Calycosin promotes axon growth by inhibiting PTPRS and alleviates spinal cord injury.

Our former studies have identified the alleviating effect of Calycosin (CA) on spinal cord injury (SCI). In this study, our purpose is to explore the influence of CA on SCI from the perspective of promoting axon growth. The SCI animal model was constructed by spinal cord compression, wherein rat primary cortex neuronal isolation was performed, and the axonal growth restriction cell model was established via chondroitin sulfate proteoglycan (CSPG) treatment. The expressions of axon regeneration markers were measured via immunofluorescent staining and western blot, and the direct target of CA was examined using silver staining. Finally, the expression of the protein tyrosine phosphatase receptor type S (PTPRS) was assessed using western blot. CA treatment increased neuronal process outgrowth and the expressions of axon regeneration markers, such as neurofilament H (NF-H), vesicular glutamate transporter 1 (vGlut1), and synaptophysin (Syn) in both SCI model rats and CSPG-treated primary cortical neurons, and PTPRS levels were elevated after SCI induction. In addition, PTPRS was the direct target of CA, and according to in vivo findings, exposure to CA reduced the PTPRS content. Furthermore, PTPRS overexpression inhibited CA's enhancement of axon regeneration marker content and neuronal axon lengths. CA improves SCI by increasing axon development through regulating PTPRS expression.

The application of functional imaging in visual field defects: a brief review.

Visual field defects (VFDs) represent a prevalent complication stemming from neurological and ophthalmic conditions. A range of factors, including tumors, brain surgery, glaucoma, and other disorders, can induce varying degrees of VFDs, significantly impacting patients' quality of life. Over recent decades, functional imaging has emerged as a pivotal field, employing imaging technology to illustrate functional changes within tissues and organs. As functional imaging continues to advance, its integration into various clinical aspects of VFDs has substantially enhanced the diagnostic, therapeutic, and management capabilities of healthcare professionals. Notably, prominent imaging techniques such as DTI, OCT, and MRI have garnered widespread adoption, yet they possess unique applications and considerations. This comprehensive review aims to meticulously examine the application and evolution of functional imaging in the context of VFDs. Our objective is to furnish neurologists and ophthalmologists with a systematic and comprehensive comprehension of this critical subject matter.

Soft Robotic Finger with Energy-Coupled Quadrastability.

The performance of the human finger is a significant inspiration for designing soft robotic fingers that can achieve high speed and high force or perform delicate and complex tasks. Existing soft grippers and actuators can be excellent in specific capabilities. However, it is still challenging for them to meet an all-around performance as the human finger, characterized by high actuation speed, wide grasping range, sensing ability, and gentle and high-load grasping capability. The proposed tendon pulley quadrastable (TPQ) finger has combined these qualities in the conducted gripping tasks. A pair of elastic tendons is utilized as the sole energy reservoir to create a novel energy distribution pattern: energy-coupled quadrastability. An energy model is built to analyze and predict the behaviors of the TPQ finger. Mechanical instability is utilized to enhance the actuation speed. The proposed soft lever mechanism endows the TPQ finger with sensing ability. The energy barrier adjusting plates control the energy barrier, adjusting the sensitivity of both active and passive actuation mechanisms. The transition of four stable states forms preplanned trajectories that are applied to create multiple grasping manners. Experiments show that it can respond to stimuli and finish a grasping task in merely 31 ms, and its payload can reach 33.25 kg. At the same time, it can also handle fragile objects such as a piece of rose and grasp a wide range of objects ranging from a thin nut (3.3 mm in height) or a thin card (0.76 mm thick) to a football (220 mm).

Rehabilitation training enhanced the therapeutic effect of calycosin on neurological function recovery of rats following spinal cord injury.

BACKGROUND: Calycosin (CA), a flavonoids component, has demonstrated potential neuroprotection effects by inhibiting oxidative stress in spinal cord injury (SCI) models. This study aims to investigate the impact of combined rehabilitation training (RT) and calycosin therapy on neurological function following SCI, primarily by assessing changes in motor function recovery, neuronal survival, neuronal oxidative stress levels, and neural proliferation, in order to provide novel insights for the treatment of SCI. MATERIALS AND METHODS: The SCI model was constructed by compressing the spinal cord using vascular clamps. Calycosin was injected intraperitoneally into the SCI model rats, and a group of 5 rats underwent RT. The motor function of rats after SCI was evaluated using the Basso Beattle Bresnaha (BBB) score and the inclined plate test. Histopathological changes were evaluated by NeuN immunohistochemistry, HE and Nissl staining. Apoptosis was detected by TUNEL staining. The antioxidant effect of combined treatment was assessed by measuring changes in oxidative stress markers after SCI. Western blot analysis was conducted to examine changes in Hsp90-Akt/ASK1-p38 pathway-related proteins. Finally, cell proliferation was detected by BrdU and Ki67 assays. RESULTS: RT significantly improved the BBB score and angle of incline promoted by calycosin, resulting in enhanced motor function recovery in rats with SCI. Combining rehabilitation training with calycosin has a positive effect on morphological recovery. Similarly, combined RT enhanced the Nissl and NeuN staining signals of spinal cord neurons increased by calycosin, thereby increasing the number of neurons. TUNEL staining results indicated that calycosin treatment reduced the apoptosis signal in SCI, and the addition of RT further reduced the apoptosis. Moreover, RT combined with calycosin reduced oxidative stress by increasing SOD and GSH levels, while decreasing MDA, NO, ROS, and LDH expressions compared to the calycosin alone. RT slightly enhanced the effect of calycosin in activating Hsp90 and Akt and inhibiting the activation of ASK1 and p38, leading to enhanced inhibition of oxidative stress by calycosin. Additionally, the proliferation indexes (Ki67 and BrdU) assays showed that calycosin treatment alone increased both, whereas the combination treatment further promoted cell proliferation. CONCLUSION: Our research findings demonstrate that rehabilitation training enhances the ability of calycosin to reduce oxidative stress, resulting in a decrease in neuronal apoptosis and an increase in proliferation, ultimately promoting neuronal survival.

Effect of adipose tissue-derived stem cells therapy on clinical response in patients with primary Sjogren's syndrome.

The purpose of this trial was to clinically assess the effect and safety of Adipose Tissue-derived Stem Cells (ADSCs) treatment on primary Sjogren's Syndrome (pSS). In this 6-month randomized, triple-blind, placebo-controlled clinical trial, pSS patients were randomly assigned to two groups. After demographic characteristics and clinical examination were achieved, local injection of ADSCs into bilateral glands was performed with patients in ADSCs group (n = 35) and placebo solution was used for another group (n = 39) at three time points. Patients were followed up at 1-, 3- and 6-month. At each visit, studies of clinical and laboratory outcomes, as well as subjective symptoms, were conducted. A total of 74 subjects who met the including criteria were allocated in two groups and eventually 64 subjects (86.5%) completed the treatments and the follow-up assessments. Secretion of salivary and lachrymal glands were significantly improved in 3-month (P < 0.05). A great improvement of European League Against Rheumatism Sjögren's Syndrome Disease Activity Index (ESSDAI) was found after ADSCs treatment with intergroup comparison from baseline to follow-up (P < 0.05). There is also a significant difference of European Alliance of Associations for Rheumatology SS Patient Reported Index (ESSPRI) between the two groups in the follow-up (P < 0.05). A significant abatement of IgG, IgM, C3, C4 and ESR between two groups was observed in part of follow-up time points (P < 0.05). The ADSCs therapy can provide relief of oral and eye's dryness in our trial in a short time and has potential improvement of subjective and systemic syndromes of pSS.

Formononetin Inhibits Microglial Inflammatory Response and Contributes to Spinal Cord Injury Repair by Targeting the EGFR/MAPK Pathway.

Zhenbao Pill contains many Chinese herbal medicinal ingredients and has been proven to have therapeutic effects on the repair of spinal cord injury (SCI). This study attempts to investigate the role of formononetin (FMN), an ingredient of Zhenbao Pill, in regulating neuroinflammation after SCI and the underlying mechanism. Primary microglia isolated from the spinal cord of newborn rats and human microglial clone 3 (HMC3) cells were stimulated with IL-1ß followed by FMN incubation. The cell viability and inflammatory cytokine levels were detected. The target of FMN was predicted and screened using databases. By silencing or overexpression of epidermal growth factor receptor (EGFR), the anti-neuroinflammatory effect of FMN was assessed in vitro. In vivo, FMN was intraperitoneally injected into rats after SCI followed by the neurological function and histopathology examination. The isolated microglia were in high purity, and the different concentrations of FMN incubation had no toxic effects on primary microglia and HMC3 cells. FMN reduced the inflammatory cytokine levels (TNF-α and IL-6) in a concentration-dependent manner. EGFR silencing or FMN incubation decreased p-EGFR and p-p38 levels and down-regulated inflammatory cytokine levels in IL-1ß-stimulated cells or supernatants. Nevertheless, the effects of FMN on microglial inflammation were reversed by EGFR overexpression. In vivo, FMN treatment improved the neuromotor function, repaired tissue injury, and inhibited EGFR/p38MAPK phosphorylation. Formononetin inhibits microglial inflammatory response and contributes to SCI repair via the EGFR/p38MAPK signaling pathway.

Calycosin ameliorates spinal cord injury by targeting Hsp90 to inhibit oxidative stress and apoptosis of nerve cells.

BACKGROUND: Zhenbao pill is effective in protecting against spinal cord injury (SCI). We attempt to explore the characteristics of calycosin (a main monomer of Zhenbao pill) in SCI and its relative mechanism. METHODS: The target of calycosin was screened using pharmacological network analysis. The SCI cell model was constructed using hydrogen peroxide (H2O2), and the animal model was developed by compressing spinal cord with a vascular clamp. Flow cytometry was conducted to test reactive oxygen species (ROS) levels and cell apoptosis. Detection of malondialdehyde (MDA) activity and Superoxide dismutase (SOD) activity were performed using relative kits. Heat shock protein 90 (HSP90) was examined using western blot and quantitative real-time PCR. Motor function tests were carried out. The hematoxylin-eosin and Nissl staining were conducted. RESULTS: In SCI models, ROS, MDA, and cell apoptosis were elevated, SOD and HSP90 levels were restrained, while calycosin addition reversed the above results. Besides, calycosin application or HSP90 overexpression enhanced phosphorylation of protein kinase B (Akt) but weakened that of apoptosis signal-regulating kinase 1 (ASK1) and p38, while HSP90 inhibitor 17-AAG treatment restrained the above results. Meanwhile, the injection of calycosin improved the motor function in SCI model rats. Furthermore, the pathologic results also clarified the positive effect of calycosin on SCI. CONCLUSION: HSP90 was lowly expressed in SCI models. Calycosin alleviated SCI by promoting HSP90 up-regulation and inhibiting oxidative stress and apoptosis of nerve cells.

Induction of alternative NF-κB within TAg-induced basal mammary tumors in activation-resistant inhibitor of κ-B kinase (IKKα) mutant mice.

BACKGROUND: The alternative NF-κB pathway is activated by the NF-κB-inducing kinase (NIK) mediated phosphorylation of the inhibitor of κ-B kinase α (IKKα). IKKα then phosphorylates p100/NFKB2 to result in its processing to the active p52 subunit. Evidence suggests that basal breast cancers originate within a subpopulation of luminal progenitor cells which is expanded by signaling to IKKα. OBJECTIVE: To determine the role of IKKα in the development of basal tumors. METHODS: Kinase dead IkkαAA/AA mice were crossed with the C3(1)-TAg mouse model of basal mammary cancer. Tumor growth and tumor numbers in WT and IkkαAA/AA mice were assessed and immunopathology, p52 expression and stem/progenitor 3D colony forming assays were performed. Nik-/- mammary glands were isolated and mammary colonies were characterized. RESULTS: While tumor growth was slower than in WT mice, IkkαAA/AA tumor numbers and pathology were indistinguishable from WT tumors. Both WT and IkkαAA/AA tumors expressed p52 except those IkkαAA/AA tumors where NIK, IKKαAA/AA and ErbB2 were undetectable. Colonies formed by WT and IkkαAA/AA mammary cells were nearly all luminal/acinar however, colony numbers and sizes derived from IkkαAA/AA cells were reduced. In contrast to IkkαAA/AA mice, virgin Nik-/- mammary glands were poorly developed and colonies were primarily derived from undifferentiated bipotent progenitor cells. CONCLUSIONS: C3(1)-TAg induced mammary tumors express p100/p52 even without functional IKKα. Therefore the development of basal-like mammary cancer does not strictly rely on IKKα activation. Signal-induced stabilization of NIK may be sufficient to mediate processing of p100NFKB2 which can then support basal-like mammary tumor formation. Lastly, in contrast to the pregnancy specific role of IKKα in lobuloalveogenesis, NIK is obligatory for normal mammary gland development.

Multiple roles for Bcl-3 in mammary gland branching, stromal collagen invasion, involution and tumor pathology.

BACKGROUND: The Bcl-3 protein is an atypical member of the inhibitor of -κB family that has dual roles as a transcriptional repressor and a coactivator for dimers of NF-κB p50 and p52. Bcl-3 is expressed in mammary adenocarcinomas and can promote tumorigenesis and survival signaling and has a key role in tumor metastasis. In this study, we have investigated the role of Bcl-3 in the normal mammary gland and impact on tumor pathology. METHODS: We utilized bcl-3-/- mice to study mammary gland structure in virgins and during gestation, lactation and early involution. Expression of involution-associated genes and proteins and putative Bcl-3 target genes was examined by qRT-PCR and immunoblot analysis. Cell autonomous branching morphogenesis and collagen I invasion properties of bcl-3-/- organoids were tested in 3D hydrogel cultures. The role of Bcl-3 in tumorigenesis and tumor pathology was also assessed using a stochastic carcinogen-induced mammary tumor model. RESULTS: Bcl-3-/- mammary glands demonstrated reduced branching complexity in virgin and pregnant mice. This defect was recapitulated in vitro where significant defects in bud formation were observed in bcl-3-/- mammary organoid cultures. Bcl-3-/- organoids showed a striking defect in protrusive collective fibrillary collagen I invasion associated with reduced expression of Fzd1 and Twist2. Virgin and pregnant bcl-3-/- glands showed increased apoptosis and rapid increases in lysosomal cell death and apoptosis after forced weaning compared to WT mice. Bcl-2 and Id3 are strongly induced in WT but not bcl-3-/- glands in early involution. Tumors in WT mice were predominately adenocarcinomas with NF-κB activation, while bcl-3-/- lesions were largely squamous lacking NF-κB and with low Bcl-2 expression. CONCLUSIONS: Collectively, our results demonstrate that Bcl-3 has a key function in mammary gland branching morphogenesis, in part by regulation of genes involved in extracellular matrix invasion. Markedly reduced levels of pro-survival proteins expression in bcl-3 null compared to WT glands 24 h post-weaning indicate that Bcl-3 has a role in moderating the rate of early phase involution. Lastly, a reduced incidence of bcl-3-/- mammary adenocarcinomas versus squamous lesions indicates that Bcl-3 supports the progression of epithelial but not metaplastic cancers.

Recent Advances in the Role of Nuclear Factor Erythroid-2-Related Factor 2 in Spinal Cord Injury: Regulatory Mechanisms and Therapeutic Options.

Nuclear factor erythroid-2-related factor 2 (Nrf2) is a pleiotropic transcription factor, and it has been documented that it can induce defense mechanisms both oxidative stress and inflammatory injury. At present, more and more evidences show that the Nrf2 signaling pathway is a key pharmacological target for the treatment of spinal cord injury (SCI), and activating the Nrf2 signaling pathway can effectively treat the inflammatory injury and oxidative stress after SCI. This article firstly introduces the biological studies of the Nrf2 pathway. Meanwhile, it is more powerful to explain that activating the Nrf2 signaling pathway can effectively treat SCI by deeply exploring the relationship between Nrf2 and oxidative stress, inflammatory injury, and SCI. In addition, several potential drugs for the treatment of SCI by promoting Nrf2 activation and Nrf2-dependent gene expression are reviewed. And some other treatment strategies of SCI by modulating the Nrf2 pathway are also summarized. It will provide new ideas and directions for the treatment of SCI.

IL-27 signaling negatively regulates FcɛRI-mediated mast cell activation and allergic response.

IL-27 is a member of the IL-12 family, exerting both anti- and pro-inflammatory activity in a cell-dependent and disease context-specific manner. Antigen-mediated cross-linking of IgE on mast cells triggers a signaling cascade that results in mast cell degranulation and proinflammatory cytokine production, which are key effectors in allergic reactions. Here, we show that the activation of mast cells is negatively regulated by IL-27 signaling. We found that mice lacking IL-27Rα (WSX-1) displayed increased sensitivity to IgE-mediated skin allergic response and chronic airway inflammation. The bone marrow-derived mast cells (BMMCs) of IL-27Rα-deficient mouse showed greater high-affinity receptor Fc epsilon RI (FcεRI)-mediated activation with significantly enhanced degranulation and cytokine production. Mechanistically, the dysregulated signaling in IL-27Rα-/- mast cells is associated with increased activation of Grb2-PLC-γ1-SLP-76, PI3K/Akt/IκBα signaling and decreased phosphorylation level of SH2 domain-containing protein phosphatase1 (SHP1). Furthermore, IL-27 treatment could inhibit mast cell activation directly, and retrovirus-based IL-27 expression in lung attenuated the airway inflammation in mice. Collectively, our findings reveal that IL-27 signaling negatively regulates mast cell activation and its mediated allergic response.

Analysis of expression profiles of long noncoding RNAs and mRNAs in brains of mice infected by rabies virus by RNA sequencing.

Rabies, caused by rabies virus (RABV), is still the deadliest infectious disease. Mechanism of host immune response upon RABV infection is not yet fully understood. Accumulating evidences suggest that long noncoding RNAs (lncRNAs) plays key roles in host antiviral responses. However, expression profile and function of lncRNAs in RABV infection remain unclear. In the present study, expression profile of lncRNAs and mRNAs profiles were investigated in RABV-infected brain tissues of mice by RNA sequencing. A total of 140 lncRNAs and 3,807 mRNAs were differentially expressed in RABV-infected animals. The functional annotation and enrichment analysis using Gene Oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that differentially expressed transcripts were predominantly involved in signaling pathways related to host immune response. The expression profiles of the selected lncRNAs in brains of mice during RABV infections were verified by quantitative real time polymerase chain reaction (qRT-PCR). To our knowledge, this is the first report to profile the lncRNA expression in RABV infected mice. Our findings provide insights into understanding the role of lncRNAs in host immune response against RABV infection.

Inhibition of rabies virus replication by interferon-stimulated gene 15 and its activating enzyme UBA7.

It was reported that ISG15 and its activating enzyme UBA7 have antiviral functions. However, there is no study to demonstrate whether ISG15 and UBA7 have anti-rabies virus function. In the current study, In vivo and in vitro anti-rabies virus function of ISG15 and UBA7 were investigated using RNAi technology. The results showed that shRNA knock-down of expression of ISG15 and UBA7 increased the titers of RABV in neuroblastoma cell line NA and microglial cell line BV-2 cells and shRNA knockdown of ISG15 conjugation alleviates the IFN-induced inhibition of RABV gene expression in vitro. Lentiviral vector mediated-shRNA knock-down of expression of ISG15 and UBA7 increased the titers of RABV in mouse brains and decreased the survivorship of mice. The study showed that ISG15 and UBA7 inhibit RABV replication in vitro and in vivo. To our knowledge, we for the first time documented the anti-RABV function of ISG15 and UBA7, which may provide a means of understanding the pathogenesis of rabies and improving therapeutic methods.

Development and characterization of a novel long-acting recombinant follicle stimulating hormone agonist by fusing Fc to an FSH-ß subunit.

STUDY QUESTION: Does a novel long-acting recombinant human FSH, KN015, a heterodimer composed of FSHα and FSHß-Fc/Fc, offer a potential FSH alternative? SUMMARY ANSWER: KN015 had in vitro activity and superior in vivo bioactivity than recombinant human FSH (rhFSH), suggesting KN015 could serve as a potential FSH agonist for clinical therapy. WHAT IS KNOWN ALREADY: rhFSH has very short half-life so that repeat injections are needed, resulting in discomfort and inconvenience for patients. The longest-acting rhFSH available in clinics is corifollitropin alpha (FSH-CTP), but its half-life is not long enough to sustain the whole therapy period, and additional injections of rhFSH are needed. STUDY DESIGN, SIZE, DURATION: Plasmids containing FSHα, FSHß-Fc and Fc cDNA were transfected into Chinese hamster ovary (CHO) cells for KN015 production. The pharmacokinetics of KN015 was investigated in 6-week-old SD rats (n = 6/group) and healthy Cynomolgus monkeys in two different dose groups (n = 2/group). A series of experiments were designed for in vitro and in vivo characterization of the bioactivity of KN015 relative to rhFSH. PARTICIPANTS/MATERIALS, SETTING, METHODS: The purity and molecular weight of KN015 were determined by reducing and non-reducing SDS-PAGE. To measure KN015 half-life, sera were collected at increasing time points and the remaining FSH concentration was measured by enzyme-linked immunosorbent assay. To assess the bioactivity of KN015 versus rhFSH in vitro, firstly cAMP production was assessed in CHO cells expressing FSH receptor (FSHR) with the treatment of Fc/Fc, rhFSH or KN015 at eight different doses (0.03, 0.09, 0.28, 0.83, 2.5, 7.5, 22.5, 67.5 nM), and secondly cumulus oocyte complexes (COCs; n = 20/group) of ICR mice (primed-PMSG 44 h before sacrificed) were collected and cultured in medium containing 1.25 pM Fc/Fc, rhFSH or KN015 at 37°C and then germinal vesicle breakdown (GVBD) and COC expansion were observed at 4 and 16 h, respectively. The in vivo activity of KN015 was compared with rhFSH by ovary weight gain and ovulation assays. In the former, ovary weight gains in 21-day-old female SD rats, after a single subcutaneous injection of KN015, were compared with those after several injections of rhFSH over a range of doses (n = 8/group). Sera were harvested for estradiol (E2) analysis, and the ovaries were processed for hematoxylin and eosin (HE) staining, immunohistochemistry (IHC), TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labeling (TUNEL), RT-PCR and western blot. In the latter, 26-day-old female SD rats (n = 8/group) were injected with different doses of KN015 or rhFSH, and were sacrificed at 24 h after an injection of hCG (20 IU/rat). Moreover, the molecular responses stimulated by KN015 or rhFSH in the ovary were also analyzed through detecting expression of the FSH target genes (Cyp19a1, Fshr and Lhcgr) and phosphatidylinositide 3-kinase (PI3K) pathway activation. MAIN RESULTS AND THE ROLE OF CHANCE: KN015 has a molecular weight of 82 kD and its half-life is 84 h in SD rats (10-fold longer than that of rhFSH) and 215 h in Cynomolgus monkeys. The EC50 value of the cAMP induction in CHO cells (KN015 versus rhFSH, 1.84 versus 0.87 nM), COC expansion and oocyte maturation assays showed KN015 had approximately half of rhFSH's activity in vitro. A single dose of KN015 (1.5 pmol/rat, 166.1 ± 19.7 mg, P < 0.01) stimulated significantly larger ovary weight gain than several injections of rhFSH (1.5 pmol/rat, 59.3 ± 28.1 mg, P < 0.01). The serum E2 level in the KN015 group was significantly higher than that in rhFSH group. The number of oocytes obtained by ovulation induction was comparable with or higher in the KN015 group than in the rhFSH group. KN015 was more effective than rhFSH in inducing FSH target genes (Cyp19a1, Fshr, Lhcgr) or activating the PI3K pathway in vivo. Moreover, a single injection of KN015 promoted granulosa cell proliferation and prevented follicle atresia to the same extent as several injections of rhFSH. LIMITATIONS, REASONS FOR CAUTION: All assays in this study were operated only in animals and clinical trials are needed to confirm they can be extrapolated to humans. WIDER IMPLICATIONS OF THE FINDINGS: KN015 is a valuable alternative to FSH and may have great potential for therapeutic applications. STUDY FUNDING/COMPETING INTERESTS: This study was supported by National Basic Research Program of China (2011|CB944504, 2012CB944403) and National Natural Science Foundation of China (81172473, 31371449). The authors have no conflicts of interest to declare.