9za plays cytotoxic and proapoptotic roles and induces cytoprotective autophagy through the PDK1/Akt/mTOR axis in non-small-cell lung cancer.

Non-small-cell lung cancer (NSCLC) is an aggressive subtype of pulmonary carcinomas with high mortality. However, chemotherapy drug resistance and high recurrence rates hinder the curative effect of platinum-based first-line chemotherapy, which makes it urgent to develop new antitumor drugs for NSCLC. 9za, a new candidate drug synthesized by our research group, has been verified with potent antilung cancer activity in preliminary experiments. However, the underlying molecular mechanism of 9za remains largely vague. This work revealed that 9za could play important cytotoxic and proapoptotic roles in NSCLC cells. Moreover, 9za could induce autophagy and promote autophagy flux. Interestingly, the cytotoxic and proapoptotic roles were significantly dependent on 9za-induced cytoprotective autophagy. That is, the coadministration of 9za with an autophagy inhibitor such as chloroquine or 3-methyladenine exhibited increased cytotoxic and proapoptotic effects compared with 9za treatment alone. In addition, 9za exposure suppressed the phosphorylation of phosphoinositide-dependent protein kinase 1 (PDK1), protein kinase B (Akt), mammalian targets of rapamycin (mTOR), p70 S6 kinase, and 4E binding protein 1 by a dose-dependent way, manifesting that the Akt/mTOR axis was implicated in 9za-induced autophagy. In addition, the overexpression of PDK1 resulted in increased phosphorylation of PDK1 and Akt and blocking of 9za-mediated autophagy. These data showed that the PDK1/Akt/mTOR pathway was involved in 9za-induced autophagy. Hence, this work provides a theoretical basis for exploiting 9za as a new antilung cancer candidate drug and hints that the combination of 9za with an autophagy inhibitor is a feasible alternative approach for the therapy of NSCLC.

Down-regulation of IFITM1 and its growth inhibitory role in cervical squamous cell carcinoma.

BACKGROUND: Cervical cancer is a major cause of death in women worldwide. Interferon-induced transmembrane protein 1 (IFITM1) is involved in antivirus defense, cell adhesion, and carcinogenesis in different tissues. However, the role of IFITM1 gene in cervical squamous cell cancer is unclear. METHODS: To explore the role of IFITM1 in carcinogenesis of cervical cancer, we investigated the expression of IFITM1 gene in cervical squamous cell carcinoma. IFITM1 mRNA level was measured by real-time quantitative RT-PCR in cervical cancer tissues and their adjacent normal tissues. IFITM1 protein level was measured by immunohistochemistry. Methylation in the IFITM1 gene promoter was detected by methylation-specific PCR. We then transfected HeLa cells with IFITM1 expression vector or control vector. IFITM1 expression was examined; cell migration and invasion were analyzed by wound healing assay and matrigel-coated transwell migration assays, respectively. HeLa cell proliferation was measured by cell counting kit-8 assay and cell cycle analysis. Cell apoptosis was analyzed by Annexin V/propidium iodide double staining assay. RESULTS: The difference in IFITM1 protein expression between samples from chronic cervicitis and cervical carcinoma was statistically significant (P < 0.01). Ki-67 and PCNA protein expression levels were significantly higher in cervical cancer tissues than in their corresponding cervicitis tissues (P < 0.05 and P < 0.001, respectively). IFITM1 mRNA level was significantly lower in cervical cancer tissues than in normal cervical tissues (P < 0.05). Methylation of the IFITM1 gene promoter was significantly higher in cervical cancer than in normal cervical tissues (P < 0.05). Transfection of the IFITM1 pcDNA3.1 construct decreased cell migration and invasion of HeLa cells, inhibited cell proliferation, and increased cell apoptosis. CONCLUSION: IFITM1 gene expression may reduce the proliferation, migration, and invasion of cervical squamous cancer cells.

Experimental study on the effect and mechanism of adipose stem cell-derived exosomes combined with botulinum toxin A on skin trauma in rats.

BACKGROUND: Adipose stem cell-derived exosomes (ADSC-EXO) and botulinum toxin type A (BTX-A) individually showed a therapeutic effect on skin wound repair. AIMS: This study investigated their synergistic effect on promoting skin wound healing in vitro and in vivo and the underlying molecular events. METHODS: ADSCs were isolated from Sprague-Dawley (SD) rats to obtain ADSC-EXO by ultrafiltration and ultracentrifugation and were confirmed using nanoparticle tracking analysis and transmission electron microscopy. Human skin fibroblasts (HSF) were cultured and treated with or without ADSC-EXO, BTX-A, or their combination. Changes in cell phenotypes and protein expression were analyzed using different in vitro assays, and a rat skin wound model was used to assess their in vivo effects. RESULTS: The isolated ADSC-EXO from primarily cultured ADSCs had a circular vesicle shape with a 30-180 nm diameter. Treatment of HSF with ADSC-EXO and/or BTX-A significantly accelerated HSF migration in vitro and skin wound healing in a rat model. Moreover, ADSC-EXO plus BTX-A treatment dramatically induced VEGFA expression but reduced COL III and COL I levels in vivo. ADSC-EXO and/or BTX-A treatment significantly upregulated TGF-ß3 expression on Day 16 after surgery but downregulated TGF-ß1 expression, suggesting that ADSC-EXO plus BTX-A promoted skin wound healing and reduced inflammatory cell infiltration. CONCLUSIONS: The ADSC-EXO plus BTX-A treatment demonstrated a synergistic effect on skin wound healing through upregulation of VEGF expression and the TGF-ß3/TGF-ß1 and COL III/COL I ratio.

Collateral projections from the ventral tegmental area/substantia nigra pars compacta to the nucleus accumbens and insular cortex in the rat.

Midbrain dopaminergic (DAergic) regions including ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) are involved in diverse brain functions. Previous studies demonstrated that the VTA/SNc to nucleus accumbens (NAc) pathway is critical in reward and motivation. Moreover, DAergic innervations within the insular cortex (IC) are reported to play important roles in pain regulation. To investigate whether VTA/SNc sends collateral projections to NAc and IC, we injected retrograde tracer Fluoro-Gold (FG) into the NAc and Fluorescent retrograde tracer beads (RetroBeads) into the ipsilateral IC in rats. Then, to detect whether collateral projection neurons participate in neuropathic pain, parts of the rats received the spare nerve injury (SNI) surgery. The immunofluorescence staining results showed that FG, RetroBeads, and FG/RetroBeads double-labeled neurons were distributed in the VTA/SNc bilaterally with an ipsilateral predominance. The proportion of FG/RetroBeads double-labeled neurons to the total number of FG and RetroBeads-labeled neurons was 16.7% and 30.3%, respectively. About 90.3% of FG/RetroBeads double-labeled neurons showed DAergic neuron marker tyrosine hydroxylase (TH)-immunoreactive (IR), whereas, only 7.5% exhibited a subset of GABAergic inhibitory projection neuron marker parvalbumin (PV)-IR. One week after SNI, about 53.1% and 33.6% of FG- and RetroBeads-labeled neurons were FG/Fos- and RetroBeads/Fos-IR neurons, respectively. Finally, about 35.9% of the FG/RetroBeads double-labeled neurons showed Fos-IR. The present study indicates that parts of DAergic and PV-IR GABAergic neurons in the VTA/SNc send collateral projections to both NAc and IC, which are activated under SNI-induced neuropathic pain, and probably contribute to the regulation of nociception.

Exosomes combined with biosynthesized cellulose conduits improve peripheral nerve regeneration.

Peripheral nerve injury is one of the more common forms of peripheral nerve disorders, and the most severe type of peripheral nerve injury is a defect with a gap. Biosynthetic cellulose membrane (BCM) is a commonly used material for repair and ligation of nerve defects with gaps. Meanwhile, exosomes from mesenchymal stem cells can promote cell growth and proliferation. We envision combining exosomes with BCMs to leverage the advantages of both to promote repair of peripheral nerve injury. Prepared exosomes were added to BCMs to form exosome-loaded BCMs (EXO-BCM) that were used for nerve repair in a rat model of sciatic nerve defects with gaps. We evaluated the repair activity using a pawprint experiment, measurement and statistical analyses of sciatica function index and thermal latency of paw withdrawal, and quantitation of the number and diameter of regenerated nerve fibers. Results indicated that EXO-BCM produced comprehensive and durable repair of peripheral nerve defects that were similar to those for autologous nerve transplantation, the gold standard for nerve defect repair. EXO-BCM is not predicted to cause donor site morbidity to the patient, in contrast to autologous nerve transplantation. Together these results indicate that an approach using EXO-BCM represents a promising alternative to autologous nerve transplantation, and could have broad applications for repair of nerve defects.

Prenatal expression of purinergic receptor P2X3 in human dorsal root ganglion.

The dorsal root ganglion (DRG) is consisted of neurons that relay multiple types of spinal sensory stimuli to the central nervous system. Several neuroactive molecules may be involved in sensory modulation especially pain processing at the DRG, including the purinergic receptor P2X3 and calcitonin-gene-related peptide (CGRP). P2X3 receptor has been considered a promising pharmaceutical target for the development of new pain medicine. Currently, litter is known about the expression of P2X3 in the human DRG. The present study characterized the localization of P2X3 in prenatal human DRG obtained from fetuses at 4-8 gestational months, by comparing to CGRP expression as well as binding pattern of isolectin-B4 (IB4), a marker of small DRG neurons presumably relevant to nociception. P2X3 immunoreactivity (IR) appeared in most neuron-like perikarya, with their numerical density reduced during the gestational period studied. P2X3 IR was co-labeled very commonly with IB4 binding and infrequently with CGRP IR and was not colocalized with IR for the gliocyte marker glutamine synthetase. Together, the data show an early and broad expression of P2X3 in prenatal human DRG neurons, pointing to a biological role of purinergic signaling during the development of spinal sensory system.

Vascular endothelial growth factor-165b protects the blood-retinal barrier from damage after acute high intraocular pressure in rats.

AIM: To elucidate the role of vascular endothelial growth factor-165b (VEGF-165b) in blood-retinal barrier (BRB) injury in the rat acute glaucoma model. METHODS: In this study, the rat acute high intraocular pressure (HIOP) model was established before and after intravitreous injection of anti-VEGF-165b antibody. The expression of VEGF-165b and zonula occludens-1 (ZO-1) in rat retina was detected by double immunofluorescence staining and Western blotting, and the breakdown of BRB was detected by Evans blue (EB) dye. RESULTS: The intact retina of rats expressed VEGF-165b and ZO-1 protein, which were mainly located in the retinal ganglion cell layer and the inner nuclear layer and were both co-expressed with vascular endothelial cell markers CD31. After acute HIOP, the expression of VEGF-165b was up-regulated; the expression of ZO-1 was down-regulated at 12h and then recovered at 3d; EB leakage increased, peaking at 12h. After intravitreous injection of anti-VEGF-165b antibody, the expression of VEGF-165b protein was no significantly changed; and the down-regulation of the expression of ZO-1 was more obvious; EB leakage became more serious, peaking at 3d. EB analysis also showed that EB leakage in the peripheral retina was greater than that in the central retina. CONCLUSION: The endogenous VEGF-165b protein may protect the BRB from acute HIOP by regulating the expression of ZO-1. The differential destruction of BRB after acute HIOP may be related to the selective loss of retinal ganglion cells.

Dynamic changes of inducible nitric oxide synthase expression in rat's retina and its role on blood-retinal barrier injury after acute high intraocular pressure.

AIM: To clarify the role of inducible nitric oxide synthase (iNOS) in blood-retinal barrier (BRB) injury after acute high intraocular pressure (IOP) in rats. METHODS: Forty-two Sprague-Dawley (SD) rats were randomized into 7 groups [control (Cont), 3, 6, 12, 24, 48, and 72h, n=6]. Except Cont group, other groups' retina tissue was obtained at corresponding time points after a model of acute high IOP have been established in rats. The expression of iNOS and tight junction protein zonula occludens (ZO)-1 was detected by Western blotting. Evans blue (EB; 3% ) was injected into the great saphenous vein to detect the leakage of EB by spectrophotometer. Nine rats were divided into Cont, 6h, 12h groups, the expression of iNOS was localized by immunofluorescence. In order to verify the role of iNOS in the damage to BRB, thirty-six rats were randomly divided into 4 groups [Cont, Cont+inhibitor (Inh), 6h and 6h+Inh, n=9]. After treatment with the iNOS-specific inhibitor 1400W, the expression of iNOS and ZO-1 and the leakage of BRB were detected again. RESULTS: The immunofluorescence results showed that the expression of iNOS was observed in the Cont group and 6h group, but not in the 12h group. iNOS was mainly expressed in the retinal nerve fiber layer, ganglion cell layer and inner nuclear layer and that it did not colocalize with the retinal ganglion cell marker NeuN but was co-expressed with the vascular endothelial cell marker CD31. Western blotting showed that in the early period (3h, 6h) after acute high IOP, the expression of iNOS was upregulated, then the down-regulation of iNOS were tested in the follow-up timing spots. ZO-1 expression showed a continuous down-regulation after 6h. The quantitative results for EB showed that the amount of EB leakage began to increase at 3h after acute high IOP. At 6h, the leakage of EB was lower, but at 12h, the leakage of EB was highest, after which it gradually recovered but remained higher than that in the Cont group. The expression of iNOS was down-regulated after 1400W treatment. ZO-1 expression was not significantly changed in the Cont+Inh group and the 6h group, and significantly down-regulated in the 6h+Inh group, and the leakage of EB was significantly increased after 1400W treatment. CONCLUSION: These results suggest that the upregulation of iNOS expression in the early stage after acute high IOP may have a protective effect on BRB injury.

Morphological features of endomorphin-2-immunoreactive ultrastructures in the dorsal root ganglion and spinal dorsal horn of the rat.

Endomorphin-2 (EM2)-immunoreactive (ir) fibers and terminals in the superficial laminae (lamina I and II) of the spinal dorsal horn (SDH) primarily come from neurons in the ipsilateral dorsal root ganglion (DRG), which are important for nociceptive information transmission and modulation. However, the morphological features of EM2-ir neurons and fibers in the DRG and terminals in the SDH under ultrastructural levels have not been completely revealed. The present study observed the distributions of EM2-ir neurons, fibers and terminals in the DRG and SDH and detected their ultrastructural features using immunoelectron microscopy. EM2-ir neurons in the DRG are primarily small or medium in size and account for 17.2% of all neurons in the DRG. EM2-ir large dense-core granule vesicles (LDCVs) are dispersed in the cytoplasm and fibers. Most of the central processes of DRG neurons were thin myelinated and unmyelinated fibers and contained a few EM2-ir LDCVs. An intensive string of EM2-ir fibers with beads and terminals were observed in the superficial laminae of the SDH, other than EM2-ir neurons. EM2-ir products were also detected sparsely in the fibers and terminals. The average diameter of terminals was 94.41 ± 18.13 nm. EM2-ir terminals formed different types of synapses, most of which were asymmetrical (91%). EM2-ir LDCVs colocalized primarily with spherical small clear vesicles in asymmetrical synapses and flat vesicles in symmetrical synapses. The average length of postsynaptic dense zones (PSDs) measured in the asymmetrical synapses was 317.00 ± 31.67 nm. These results indicate that EM2-containing structures are distributed in the cytoplasm of DRG neurons, the central processes and terminals in the SDH and provide morphological evidence for the antinociceptive effects of EM2 in the SDH.

Construction of Exosomes that Overexpress CD47 and Evaluation of Their Immune Escape.

Our general purpose was to provide a theoretical and practical foundation for the use of exosomes (EXOs) that have high levels of CD47 as stable and efficient drug carriers. Thus, we prepared EXOs from adipose tissue-derived mesenchymal stromal cells (ADMSCs) that had high levels of CD47 (EXOsCD47) and control EXOs (without CD47), and then compared their immune escape in vivo and their resistance to phagocytosis in vitro. Nanoflow cytometry was used to determine the CD47 level in these EXOs, and the amount of EXOsCD47 that remained in rat plasma at 3 h after intraperitoneal injection. Phagocytosis of the EXOs was also determined using in vitro rat macrophage bone marrow (RMA-BM) experiments. Our in vitro results showed that macrophages ingested significantly more control EXOs than EXOsCD47 (p < 0.01), with confirmation by ultra-high-definition laser confocal microscopy. Consistently, our in vivo results showed that rats had 1.377-fold better retention of EXOsCD47 than control EXOs (p < 0.01). These results confirmed that these engineered EXOsCD47 had improved immune escape. Our results therefore verified that EXOsCD47 had increased immune evasion relative to control EXOs, and have potential for use as drug carriers.

Untangle the Multi-Facet Functions of Auts2 as an Entry Point to Understand Neurodevelopmental Disorders.

Neurodevelopmental disorders are psychiatric diseases that are usually first diagnosed in infancy, childhood and adolescence. Autism spectrum disorder (ASD) is a neurodevelopmental disorder, characterized by core symptoms including impaired social communication, cognitive rigidity and repetitive behavior, accompanied by a wide range of comorbidities such as intellectual disability (ID) and dysmorphisms. While the cause remains largely unknown, genetic, epigenetic, and environmental factors are believed to contribute toward the onset of the disease. Autism Susceptibility Candidate 2 (Auts2) is a gene highly associated with ID and ASD. Therefore, understanding the function of Auts2 gene can provide a unique entry point to untangle the complex neuronal phenotypes of neurodevelpmental disorders. In this review, we discuss the recent discoveries regarding the molecular and cellular functions of Auts2. Auts2 was shown to be a key-regulator of transcriptional network and a mediator of epigenetic regulation in neurodevelopment, the latter potentially providing a link for the neuronal changes of ASD upon environmental risk-factor exposure. In addition, Auts2 could synchronize the balance between excitation and inhibition through regulating the number of excitatory synapses. Cytoplasmic Auts2 could join the fine-tuning of actin dynamics during neuronal migration and neuritogenesis. Furthermore, Auts2 was expressed in developing mouse and human brain regions such as the frontal cortex, dorsal thalamus, and hippocampus, which have been implicated in the impaired cognitive and social function of ASD. Taken together, a comprehensive understanding of Auts2 functions can give deep insights into the cause of the heterogenous manifestation of neurodevelopmental disorders such as ASD.

Expression of HGF/c-Met is dynamically regulated in the dorsal root ganglions and spinal cord of adult rats following sciatic nerve ligation.

Hepatocyte growth factor (HGF) and its receptor c-Met play pivotal roles in post-traumatic regeneration of the nervous system. However, following peripheral nerve injury, the role and regulation of the HGF/c-Met system is less clear. Therefore, using a sciatic nerve ligation (SNL) model, spatiotemporal changes in HGF and c-Met expression were detected in the dorsal root ganglions (DRGs) and lumbar spinal cords of adult rats. HGF expression following SNL was found to be significantly decreased in ipsilateral L4-L5 DRGs from day 3 to day 14, with the lowest levels of expression detected on days 5 and 7. In contrast, no significant change in HGF expression was detected in the lumbar spinal cords. c-Met expression in ipsilateral L4-L5 DRGs and within the ipsilateral dorsal horn was found to be significantly up-regulated following SNL, particularly from day 5 to day 14, with peak levels of expression detected on days 7 and 14. In contrast, c-Met levels following SNL consistently remained stable in the spinal ventral horn. These findings suggest that the HGF/c-Met system is spatiotemporally regulated by a unique pattern of signaling pathways induced by peripheral nerve injury, and these pathways have a role in promoting the survival of injured neurons, especially adult DRG sensory neurons.

[The protective effect of puerarin on Abeta(25-35)-induced PC12 cell injury].

OBJECTIVE: To study The protective effect of puerarin on Abeta(25-35)-induced PC12 cell injury. METHODS: PC12 cells were treated with puerarin for 0.5 h, then incubated with Abeta(25-35) (50 micromol/L) for 24 h to investigate the production of reactive oxygen species (ROS), mitochondrial membrane potential levels and Caspase-3 activation; The expressions of Bax, bcl-2 were measured by Western Blotting. RESULTS: Preincubation of the cell with puerarin could inhibit the ROS and increase mitochondrial membrane potential levels. Puerarin was also found to increase the Bcl-2/Bax ratio and reduce Caspase-3 activation. CONCLUSION: Puerarin may act as an intracellular ROS scavenger, and its antioxidant properties may protect against Abeta(25-35)-induced cell injury.

Design and evaluation of a biosynthesized cellulose drug releasing duraplasty.

Decompressive craniectomy (DC) is a standard surgical procedure performed on stroke patients in which a portion of a skull is removed and a duraplasty membrane is applied onto the brain. While DC can significantly reduce the risk of death, it does not reverse the stroke damage. In this study, a novel biosynthesized cellulose (BC)-based drug releasing duraplasty was developed and studied. The BC duraplasty fabrication process allowed readily incorporation of growth factors (GFs) in a sterile manner and control of physical and mechanical properties of the resulting duraplasty. Our results showed that BC duraplasty containing the highest amount of dry cellulose presented swelling ratio of 496 ± 27%, Young's modulus of 0.37 ± 0.02 MPa, ultimate tensile strength of 0.96 ± 0.02 MPa, while releasing GFs for over 10 days. In addition, neural stem/progenitor cell (NSPC) cultures demonstrated that the GFs released from the BC duraplasty promoted NSPC proliferation and differentiation in vitro. Finally, animal studies revealed that the BC duraplasty did not cause any inflammatory reactions after the DC procedure in vivo. In summary, this newly developed GF loaded BC membrane demonstrates a promising potential as drug releasing duraplasty, not only for stroke treatments but also for traumatic brain injuries and spinal cord injuries.

Butein attenuates the cytotoxic effects of LPS-stimulated microglia on the SH-SY5Y neuronal cell line.

Neuroinflammation is involved in brain aging and neuronal cell death in neurodegenerative diseases such as Alzheimer's disease (AD). Butein has been suggested to have anti-inflammatory, anti-apoptotic, and anti-cancer effects. However, few studies have been done to evaluate whether butein exerts protective effects on neurons, and the potential mechanism for this effect has not been studied. Here, we examined the effect of butein on SH-SY5Y neuroblastoma cells grown with conditioned medium from BV2 microglia cells that had been activated by lipopolysaccharide (LPS) as a neuroinflammation model. We found butein pretreatment significantly increased SH-SY5Y cell viability in a dose-dependent manner by inhibiting the apoptosis normally induced by microglia-conditioned medium. SH-SY5Y cells treated with microglia-conditioned medium showed upregulated ERK signaling pathway-related mRNA expression and protein phosphorylation, which was dose-dependently reversed by butein. Immunocytochemistry and Western blot results showed that BV2-LPS conditioned medium-induced Nuclear factor kappaB (NF-κB) transactivational activity in SH-SY5Y cells, but this was attenuated by butein treatment of the BV2 cells prior to their exposure to LPS. Collectively, our results indicate that butein effectively mitigates inflammatory injury caused by LPS-conditioned medium from microglia, possibly due to reductions in the transactivational activity of NF-κB p65 and ERK signaling pathway activation, and provide evidence for a neuroprotective role of butein through blocking negative consequences of microglial activation.

A novel dual MEK/PDK1 inhibitor 9za retards the cell cycle at G0/G1 phase and induces mitochondrial apoptosis in non-small cell lung cancer cells.

BACKGROUND: A novel dual MEK/PDK1 inhibitor named 9za has been synthesized by our research team. Preliminary study showed that 9za possessed potent cytotoxicity and proapoptosis in non-small cell lung cancer (NSCLC) cells. Nevertheless, the precise underlying mechanism is vague. METHODS: In this work, we adopted the MTT assay, the Cell Cycle Detection Kit, and the JC-1 staining assay to detect the cell viability, the cell cycle distribution and the mitochondrial membrane potential (MMP), respectively. Cell apoptosis was measured by the morphology observation under a light microscope, Annexin V-FITC/propidium iodide (PI) apoptosis detection and the colorimetric TUNEL assay. Western blot was used to monitor the cell cycle-, apoptosis-related proteins and relevant proteins involved in the signaling pathways. RESULTS: The MTT assay demonstrated that 9za sharply decreased the viability of NSCLC cells. Cell cycle analysis revealed that low concentrations of 9za arrested the cell cycle at the G0/G1 phase , which was further confirmed by the decreased levels of Cyclin D1, cyclin-dependent kinase 4 (CDK4) and cyclin-dependent kinase 6 (CDK6). Additionally, morphological observations, Annexin V-FITC/propidium iodide (PI) apoptosis analysis and TUNEL assays indicated that high concentrations of 9za induced cell apoptosis. Furthermore, the JC-1 staining assay revealed that the mitochondrial membrane potential was downregulated following 9za exposure. Western blot also showed that 9za markedly decreased the expression levels of total Bcl-2, Cytochrome C in the mitochondria and BCL2 associated X (BAX) in the cytoplasm. However, the levels of BAX in the mitochondria, Cytochrome C in the cytoplasm, active caspase-9, active caspase-3 and cleaved-PARP showed the opposite changes. Moreover, the dose-dependent decreased phosphorylation levels of PDK1, protein kinase B (Akt), MEK and extracellular signal regulated kinase 1/2 (ERK1/2) after 9za treatment verified that 9za was indeed a dual MEK/PDK1 inhibitor, as we expected. Compared with a single MEK inhibitor PD0325901 or a single PDK1 inhibitor BX517, the dual MEK/PDK1 inhibitor 9za could strengthen the cytotoxic and proapoptotic effect, indicating that the double blocking of the MEK and PDK1 signaling pathways plays stronger cell growth inhibition and apoptosis induction roles than the single blocking of the MEK or PDK1 signaling pathway in NSCLC cells. Our work elucidated the molecular mechanisms for 9za as a novel drug candidate against NSCLC.

Dose-effect relationship and molecular mechanism by which BMSC-derived exosomes promote peripheral nerve regeneration after crush injury.

BACKGROUND: The development of new treatment strategies to improve peripheral nerve repair after injury, especially those that accelerate axonal nerve regeneration, is very important. The aim of this study is to elucidate the molecular mechanisms of how bone marrow stromal cell (BMSC)-derived exosomes (EXOs) participate in peripheral nerve regeneration and whether the regenerative effect of EXOs is correlated with dose. METHOD: BMSCs were transfected with or without an siRNA targeting Ago2 (SiAgo2). EXOs extracted from the BMSCs were administered to dorsal root ganglion (DRG) neurons in vitro. After 48 h of culture, the neurite length was measured. Moreover, EXOs at four different doses were injected into the gastrocnemius muscles of rats with sciatic nerve crush injury. The sciatic nerve functional index (SFI) and latency of thermal pain (LTP) of the hind leg sciatic nerve were measured before the operation and at 7, 14, 21, and 28 days after the operation. Then, the number and diameter of the regenerated fibers in the injured distal sciatic nerve were quantified. Seven genes associated with nerve regeneration were investigated by qRT-PCR in DRG neurons extracted from rats 7 days after the sciatic nerve crush. RESULTS: We showed that after 48 h of culture, the mean number of neurites and the length of cultured DRG neurons in the SiAgo2-BMSC-EXO and SiAgo2-BMSC groups were smaller than that in the untreated and siRNA control groups. The average number and diameter of regenerated axons, LTP, and SFI in the group with 0.9 × 1010 particles/ml EXOs were better than those in other groups, while the group that received a minimum EXO dose (0.4 × 1010 particles/ml) was not significantly different from the PBS group. The expression of PMP22, VEGFA, NGFr, and S100b in DRGs from the EXO-treated group was significantly higher than that in the PBS control group. No significant difference was observed in the expression of HGF and Akt1 among the groups. CONCLUSIONS: These results showed that BMSC-derived EXOs can promote the regeneration of peripheral nerves and that the mechanism may involve miRNA-mediated regulation of regeneration-related genes, such as VEGFA. Finally, a dose-effect relationship between EXO treatment and nerve regeneration was shown.

The ATP-P2X7 Signaling Pathway Participates in the Regulation of Slit1 Expression in Satellite Glial Cells.

Slit1 is one of the known signaling factors of the slit family and can promote neurite growth by binding to its receptor, Robo2. Upregulation of Slit1 expression in dorsal root ganglia (DRG) after peripheral nerve injury plays an important role in nerve regeneration. Each sensory neuronal soma in the DRG is encapsulated by several surrounding satellite glial cells (SGCs) to form a neural structural unit. However, the temporal and spatial patterns of Slit1 upregulation in SGCs in DRG and its molecular mechanisms are not well understood. This study examined the spatial and temporal patterns of Slit1 expression in DRG after sciatic nerve crush by immunohistochemistry and western blotting. The effect of neuronal damage signaling on the expression of Slit1 in SGCs was studied in vivo by fluorescent gold retrograde tracing and double immunofluorescence staining. The relationship between the expression of Slit1 in SGCs and neuronal somas was also observed by culturing DRG cells and double immunofluorescence labeling. The molecular mechanism of Slit1 was further explored by immunohistochemistry and western blotting after intraperitoneal injection of Bright Blue G (BBG, P2X7R inhibitor). The results showed that after peripheral nerve injury, the expression of Slit1 in the neurons and SGCs of DRG increased. The expression of Slit1 was presented with a time lag in SGCs than in neurons. The expression of Slit1 in SGCs was induced by contact with surrounding neuronal somas. Through injured cell localization, it was found that the expression of Slit1 was stronger in SGCs surrounding injured neurons than in SGCs surrounding non-injured neurons. The expression of vesicular nucleotide transporter (VNUT) in DRG neurons was increased by injury signaling. After the inhibition of P2X7R, the expression of Slit1 in SGCs was downregulated, and the expression of VNUT in DRG neurons was upregulated. These results indicate that the ATP-P2X7R pathway is involved in signal transduction from peripheral nerve injury to SGCs, leading to the upregulation of Slit1 expression.

Bone marrow mesenchymal stem cell-derived exosome uptake and retrograde transport can occur at peripheral nerve endings.

We investigated the occurrence of mesenchymal stem cell (MSC)-derived exosome uptake and retrograde transport at peripheral nerve endings using bone marrow MSCs (bMSCs) transduced with recombinant CD63-green fluorescent protein (GFP) lentiviral plasmid. GFP was used to track the release of bMSC-derived exosomes and the uptake and transport at peripheral nerve terminals, the dorsal root ganglion (DRG), and the spinal cord. In vitro cell culture and injection of a CD63-GFP exosome suspension into the right gastrocnemius muscle of an in vivo rat model were also performed. Fluorescence microscopy of co-cultured CD63-GFP exosomes and SH-SY5Y or BV2 cell lines and primary cultured DRG cells in a separate experiment demonstrated exosome uptake into DRG neurons and glia. Moreover, we observed both retrograde axoplasmic transport and hematogenous transport of exosomes injected into rat models at the DRG and the ipsilateral side of the anterior horn of the spinal cord using fluorescence microscopy, immunohistochemistry, and Western blot analyses. In conclusion, we showed that exosome uptake at peripheral nerve endings and retrograde transport of exosomes to DRG neurons and spinal cord motor neurons in the anterior horn can occur. In addition, our findings propose a novel drug delivery approach for treating neuronal diseases.

Anterior insular cortex mediates hyperalgesia induced by chronic pancreatitis in rats.

Central sensitization plays a pivotal role in the maintenance of chronic pain induced by chronic pancreatitis (CP), but cortical modulation of painful CP remains elusive. This study was designed to examine the role of anterior insular cortex (aIC) in the pathogenesis of hyperalgesia in a rat model of CP. CP was induced by intraductal administration of trinitrobenzene sulfonic acid (TNBS). Abdomen hyperalgesia and anxiety were assessed by von Frey filament and open field tests, respectively. Two weeks after surgery, the activation of aIC was indicated by FOS immunohistochemical staining and electrophysiological recordings. Expressions of VGluT1, NMDAR subunit NR2B and AMPAR subunit GluR1 were analyzed by immunoblottings. The regulatory roles of aIC in hyperalgesia and pain-related anxiety were detected via pharmacological approach and chemogenetics in CP rats. Our results showed that TNBS treatment resulted in long-term hyperalgesia and anxiety-like behavior in rats. CP rats exhibited increased FOS expression and potentiated excitatory synaptic transmission within aIC. CP rats also showed up-regulated expression of VGluT1, and increased membrane trafficking and phosphorylation of NR2B and GluR1 within aIC. Blocking excitatory synaptic transmission significantly attenuated abdomen mechanical hyperalgesia. Specifically inhibiting the excitability of insular pyramidal cells reduced both abdomen hyperalgesia and pain-related anxiety. In conclusion, our findings emphasize a key role for aIC in hyperalgesia and anxiety of painful CP, providing a novel insight into cortical modulation of painful CP and shedding light on aIC as a potential target for neuromodulation interventions in the treatment of CP.