorescent staining and western blot were applied to evaluate astrocytes and microglia polarization. RESULTS: Administration of BYHWD significantly alleviated infarct volume and brain tissue injuries and ameliorated microstructural damages, accompanied with improved axonal/synaptic plasticity-related factors, axonal growth guidance factors and decreased axonal growth inhibitors. Meanwhile, BYHWD remarkably improved cerebral blood flow, cerebral vascular signal and promoted the expression of proangiogenic factors. Particularly, treatment with BYHWD obviously suppressed astrocytes A1 and microglia M1 polarization accompanied with promoted astrocyte A2 and microglia M2 polarization. Furthermore, BYHWD effectively improved energy transporters. Especially, BYHWD markedly increased expression of phosphorylated AMPK, cyclic AMP-response element binding protein (CREB) and brain-derived neurotrophic factor (BDNF) accompanied by inactivation of the NF-κB. CONCLUSION: Taken together, these findings identified that the beneficial roles of BYHWD on neurovascular remodeling were related to AMPK pathways -mediated energy transporters and NFκB/CREB pathways.

Three-phase Enriched Environment Improves Post-stroke Gait Dysfunction via Facilitating Neuronal Plasticity in the Bilateral Sensorimotor Cortex: A Multimodal MRI/PET Analysis in Rats.

The three-phase Enriched Environment (EE) paradigm has been shown to promote post-stroke functional improvement, but the neuronal mechanisms are still unclear. In this study, we applied a multimodal neuroimaging protocol combining magnetic resonance imaging (MRI) and positron emission tomography (PET) to examine the effects of post-ischemic EE treatment on structural and functional neuroplasticity in the bilateral sensorimotor cortex. Rats were subjected to permanent middle cerebral artery occlusion. The motor function of the rats was examined using the DigiGait test. MRI was applied to investigate the EE-induced structural modifications of the bilateral sensorimotor cortex. [18F]-fluorodeoxyglucose PET was used to detect glucose metabolism. Blood oxygen level-dependent (BOLD)-functional MRI (fMRI) was used to identify the regional brain activity and functional connectivity (FC). In addition, the expression of neuroplasticity-related signaling pathways including neurotrophic factors (BDNF/CREB), axonal guidance proteins (Robo1/Slit2), and axonal growth-inhibitory proteins (NogoA/NgR) as well as downstream proteins (RhoA/ROCK) in the bilateral sensorimotor cortex were measured by Western blots. Our results showed the three-phase EE improved the walking ability. Structural T2 mapping imaging and diffusion tensor imaging demonstrated that EE benefited structure integrity in the bilateral sensorimotor cortex. PET-MRI fused images showed improved glucose metabolism in the corresponding regions after EE intervention. Specifically, the BOLD-based amplitude of low-frequency fluctuations showed that EE increased spontaneous activity in the bilateral motor cortex and ipsilateral sensory cortex. In addition, FC results showed increased sensorimotor connectivity in the ipsilateral hemisphere and increased interhemispheric motor cortical connectivity and motor cortical-thalamic connectivity following EE intervention. In addition, a strong correlation was found between increased functional connectivity and improved motor performance of limbs. Specifically, EE regulated the expression of neuroplasticity-related signaling, involving BDNF/CREB, Slit2/Robo1, as well as the axonal growth-inhibitory pathways Nogo-A/Nogo receptor and RhoA/ROCK in the bilateral sensorimotor cortex. Our results indicated that the three-phase enriched environment paradigm enhances neuronal plasticity of the bilateral sensorimotor cortex and consequently ameliorates post-stroke gait deficits. These findings might provide some new clues for the development of EE and thus facilitate the clinical translation of EE.

Proposing a novel molecular subtyping scheme for predicting distant recurrence-free survival in breast cancer post-neoadjuvant chemotherapy with close correlation to metabolism and senescence.

Background: High relapse rates remain a clinical challenge in the management of breast cancer (BC), with distant recurrence being a major driver of patient deterioration. To optimize the surveillance regimen for distant recurrence after neoadjuvant chemotherapy (NAC), we conducted a comprehensive analysis using bioinformatics and machine learning approaches. Materials and methods: Microarray data were retrieved from the GEO database, and differential expression analysis was performed with the R package 'Limma'. We used the Metascape tool for enrichment analyses, and 'WGCNA' was utilized to establish co-expression networks, selecting the soft threshold power with the 'pickSoftThreshold' algorithm. We integrated ten machine learning algorithms and 101 algorithm combinations to identify key genes associated with distant recurrence in BC. Unsupervised clustering was performed with the R package 'ConsensusCluster Plus'. To further screen the key gene signature of residual cancer burden (RCB), multiple knockdown studies were analyzed with the Genetic Perturbation Similarity Analysis (GPSA) database. Single-cell RNA sequencing (scRNA-seq) analysis was conducted through the Tumour Immune Single-cell Hub (TISCH) database, and the XSum algorithm was used to screen candidate small molecule drugs based on the Connectivity Map (CMAP) database. Molecular docking processes were conducted using Schrodinger software. GMT files containing gene sets associated with metabolism and senescence were obtained from GSEA MutSigDB database. The GSVA score for each gene set across diverse samples was computed using the ssGSEA function implemented in the GSVA package. Results: Our analysis, which combined Limma, WGCNA, and machine learning approaches, identified 16 RCB-relevant gene signatures influencing distant recurrence-free survival (DRFS) in BC patients following NAC. We then screened GATA3 as the key gene signature of high RCB index using GPSA analysis. A novel molecular subtyping scheme was developed to divide patients into two clusters (C1 and C2) with different distant recurrence risks. This molecular subtyping scheme was found to be closely associated with tumor metabolism and cellular senescence. Patients in cluster C2 had a poorer DRFS than those in cluster C1 (HR: 4.04; 95% CI: 2.60-6.29; log-rank test p < 0.0001). High GATA3 expression, high levels of resting mast cell infiltration, and a high proportion of estrogen receptor (ER)-positive patients contributed to better DRFS in cluster C1. We established a nomogram based on the N stage, RCB class, and molecular subtyping. The ROC curve for 5-year DRFS showed excellent predictive value (AUC=0.91, 95% CI: 0.95-0.86), with a C-index of 0.85 (95% CI: 0.81-0.90). Entinostat was identified as a potential small molecule compound to reverse high RCB after NAC. We also provided a comprehensive review of the EDCs exposures that potentially impact the effectiveness of NAC among BC patients. Conclusion: This study established a molecular classification scheme associated with tumor metabolism and cancer cell senescence to predict RCB and DRFS in BC patients after NAC. Furthermore, GATA3 was identified and validated as a key gene associated with BC recurrence.

Tetramethylpyrazine promotes axonal remodeling and modulates microglial polarization via JAK2-STAT1/3 and GSK3-NFκB pathways in ischemic stroke.

Ischemic stroke results in demyelination that underlies neurological disfunction. Promoting oligodendrogenesis will rescue the injured axons and accelerate remyelination after stroke. Microglia react to ischemia/hypoxia and polarize to M1/M2 phenotypes influencing myelin injury and repair. Tetramethylpyrazine (TMP) has neuroprotective effects in treating cerebrovascular disorders. This study aims to evaluate whether TMP promotes the renovation of damaged brain tissues especially on remyelination and modulates microglia phenotypes following ischemic stroke. Here magnetic resonance imaging (MRI)-diffusion tensor imaging (DTI) and histopathological evaluation are performed to characterize the process of demyelination and remyelination. Immunofluorescence staining is used to prove oligodendrogenesis and microglial polarization. Western blotting is conducted to examine interleukin (IL)-6, IL-10, transforming growth factor ß (TGF-ß) and Janus protein tyrosine kinase (JAK) 2-signal transducer and activator of transcription (STAT) 1/3-glycogen synthase kinase (GSK) 3-nuclear transcription factor κB (NFκB) signals. Results show TMP alleviates the injury of axons and myelin sheath, increases NG2+, Ki67+/NG2+, CNPase+, Ki67+/CNPase+, Iba1+/Arg-1+ cells and decreases Iba1+ and Iba1+/CD16+ cells in periinfarctions of rats. Particularly, TMP downregulates IL-6 and upregulates IL-10 and TGF-ß expressions, besides, enhances JAK2-STAT3 and suppresses STAT1-GSK3-NFκB activation in middle cerebral artery occlusion (MCAo) rats. Then we demonstrate that TMP reverses M1/M2 phenotype via JAK2-STAT1/3 and GSK3-NFκB pathways in lipopolysaccharide (LPS) plus interferon-γ (IFN-γ)-stimulated BV2 microglia. Blocking JAK2 with AG490 counteracts TMP's facilitation on M2 polarization of microglia. This study warrants the promising therapy for stroke with TMP treatment.

Lanosterol synthase loss of function decreases the malignant phenotypes of HepG2 cells by deactivating the Src/MAPK signaling pathway.

Cholesterol is critical for tumor cells to maintain their membrane components, cell morphology and activity functions. The inhibition of the cholesterol pathway may be an efficient strategy with which to limit tumor growth and the metastatic process. In the present study, lanosterol synthase (LSS) was knocked down by transfecting LSS short hairpin RNA into HepG2 cells, and cell growth, apoptosis and migratory potential were then detected by Cell Counting Kit-8 cell proliferation assay, flow cytometric analysis and wound healing assay, respectively. In addition, proteins associated with the regulation of the aforementioned cell biological behaviors were analyzed by western blot analysis. The activity of the Src/MAPK signaling pathway was measured by western blotting to elucidate the possible signal transduction mechanisms. LSS knockdown in the HepG2 liver cancer cell line inhibited cell proliferation, with cell cycle arrest at the S phase; it also decreased cell migratory ability and increased apoptosis. The expression proteins involved in the regulation of cell cycle, cell apoptosis and migration was altered by LSS knockdown in HepG2 cells. Furthermore, a decreased Src/MAPK activity was observed in the HepG2 cells subjected to LSS knockdown. LSS loss of function decreased the malignant phenotypes of HepG2 cells by deactivating the Src/MAPK signaling pathway and regulating expression of genes involved in cell cycle regulation, cell apoptosis and migration.

Tetramethylpyrazine promotes stroke recovery by inducing the restoration of neurovascular unit and transformation of A1/A2 reactive astrocytes.

2,3,5,6-Tetramethylpyrazine (TMP) as an active ingredient extracted from a traditional Chinese herbal medicine Ligusticum chuanxiong Hort. has been proved to penetrate blood-brain barrier (BBB) and show neuroprotective effects on cerebral ischemia. However, whether TMP could regulate astrocytic reactivity to facilitate neurovascular restoration in the subacute ischemic stroke needs to be urgently verified. In this research, permanent occlusion of the middle cerebral artery (MCAO) model was conducted and TMP (10, 20, 40 mg/kg) was intraperitoneally administrated to rats once daily for 2 weeks. Neurological function was evaluated by motor deficit score (MDS). Magnetic resonance imaging (MRI) was implemented to analyze tissue injury and cerebral blood flow (CBF). Magnetic resonance angiography (MRA) was applied to exhibit vascular signals. Transmission electron microscopy (TEM) was performed to detect the neurovascular unit (NVU) ultrastructure. Haematoxylin and eosin (HE) staining was utilized to evaluate cerebral histopathological lesions. The neurogenesis, angiogenesis, A1/A2 reactivity, aquaporin 4 (AQP4) and connexin 43 (Cx43) of astrocytes were observed with immunofluorescent staining. Then FGF2/PI3K/AKT signals were measured by western blot. Findings revealed TMP ameliorated neurological functional recovery, preserved NVU integrity, and enhanced endogenous neurogenesis and angiogenesis of rats with subacute ischemia. Shifting A1 to A2 reactivity, suppressing excessive AQP4 and Cx43 expression of astrocytes, and activating FGF2/PI3K/AKT pathway might be potential mechanisms of promoting neurovascular restoration with TMP after ischemic stroke.

[Content analysis and quality evaluation of main active components and mineral elements of Cynomorium songaricum in different habitats].

To clarify the content characteristics of the main active components and mineral elements of Cynomorium songaricum under different habitat conditions, and further explore the relationship between the quality of C. songaricum and habitats, this study took C. songaricum from 25 different habitats in China as the research object, and measured the contents of 8 main active components and 12 mineral elements separately. Diversity analysis, correlation analysis, principal component analysis and cluster analysis were carried out. The results showed that the genetic diversity of total flavonoids, ursolic acid, ether extract, potassium(K), phosphorus(P) and zinc(Zn) in C. songaricum was high. The coefficient of variation of crude polysaccharide, ether extract, gallic acid, protocatechuic aldehyde, catechin, epicatechin, calcium(Ca), sodium(Na), magnesium(Mg), sulfur(S), iron(Fe), manganese(Mn), selenium(Se) and nickel(Ni) were all over 36%, indicating that the quality of C. songaricum was significantly affected by habitats. There were strong synergistic and weak antagonistic effects among the contents of the 8 active components, and complex antagonistic and synergistic effects among the contents of the 12 mineral elements. Principal component analysis revealed that crude polysaccharide, ursolic acid, catechin, epicatechin and total flavonoids could be used as the characteristic components to evaluate the quality of C. songaricum, and Na, copper(Cu), Mn and Ni were the characteristic elements to evaluate the quality of C. songaricum. In cluster ana-lysis, the second group with the main active components as cluster center had better quality in terms of the content of active substances, and the second group with the mineral elements as cluster center had higher utilization potential in the exploitation of mineral elements. This study could provide a basis for resource evaluation and breeding of excellent varieties of C. songaricum in different habitats, and provide a reference for cultivation and identification of C. songaricum.

Nitrogen deposition enhances the deterministic process of the prokaryotic community and increases the complexity of the microbial co-network in coastal wetlands.

Global nitrogen deposition has increased significantly in recent years. At present, research on the effects of different amounts and types of nitrogen deposition on soil microorganisms in coastal wetlands is scarce. In this study, based on 7 years of simulated nitrogen deposition at multiple levels (low, medium, high) and of multiple types (NH4NO3, NH4Cl, KNO3), the effects of different nitrogen deposition conditions on the diversity, community assembly processes, co-networks, and community function of soil prokaryotes in coastal wetlands were examined. The results showed that, compared with that in control, the microbial α diversity increased significantly under nitrogen deposition (P < 0.05). However, it decreased significantly in the high-NH4NO3 and high-NH4Cl treatments (P < 0.05). The deterministic process of community assembly was strengthened under the different types of nitrogen deposition. Compared with that under NH4+-N deposition, the microbial co-network under NO3--N deposition was more complex. Network stability significantly decreased under different NH4+-N deposition levels. In addition, the results of FAPROTAX functional prediction showed that microbial community functional groups associated with carbon and nitrogen cycling changed significantly (P < 0.05). In conclusion, our results emphasize that nitrogen deposition environments cause changes in soil microbial community structure and interactions, and may also affect soil carbon and nitrogen cycling, but the effects of different forms and levels of nitrogen deposition are not consistent. This study provides new insights for evaluating the changes in soil microbial communities in coastal wetlands caused by different types of long-term nitrogen deposition, and has scientific significance for assessing the ecological effects of long-term nitrogen deposition.

Chimeric Oncolytic Adenovirus Armed Chemokine Rantes for Treatment of Breast Cancer.

The immunosuppressive state in the tumor microenvironment (TME) of breast cancer makes it difficult to treat with immunotherapy. Oncolytic viruses not only lyse tumor cells but also reshape the TME. Therefore, they can play a multi-mechanism synergistic effect with immunotherapy. In this study, an oncolytic adenovirus Ad5F11bSP-Rantes was constructed and used as a vector to express the chemokine Rantes. The objective of this study was to test the dual mechanisms of the oncolytic effect mediated by virus replication and the enhanced anticancer immune response mediated by Rantes chemotaxis of immune cells. It was found that Ad5F11bSP-Rantes has strong infectivity and effective killing activity against breast cancer cells. In the established triple negative breast cancer (TNBC) xenograft model in NCG mice whose immune system was humanized with human peripheral blood mononuclear cells (PBMCs), Ad5F11bSP-Rantes achieved 88.33% tumor inhibition rate. Rantes expression was high in mouse blood, a large number of CD3+ lymphocytes infiltrated in tumor tissues and E-cadherin was up-regulated in cancer cells, suggesting that Ad5F11bSP-Rantes altered the TME and induced a reversal of cancer cell epithelial-mesenchymal transition (EMT). In conclusion, oncolytic adenovirus can exert the oncolytic effect and the chemotactic effect of immune cells and realize the synergy of multiple anticancer effects. This strategy creates a candidate treatment for the optimization of breast cancer, especially TNBC, combination therapy.

Magnetic Resonance Imaging Investigation of Neuroplasticity After Ischemic Stroke in Tetramethylpyrazine-Treated Rats.

Ischemic stroke elicits white matter injury typically signed by axonal disintegration and demyelination; thus, the development of white matter reorganization is needed. 2,3,5,6-Tetramethylpyrazine (TMP) is widely used to treat ischemic stroke. This study was aimed to investigate whether TMP could protect the white matter and promote axonal repair after cerebral ischemia. Male Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion (MCAO) and treated with TMP (10, 20, 40 mg/kg) intraperitoneally for 14 days. The motor function related to gait was evaluated by the gait analysis system. Multiparametric magnetic resonance imaging (MRI) was conducted to noninvasively identify gray-white matter structural integrity, axonal reorganization, and cerebral blood flow (CBF), followed by histological analysis. The expressions of axonal growth-associated protein 43 (GAP-43), synaptophysin (SYN), axonal growth-inhibitory signals, and guidance factors were measured by Western blot. Our results showed TMP reduced infarct volume, relieved gray-white matter damage, promoted axonal remodeling, and restored CBF along the peri-infarct cortex, external capsule, and internal capsule. These MRI findings were confirmed by histopathological data. Moreover, motor function, especially gait impairment, was improved by TMP treatment. Notably, TMP upregulated GAP-43 and SYN and enhanced axonal guidance cues such as Netrin-1/DCC and Slit-2/Robo-1 but downregulated intrinsic growth-inhibitory signals NogoA/NgR/RhoA/ROCK-2. Taken together, our data indicated that TMP facilitated poststroke axonal remodeling and motor functional recovery. Moreover, our findings suggested that TMP restored local CBF, augmented guidance cues, and restrained intrinsic growth-inhibitory signals, all of which might improve the intracerebral microenvironment of ischemic areas and then benefit white matter remodeling.

The Impact of Ischemic Stroke on Gray and White Matter Injury Correlated With Motor and Cognitive Impairments in Permanent MCAO Rats: A Multimodal MRI-Based Study.

Background: Identifying the alterations of the cerebral gray and white matter is an important prerequisite for developing potential pharmacological therapy for stroke. This study aimed to assess the changes of gray and white matter after permanent middle cerebral artery occlusion (pMCAO) in rats using magnetic resonance imaging (MRI), and to correlate them with the behavior performance. Methods: Rats were subjected to pMCAO or sham surgery and reared for 30 days. Motor and cognitive function of the rats were examined by gait and Morris water maze (MWM) tests, respectively. Multimodal MRI was conducted to examine the functional and structural changes of the gray and white matter followed with luxol fast blue (LFB) staining. Results: The gait and MWM tests revealed significant motor and cognitive dysfunction in pMCAO rats, respectively. Magnetic resonance angiography presented abnormal intracranial arteries in pMCAO rats with reduced signal intensity of the anterior cerebral artery, anterior communicating cerebral artery, internal carotid artery, and increased basilar artery vessel signal compared with sham rats. Arterial spin labeling confirmed the decreased cerebral blood flow in the infarcted sensorimotor cortex and striatum. Structural T2-weighted imaging and T2 mapping showed brain atrophy and elevation of T2 value in the gray (sensorimotor cortex, striatum) and white (external capsule, internal capsule) matter of pMCAO rats. The results from diffusion tensor imaging (DTI) corresponded well with LFB staining showing reduced relative FA accompanied with increased relative AD and RD in the gray and white matter of pMCAO rats compared with sham rats. Fiber tracking derived from DTI further observed significantly reduced fiber density and length in the corresponding brain regions of pMCAO rats compared with sham rats. Specially, the DTI parameters (especially FA) in the relevant gray matter and white matter significantly correlated with the behavior performance in the gait and MWM tests. Conclusion: Collectively, the gray and white matter damages could be non-invasively monitored in pMCAO rats by multimodal MRI. DTI-derived parameters, particularly the FA, might be a good imaging index to stage gray and white matter damages associated with post-stroke motor and cognitive impairments.

Line-of-sight-based global finite-time stable path following control of unmanned surface vehicles with actuator saturation.

This paper focuses on the path following problem of unmanned surface vehicles (USVs) with unknown velocities, model uncertainties, and actuator saturation. To steer a USV rapidly and accurately follow the desired parameterized path, a line-of-sight (LOS)-based finite-time path following scheme is constructed in which the finite-time technique can ensure the fast error convergence, such that some intelligent operations, including patrolling, fuel supplying, and formation control, can be promptly performed. First, USV kinematic and kinetic models are established, and finite-time observers are subsequently employed to identify the unmeasured USV velocities and model uncertainties. Then, an LOS guidance law is designed to achieve the finite-time convergence of the position errors. In addition, an optimized look-ahead distance is developed using a fuzzy algorithm. Meanwhile, the control subsystem is designed at the kinetic level by combining the backstepping sliding mode method and a novel auxiliary dynamic system, where the auxiliary system is applied to address actuator saturation. Subsequently, theoretical analysis is conducted to verify that the entire system is uniformly global finite-time stable (UGFTS). Finally, the simulation studies confirms the availability of the developed method.

Effect of Neurorepair for Motor Functional Recovery Enhanced by Total Saponins From Trillium tschonoskii Maxim. Treatment in a Rat Model of Focal Ischemia.

Trillium tschonoskii Maxim. (TTM), is a perennial herb from Liliaceae, that has been widely used as a traditional Chinese medicine treating cephalgia and traumatic hemorrhage. The present work was designed to investigate whether the total saponins from Trillium tschonoskii Maxim. (TSTT) would promote brain remodeling and improve gait impairment in the chronic phase of ischemic stroke. A focal ischemic model of male Sprague-Dawley (SD) rats was established by permanent middle cerebral artery occlusion (MCAO). Six hours later, rats were intragastrically treated with TSTT (120, 60, and 30 mg/kg) and once daily up to day 30. The gait changes were assessed by the CatWalk-automated gait analysis system. The brain tissues injuries, cerebral perfusion and changes of axonal microstructures were detected by multimodal magnetic resonance imaging (MRI), followed by histological examinations. The axonal regeneration related signaling pathways including phosphatidylinositol 3-kinases (PI3K)/protein kinase B (AKT)/glycogen synthase kinase-3 (GSK-3)/collapsin response mediator protein-2 (CRMP-2) were measured by western blotting. TSTT treatment significantly improved gait impairment of rats. MRI analysis revealed that TSTT alleviated tissues injuries, significantly improved cerebral blood flow (CBF), enhanced microstructural integrity of axon and myelin sheath in the ipsilesional sensorimotor cortex and internal capsule. In parallel to MRI findings, TSTT preserved myelinated axons and promoted oligodendrogenesis. Specifically, TSTT interventions markedly up-regulated expression of phosphorylated GSK-3, accompanied by increased expression of phosphorylated PI3K, AKT, but reduced phosphorylated CRMP-2 expression. Taken together, our results suggested that TSTT facilitated brain remodeling. This correlated with improving CBF, encouraging reorganization of axonal microstructure, promoting oligodendrogenesis and activating PI3K/AKT/GSK-3/CRMP-2 signaling, thereby improving poststroke gait impairments.

Downregulated expression of IL­28RA is involved in the pathogenesis of pancreatic ductal adenocarcinoma.

Pancreatic cancer ranks seventh in terms of cancer­related mortality in men and women worldwide, where the most common subtype is pancreatic ductal adenocarcinoma (PDAC). To date, the pathogenesis of PDAC remains incompletely understood and the prognosis of PDAC is poor. In the present study, the expression of interleukin­28 receptor α subunit (IL­28RA) in PDAC tissues was detected using immunofluorescence staining and western blotting. IL­28RA recombinant plasmids and control pCMV6­entrymammalian expression plasmid, short hairpin (sh)IL­28RA plasmids and control pRS scrambled shRNA vector purchased were used to produce stably transfected PANC­1 cells overexpressing IL­28RA or with IL­28RA expression knocked down. MTS assays were used to measure cell viability and wound healing assay was used to assess the cell migratory ability in vitro. Flow cytometry analysis was performed to determine the proportion of cells in each phase of the cell cycle whereas total protein and phosphorylated protein levels were assessed using western blotting. Xenograft models of subcutaneous tumors were established by injecting PANC­1 cells hypodermically into nude mice to investigate the effect of IL­28RA on tumorigenesis and tumor growth. The results showed that the expression of IL­28RA in PDAC tissues was lower compared with that in normal tissues. IL­28RA overexpression in vitro resulted in the activation of the IL­28RA pathway, which reduced cell viability and decreased the proportion of cells in the G2/M phase by reducing cyclin B1 expression. In addition, IL­28RA overexpression inhibited migration of PDAC cells. By contrast, an increased proportion of cells in G2/M phase, upregulated cyclin B1 expression and enhanced cell viability and migratory ability along with inhibition of the IL­28RA pathway were observed in PANC­1 cells following IL­28RA knockdown. The inhibitory effect of IL­28RA was observed by tumor size in a nude mouse model induced by PANC­1 cells with stable IL­28RA overexpression or knockdown. The tumor size induced by PANC­1 cells with stable IL­28RA overexpression were smaller, whilst larger tumors induced by PANC­1 cells were observed following stable IL­28RA knockdown, when compared to control. Further studies showed that the effect of IL­28RA on PDAC cells was exerted by regulating the phosphorylation levels of STAT1 and AKT. In conclusion, lower IL­28RA expression may contribute to the pathogenesis of PDAC, where results from the present may provide further insights into the progression of PDAC, in addition to highlighting potentially novel therapeutic targets for this disease.

Trillium tschonoskii rhizomes' saponins induces oligodendrogenesis and axonal reorganization for ischemic stroke recovery in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Trillium tschonoskii Maxim. is one of traditional Chinese medical herbs that has been utilized to treat brain damages and cephalalgia. The neuroprotective effect of total saponins from Trillium tschonoskii rhizome (TSTT) has been demonstrated efficacy in rats following ischemia. However, the axonal remodeling effect of TSTT and the detailed mechanisms after ischemic stroke have not been investigated. AIM OF THE STUDY: We aimed to estimate therapeutic role of TSTT in axonal remodeling using magnetic resonance imaging (MRI) technique, and explored possible mechanisms underlying this process followed by histological assays in ischemic rats. METHODS: Male Sprague-Dawley (SD) rats underwent permanently focal cerebral ischemia induced by occluding right permanent middle cerebral artery. TSTT was intragastrically administrated 6 h after surgery and once daily for consecutive 15 days. Neurological function was assessed by the motor deficit score and beam walking test. T2 relaxation mapping and diffusion tensor imaging (DTI) were applied for detecting cerebral tissues damages and microstructural integrity of axons. Luxol fast blue (LFB) and transmission electron microscope (TEM) were performed to evaluate histopathology in myelinated axons. Double immunofluorescent staining was conducted to assess oligodendrogenesis. Furthermore, the protein expressions regarding to axonal remodeling related signaling pathways were detected by Western blot assays. RESULTS: TSTT treatment (65, 33 mg/kg) markedly improved motor function after ischemic stroke. T2 mapping MRI demonstrated that TSTT decreased lesion volumes, and DTI further confirmed that TSTT preserved axonal microstructure of the sensorimotor cortex and internal capsule. Meanwhile, diffusion tensor tractography (DTT) showed that TSTT elevated correspondent density and length of fiber in the internal capsule. These MRI measurements were confirmed by histological examinations. Notably, TSTT significantly increased Ki67/NG2, Ki67/CNPase double-labeled cells along the boundary zone of ischemic cortex and striatum. Meanwhile, TSTT treatment up-regulated the phosphorylation level of Ser 9 in GSK-3ß, and down-regulated phosphorylated ß-catenin and CRMP-2 expression. CONCLUSION: Taken together, our findings indicated that TSTT (65, 33 mg/kg) enhanced post-stroke functional recovery, amplified endogenous oligodendrogenesis and promoted axonal regeneration. The beneficial role of TSTT might be correlated with GSK-3/ß-catenin/CRMP-2 modulating axonal reorganization after ischemic stroke.

Distribution of Bacterial Endophytes in the Non-lesion Tissues of Potato and Their Response to Potato Common Scab.

The response of plant endophytes to disease within infected tissues has been well demonstrated, but the corresponding response of endophytes in non-lesion tissues remains unclear. Here, we studied the composition and distribution of bacterial endophytes in potato roots (RE), stems (SE), and tubers (TE), and explored the response of endophytes in non-lesion tissues to potato common scab (PCS), which is a soil-borne disease caused by pathogenic Streptomyces and results in serious losses to the global economy every year. Via high-throughput sequencing, it was seen that the composition of endophytes in roots, stems, and tubers had significant differences (P < 0.05) and the distribution of the bacterial communities illustrated a gradient from soil to root to tuber/stem. PCS significantly reduced bacterial endophytes α-diversity indexes, including ACE and the number of observed operational taxonomic units (OTUs), of RE without significantly reducing the indexes of SE and TE. No significant effect on the composition of endophytes were caused by PCS in roots, tubers, or stems between high PCS severity (H) and low PCS severity (L) infections at the community level, but PCS did have a substantial impact on the relative abundance of several specific endophytes. Rhizobium and Sphingopyxis were significantly enriched in root endophytes with low PCS severity (REL); Delftia and Ochrobactrum were significantly enriched in stem endophytes with low PCS severity (SEL); Pedobacter, Delftia, and Asticcacaulis were significantly enriched in tuber endophytes with high PCS severity (TEH). OTU62, a potential PCS pathogen in this study, was capable of colonizing potato tubers, roots, and stems with few or no symptoms present. Co-occurrence networks showed that the number of correlations to OTU62 was higher than average in these three tissue types, suggesting the importance of OTU62 in endophytic communities. This study clarified the distribution and composition of potato endophytes in tubers, roots, and stems, and demonstrated the response of endophytes in non-lesion tissues to PCS.

Changes of gut microbiota in pregnant sows induced by 5-Aminolevulinic acid.

Sow health is related to farm productivity and sustainability, but the increased resistance of bacteria to antibiotics in the pig industry has led to a decline in resistance to disease and environmental pollution. 5-Aminolevulinic acid (5-ALA) is considered a feed additive to replace antibiotics, but the effect of 5-ALA on gut microbiota has not been studied. In this study, we fed 12 healthy Landrace × Large White two-line hybrid sows with different concentrations of 5-ALA; blood and fecal samples were obtained after 110 days of pregnancy, and 16S rRNA amplicon sequencing was performed. The alpha diversity of the gut microbiota in sows was not significant among the sows fed different concentrations of 5-ALA. PCoA revealed a significant (P < 0.05) difference in the gut microbiota composition with different 5-ALA groups. LEfSe revealed that 5-ALA increased relative abundance of Streptococcus, while Myroides was enriched in CK group. Functional prediction of Tax4Fun showed that different concentrations of 5-ALA significantly (P < 0.05) increased the mean relative abundance of KEGG pathways involved in core microbiota cellular processes, environmental information processing, and genetic information processing. In summary, 5-ALA changed bacterial community composition of gut microbiota, reduced colonization of some pathogenes and increased the relative abundance of some probiotics. These results provide a theoretical basis for the healthy breeding of pigs.

Transcriptomics Reveals Host-Dependent Differences of Polysaccharides Biosynthesis in Cynomorium songaricum.

Cynomorium songaricum is a root holoparasitic herb that is mainly hosted in the roots of Nitraria roborowskii and Nitraria sibirica distributed in the arid desert and saline-alkaline regions. The stem of C. songaricum is widely used as a traditional Chinese medicine and applied in anti-viral, anti-obesity and anti-diabetes, which largely rely on the bioactive components including: polysaccharides, flavonoids and triterpenes. Although the differences in growth characteristics of C. songaricum between N. roborowskii and N. sibirica have been reported, the difference of the two hosts on growth and polysaccharides biosynthesis in C. songaricum as well as regulation mechanism are not limited. Here, the physiological characteristics and transcriptome of C. songaricum host in N. roborowskii (CR) and N. sibirica (CS) were conducted. The results showed that the fresh weight, soluble sugar content and antioxidant capacity on a per stem basis exhibited a 3.3-, 3.0- and 2.1-fold increase in CR compared to CS. A total of 16,921 differentially expressed genes (DEGs) were observed in CR versus CS, with 2573 characterized genes, 1725 up-regulated and 848 down-regulated. Based on biological functions, 50 DEGs were associated with polysaccharides and starch metabolism as well as their transport. The expression levels of the selected 37 genes were validated by qRT-PCR and almost consistent with their Reads Per kb per Million values. These findings would provide useful references for improving the yield and quality of C. songaricum.

Cholesterol partially rescues the inhibition effect of pravastatin on keratinocytes proliferation by regulating cell cycle relative proteins through AKT and ERK pathway.

Mevalonate pathway plays a key role in skin physiological process in human. Recently, it has been reported that mutation of some genes in the mevalonate pathway cause disseminated superficial actinic porokeratosis (DSAP). But the pathogenesis is still unknown. Pravastatin (PRA), one of HMG-CoA reductase (HMGCR) inhibitors, has been found to inhibit cells proliferation, including keratinocytes (KCs). In this study, we use PRA to block the mevalonate pathway in KCs with or without the down-stream intermediate products replenishment. The results demonstrated that PRA strongly inhibited proliferation of KCs and caused the G0 /G1 arrest. When some down-stream intermediate products were added, only cholesterol (CH) could partially rescue the inhibition effect of PRA on KCs proliferation, but not other products, such as mevalonic acid, farnesyl pyrophosphate or geranylgeranyl pyrophosphate. Mechanistic analysis revealed that PRA down-regulated expression of cyclin B1, but up-regulated cyclin E and p21 expression. And PRA increased the phosphorylation level of Protein Kinase B (AKT) but decreased the phosphorylation level of Extracellular Signal Regulated Kinase (ERK1/2). CH could attenuate the elevated cyclin E and activated AKT induced by PRA. These results indicated that CH could rescue the proliferation inhibition of KCs caused by PRA, which laid a foundation for elucidating the pathogenesis of DSAP clearly.