Matrix stiffness triggers chemoresistance through elevated autophagy in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) has a signature of extremely high matrix stiffness caused by a special desmoplastic reaction, which dynamically stiffens along with the pathological process. The poor prognosis and low five-year survival rate of PDAC are partly owing to chemoresistance triggered by substrate stiffness. Understanding the potential mechanisms of matrix stiffness causing PDAC chemoresistance is of great significance. In this study, methacrylated gelatin hydrogel was used as platform for PANC-1 and MIA-PaCa2 cell culture. The results indicated that compared to soft substrate, stiff substrate distinctively reduced the gemcitabine sensitivity of pancreatic cancer. Intriguingly, transmission electron microscopy, immunofluorescence staining, western blot and qRT-PCR assay showcased that the number of autophagosomes and the expression of LC3 were elevated. The observations indicate that matrix stiffness may regulate the autophagy level, which plays a vital role during chemoresistance. In brief, soft substrate exhibited low autophagy level, while the counterpart displayed elevated autophagy level. In order to elucidate the underlying interaction between matrix stiffness-mediated cell autophagy and chemoresistance, rescue experiments with rapamycin and chloroquine were conducted. We found that inhibiting cell autophagy dramatically increased the sensitivity of pancreatic cancer cells to gemcitabine in the stiff group, while promoting autophagy-driven chemoresistance in the soft group, demonstrating that matrix stiffness modulated chemoresistance via autophagy. Furthermore, RNA-seq results showed that miR-1972 may regulate autophagy level in response to matrix stiffness. Overall, our research shed light on the synergistic therapy of PDAC combined with gemcitabine and chloroquine, which is conducive to promoting a therapeutic effect.

Exosomal miR-673-5p from fibroblasts promotes Schwann cell-mediated peripheral neuron myelination by targeting the TSC2/mTORC1/SREBP2 axis.

Peripheral myelination is a complicated process, wherein Schwann cells (SCs) promote the formation of the myelin sheath around the axons of peripheral neurons. Fibroblasts are the second resident cells in the peripheral nerves; however, the precise function of fibroblasts in SC-mediated myelination has rarely been examined. Here, we show that exosomes derived from fibroblasts boost myelination-related gene expression in SCs. We used exosome sequencing, together with bioinformatic analysis, to demonstrate that exosomal microRNA miR-673-5p is capable of stimulating myelin gene expression in SCs. Subsequent functional studies revealed that miR-673-5p targets the regulator of mechanistic target of the rapamycin (mTOR) complex 1 (mTORC1) tuberous sclerosis complex 2 in SCs, leading to the activation of downstream signaling pathways including mTORC1 and sterol-regulatory element binding protein 2. In vivo experiments further confirmed that miR-673-5p activates the tuberous sclerosis complex 2/mTORC1/sterol-regulatory element binding protein 2 axis, thus promoting the synthesis of cholesterol and related lipids and subsequently accelerating myelin sheath maturation in peripheral nerves. Overall, our findings revealed exosome-mediated cross talk between fibroblasts and SCs that plays a pivotal role in peripheral myelination. We propose that exosomes derived from fibroblasts and miR-673-5p might be useful for promoting peripheral myelination in translational medicine.

Pancreatic Extracellular Matrix/Alginate Hydrogels Provide a Supportive Microenvironment for Insulin-Producing Cells.

Type 1 diabetes mellitus (T1DM), as an autoimmune deficiency disease, is associated with an absolute deficiency of insulin subject to islet ß-cell destruction. Insulin-producing cells (IPCs) differentiated from induced pluripotent stem cells are an ideal replacement origin of ß-cells, which can be applied for cell transplantation therapies in T1DM. At present, more strategies focus on inducing and differentiating to obtain IPCs; however, the unsatisfactory differentiation efficiency and the lack of ideal carriers for in vivo transplantation limited their application. It is necessary to consider the cell microenvironment by constructing a biomimetic niche to improve the differentiation and transplantation efficiency. The main components of the extracellular matrix derived from pancreatic (the niche of ß-cells) decellularization were retained, which could provide the ideal extracellular microenvironment for IPCs. In this research, a hydrogel prepared with alginate (Alg) and the pancreatic extracellular matrix (pECM) was assessed for the beneficial outcomes on encapsulated IPCs. The results showed that pECM/Alg improved the differentiation efficiency and promoted insulin secretion and the expression of insulin-related genes as well. Besides, pECM/Alg-encapsulated IPCs exhibited obvious biocompatibility in vivo, which can prolong the transplantation effect and hypoglycemic function by reducing the inflammatory reaction. RNA-seq indicated that the PI3K/Akt pathway may be related to the improvement of the differentiation efficiency and function of IPCs. In general, the pECM/Alg hydrogel provides an ideal biomimetic microenvironment for IPCs and is suitable for in vivo transplantation.

Application of conductive PPy/SF composite scaffold and electrical stimulation for neural tissue engineering.

Electrical stimulation (ES) with conductive polymers can dramatically enhance neurite outgrowth and promote neural regeneration. However, besides ES, the practical applications of neural repair is also highly dependent on the nerve cell functionality and response to substrate conductivity. Therefore, the combination of the ES and suitable materials, such as tissue scaffolds, has been applied to facilitate treatment of neural injuries and demonstrated great potential in peripheral nerve regeneration. In this study, polypyrrole/silk fibroin (PPy/SF) conductive composite scaffold was fabricated by 3D bioprinting and electrospinning. Schwann cells seeded on these scaffolds were electrically stimulated and hence demonstrated enhanced viability, proliferation and migration, as well as upregulated expression of neurotrophic factors. Furthermore, the constructed PPy/SF conductive nerve guidance conduits accompanying with ES could effectively promote axonal regeneration and remyelination in vivo. Moreover, we found that the MAPKs signal transduction pathway was activated by ES at the conductive conduit. Our findings demonstrate that the PPy/SF conductive composite scaffolds with longitudinal guidance exhibit favorable properties for clinical use and promotes nerve regeneration and functional recovery.

Tubular scaffold with microchannels and an H-shaped lumen loaded with bone marrow stromal cells promotes neuroregeneration and inhibits apoptosis after spinal cord injury.

As a result of its complex histological structure, regeneration patterns of grey and white matter are quite different in the spinal cord. Therefore, tissue engineering scaffolds for repairing spinal cord injury must be able to adapt to varying neural regeneration patterns. The aim of the present study was to improve a previously reported spinal cord-mimicking partition-type scaffold by adding microchannels on a single tubular wall along its longitudinal axis, thus integrating the two architectures of a single H-shaped central tube and many microchannels. Next, the integrated scaffold was loaded with bone marrow stromal cells (BMSCs) and transplanted to bridge the 5-mm defect of a complete transverse lesion in the thoracic spinal cord of rats. Subsequently, effects on nerve regeneration, locomotion function recovery, and early neuroprotection were observed. After 1 year of repair, the integrated scaffold could guide the regeneration of axons appearing in the debris of degraded microchannels, especially serotonin receptor 1A receptor-positive axonal tracts, which were relatively orderly arranged. Moreover, a network of nerve fibres was present, and a few BMSCs expressed neuronal markers in tubular lumens. Functionally, electrophysiological and locomotor functions of rats were partially recovered. In addition, we found that BMSCs could protect neurons and oligodendrocytes from apoptosis during the early stage of implantation. Taken together, our results demonstrate the potential of this novel integrated scaffold loaded with BMSCs to promote spinal cord regeneration through mechanical guidance and neuroprotective mechanisms.

Bone marrow-derived neural crest precursors improve nerve defect repair partially through secreted trophic factors.

BACKGROUND: Emerging evidence suggests that neural crest-derived cells (NCCs) present important functions in peripheral nerve regeneration to correct the insufficiency of autogenous Schwann cells. Postmigratory NCCs have been successfully isolated from adult rat bone marrow in our previous work. In this study, we aim to provide neural crest-derived Schwann cell precursors (SCPs) for repair of nerve defects in adult rats, and partially reveal the mechanisms involved in neuroregeneration of cell therapy. METHODS: A clonal cell line of neural crest precursors of rat bone marrow origin (rBM-NCPs) with SCP identity was expanded in adherent monolayer culture to ensure the stable cell viability of NCPs and potentiate the repair of nerve defects after rBM-NCPs implantation based on tissue engineering nerve grafts (TENG). Here the behavioral, morphological, and electrophysiological detection was performed to evaluate the therapy efficacy. We further investigated the treatment with NCP-conditioned medium (NCP-CM) to sensory neurons after exposure to oxygen-glucose-deprivation (OGD) and partially compared the expression of trophic factor genes in rBM-NCPs with that in mesenchymal stem cells of bone marrow origin (rBM-MSCs). RESULTS: It was showed that the constructed TENG with rBM-NCPs loaded into silk fibroin fiber scaffolds/chitosan conduits repaired 10-mm long sciatic nerve defects more efficiently than conduits alone. The axonal regrowth, remyelination promoted the reinnervation of the denervated hind limb muscle and skin and thereby alleviated muscle atrophy and facilitated the rehabilitation of motor and sensory function. Moreover, it was demonstrated that treatment with NCP-CM could restore the cultured primary sensory neurons after OGD through trophic factors including epidermal growth factor (EGF), platelet-derived growth factor alpha (PDGFα), ciliary neurotrophic factor (CNTF), and vascular endothelial growth factor alpha (VEGFα). CONCLUSIONS: In summary, our findings indicated that monolayer-cultured rBM-NCPs cell-based therapy might effectively repair peripheral nerve defects partially through secreted trophic factors, which represented the secretome of rBM-NCPs differing from that of rBM-MSCs.

lncRNA Gm10451 regulates PTIP to facilitate iPSCs-derived ß-like cell differentiation by targeting miR-338-3p as a ceRNA.

iPSCs-derived insulin-producing cell transplantation is a promising strategy for diabetes therapy. Although there have been many protocols of mature, glucose-responsive ß cells induced in vitro over the past few years, many underlying problems remain to be resolved. As a crucial regulator, long noncoding RNAs (lncRNAs) participate in numerous biological processes, including the maintenance of pluripotency, and stem cell differentiation. In this study, we identified a novel lncRNA Gm10451 as a functional regulator for ß-like cell differentiation. Localized to the cytoplasm, Gm10451 regulates histone H3K4 methyltransferase complex PTIP to facilitate Insulin+/Nkx6.1+ ß-like cell differentiation by targeting miR-338-3p as a competing endogenous RNA (ceRNA). miR-338-3p has also been shown to suppress Nkx6.1+ early-stage ß-like cell differentiation by targeting PTIP. Following transplantation into streptozotocin (STZ)-mice, Gm10451 loss in ß-like cells prevented the expression of mature ß-cell makers, such as Insulin, Nkx6.1, and Mafa. Accordingly, hyperglycemia in the mice was not resolved. Taken together, this study provides an efficient epigenetic target for generating more mature and functional iPSCs-derived ß-like cells. We anticipate that pancreatic organoids, which are generated from human stem cells, biological materials, and epigenetic modifications, can be used in the future as a novel diabetes treatment option.

[Preparation and properties of fiber-based conductive composite scaffolds for peripheral nerve regeneration].

Objective: To explore the preparation method, physical and chemical properties, and biocompatibility of a conductive composite scaffold based on polypyrrole/silk fibroin (PPy/SF) fiber with "shell-core" structure, and to provide a preliminary research basis for the application in the field of tissue engineered neuroscience. Methods: The conductive fibers with "shell-core" structure were prepared by three-dimensional printing combined with in-situ polymerization. PPy/SF fiber-based conductive composite scaffolds were formed by electrospinning. In addition, core-free PPy conductive fibers and SF electrospinning fibers were prepared. The stability, biomechanics, electrical conductivity, degradation performance, and biological activity of each material were tested to analyze the comprehensive properties of fiber-based conductive composite scaffolds. Results: Compared with pure core-free PPy conductive fibers and SF electrospinning fibers, the PPy/SF fiber-based conductive composite scaffolds with "shell-core" structure could better maintain the stability performance, enhance the mechanical stretchability of the composite scaffolds, maintain long-term electrical activity, and improve the anti-degradation performance. At the same time, PPy/SF conductive composite scaffolds were suitable for NIH3T3 cells attachment, conducive to cell proliferation, and had good biological activity. Conclusion: PPy/SF fiber-based conductive composite scaffolds meet the needs of conductivity, stability, and biological activity of artificial nerve grafts, and provide a new idea for the development of a new generation of high-performance and multi-functional composite materials.

The High-Frequency Ultrasound Detection of Rat Sciatic Nerve in a Crushed Injury Model.

OBJECTIVE: This study aimed to visualize sciatic nerve injury in rats using ultrasound imaging in a crushed injury model. METHODS: Adult male Sprague-Dawley rats were subjected to a left sciatic nerve crush operation. Then, high-frequency ultrasound was used to image both sciatic nerves at 2 days and at 1, 2, 3, 4, and 6 weeks after surgery. RESULTS: Normal uninjured nerves have uniform thickness, display a smooth epineurium and inner adventitia, and are oblong in transverse sections. After the crush operation, nerve thickness increased, the inner echo signal decreased, the image of the epineurium became obscured and coarse before becoming smooth again, and transverse sections of the nerve fibers changed from being semicircular to oval in shape before becoming elliptical again. These observations were consistent with pathological changes associated with nerve injury. CONCLUSIONS: High-frequency ultrasound is capable of capturing dynamic changes in rat sciatic nerves in a crushed injury model. This can be used as an auxiliary method of evaluation in traditional peripheral nerve injury experiments.

Novel conductive polypyrrole/silk fibroin scaffold for neural tissue repair.

Three dimensional (3D) bioprinting, which involves depositing bioinks (mixed biomaterials) layer by layer to form computer-aided designs, is an ideal method for fabricating complex 3D biological structures. However, it remains challenging to prepare biomaterials with micro-nanostructures that accurately mimic the nanostructural features of natural tissues. A novel nanotechnological tool, electrospinning, permits the processing and modification of proper nanoscale biomaterials to enhance neural cell adhesion, migration, proliferation, differentiation, and subsequent nerve regeneration. The composite scaffold was prepared by combining 3D bioprinting with subsequent electrochemical deposition of polypyrrole and electrospinning of silk fibroin to form a composite polypyrrole/silk fibroin scaffold. Fourier transform infrared spectroscopy was used to analyze scaffold composition. The surface morphology of the scaffold was observed by light microscopy and scanning electron microscopy. A digital multimeter was used to measure the resistivity of prepared scaffolds. Light microscopy was applied to observe the surface morphology of scaffolds immersed in water or Dulbecco's Modified Eagle's Medium at 37°C for 30 days to assess stability. Results showed characteristic peaks of polypyrrole and silk fibroin in the synthesized conductive polypyrrole/silk fibroin scaffold, as well as the structure of the electrospun nanofiber layer on the surface. The electrical conductivity was 1 × 10-5-1 × 10-3 S/cm, while stability was 66.67%. A 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay was employed to measure scaffold cytotoxicity in vitro. Fluorescence microscopy was used to observe EdU-labeled Schwann cells to quantify cell proliferation. Immunohistochemistry was utilized to detect S100ß immunoreactivity, while scanning electron microscopy was applied to observe the morphology of adherent Schwann cells. Results demonstrated that the polypyrrole/silk fibroin scaffold was not cytotoxic and did not affect Schwann cell proliferation. Moreover, filopodia formed on the scaffold and Schwann cells were regularly arranged. Our findings verified that the composite polypyrrole/silk fibroin scaffold has good biocompatibility and may be a suitable material for neural tissue engineering.

Electrospun silk fibroin-based neural scaffold for bridging a long sciatic nerve gap in dogs.

Silk fibroin (SF)-derived silkworms represent a type of highly biocompatible biomaterial for tissue engineering. We have previously investigated biocompatibility of SF with neural cells isolated from the central nervous system or peripheral nerve system in vitro, and also developed a SF-based nerve graft conduit or tissue-engineered nerve grafts by introducing bone marrow mesenchymal stem cells, as support cells, into SF-based scaffold and evaluated the outcomes of peripheral nerve repair in a rat model. As an extension of the previous study, the electrospun technique was performed here to fabricate SF-based neural scaffold inserted with silk fibres for bridging a 30-mm-long sciatic nerve gap in dogs. Assessments including functional, histological and morphometrical analyses were applied 12 months after surgery. All the results indicated that the SF-based neural scaffold group achieved satisfactory regenerative outcomes, which were close to those achieved by autologous nerve grafts as the golden-standard for peripheral nerve repair. Overall, our results raise a potential possibility for the translation of SF-based electrospun neural scaffolds as an alternative to nerve autografts into the clinic.

Chitosan degradation products facilitate peripheral nerve regeneration by improving macrophage-constructed microenvironments.

Chitosan-based artificial nerve grafts have been widely employed to repair peripheral nerve defects. Our previous study has shown that chitosan constructed nerve graft not only provides suitable scaffolds for nerve regeneration, its degradation products, chitooligosaccharides (COS), also promote nerve repair. However, the involved mechanisms are still not fully elucidated. In the present study, we observed that pro-inflammatory cytokines, as well as macrophage infiltration, were transiently up-regulated in the injured sciatic nerves which were bridged with silicon tubes filled with COS. Based upon transcriptome analysis, the axis of miR-327/CCL2 in Schwann cells (SCs) was identified as a potential target of COS. The following experiments have confirmed that COS stimulate CCL2 expression by down-regulating miR-327 in SCs. Consequently, the resulting CCL2 induces macrophage migration at injury sites to re-construct microenvironments and thus facilitates nerve regeneration. Collectively, our data provide a theoretical basis for the clinical application of chitosan-based grafts in peripheral nerve regeneration.

[Construction and biocompatibility in vitro evaluation of electrospun-graphene/silk fibroin nanofilms].

Objective: To explore the construction and biocompatibility in vitro evaluation of the electrospun-graphene (Gr)/silk fibroin (SF) nanofilms. Methods: The electrostatic spinning solution was prepared by dissolving SF and different mass ratio (0, 5%, 10%, 15%, and 20%) of Gr in formic acid solution. The hydrophilia and hydrophobic was analyzed by testing the static contact angle of electrostatic spinning solution of different mass ratio of Gr. Gr-SF nanofilms with different mass ratio (0, 5%, 10%, 15%, and 20%, as groups A, B, C, D, and E, respectively) were constructed by electrospinning technology. The structure of nanofilms were observed by optical microscope and scanning electron microscope; electrochemical performance of nanofilms were detected by cyclic voltammetry at electrochemical workstation; the porosity of nanofilms were measured by n-hexane substitution method, and the permeability were observed; L929 cells were used to evaluate the cytotoxicity of nanofilms in vitro at 1, 4, and 7 days after culture. The primary Sprague Dawley rats' Schwann cells were co-cultured with different Gr-SF nanofilms of 5 groups for 3 days, the morphology and distribution of Schwann cells were identified by toluidine blue staining, the cell adhesion of Schwann cells were determined by cell counting kit 8 (CCK-8) method, the proliferation of Schwann cells were detected by EdU/Hoechst33342 staining. Results: The static contact angle measurement confirmed that the hydrophilia of Gr-SF electrospinning solution was decreased by increasing the mass ratio of Gr. Light microscope and scanning electron microscopy showed that Gr-SF nanofilms had nanofiber structure, Gr particles could be dispersed uniformly in the membrane, and the increasing of mass ratio of Gr could lead to the aggregation of particles. The porosity measurement showed that the Gr-SF nanofilms had high porosity (>65%). With the increasing of mass ratio of Gr, the porosity and conductivity of Gr-SF nanofilm increased gradually, the value in the group A was significantly lower than those in groups C, D, and E ( P<0.05). In vitro L929 cells cytotoxicity test showed that all the Gr-SF nanofilms had good biocompatibility. Toluidine blue staining, CCK-8 assay, and EdU/Hoechst33342 staining showed that Gr-SF nanofilms with mass ratio of Gr less than 10% could support the survival and proliferation of co-cultured Schwann cells. Conclusion: The Gr-SF nanofilm with mass ratio of Gr less than 10% have proper hydrophilia, conductivity, porosity, and other physical and chemical properties, and have good biocompatibility in vitro. They can be used in tissue engineered nerve preparation.

The Effect of Low-Intensity Ultrasound on Brain-Derived Neurotropic Factor Expression in a Rat Sciatic Nerve Crushed Injury Model.

Low-intensity ultrasound (LIU) can improve nerve regeneration and functional recovery after peripheral nerve crush injury, but the underlying mechanism is not clear. The objective of this study was to examine the effects of LIU on rat sciatic crush injury and to investigate a possible molecular mechanism. Adult male Sprague-Dawley rats underwent left sciatic nerve crush surgery and were then randomized into two groups: a treatment group that received LIU every other d, and a control group that received sham exposure. Compared with rats in the control group, rats in the treatment group had higher sciatic nerve function indexes, compound muscle action potentials, wet weight ratios of the target muscle and mRNA expression of brain-derived neurotropic factor (BDNF) in the crushed nerve and ipsilateral dorsal root ganglia. Our findings suggest that LIU might promote injured nerve regeneration by stimulating BDNF release.

AMPK Negatively Regulates Peripheral Myelination via Activation of c-Jun.

The process of Schwann cells (SCs) forming a sheath around axons is termed as myelination, which plays a pivotal role for proper physiological function in the peripheral nervous system (PNS). The molecular mechanisms regulating SC myelination in the PNS remain to be elucidated. Here, we show that AMP-activated protein kinase (AMPK) in sciatic nerves was gradually decreased during the PNS myelination process. Pharmacological interventions showed that activation of AMPK by AICAR attenuated myelin gene expression in SCs, whereas inhibition of AMPK by Compound C (ComC) or AMPKα1 knockdown stimulated myelin gene expression. Following experiments revealed that c-Jun, a negative modulator of PNS myelination, was activated by AMPK in SCs. The application of ComC in newborn rats markedly downregulated c-Jun expression in sciatic nerves. The lipid and protein synthesis in sciatic nerves was greatly potentiated by ComC. As a consequence, myelin gene expression in sciatic nerves, as well as myelin sheath thickness, were promoted in the ComC-treated rats. All together, our data identify that AMPK is an important negative regulator of Schwann cell myelination in the PNS, and this regulation role may rely on c-Jun activation.

Angiogenesis in tissue-engineered nerves evaluated objectively using MICROFIL perfusion and micro-CT scanning.

Angiogenesis is a key process in regenerative medicine generally, as well as in the specific field of nerve regeneration. However, no convenient and objective method for evaluating the angiogenesis of tissue-engineered nerves has been reported. In this study, tissue-engineered nerves were constructed in vitro using Schwann cells differentiated from rat skin-derived precursors as supporting cells and chitosan nerve conduits combined with silk fibroin fibers as scaffolds to bridge 10-mm sciatic nerve defects in rats. Four weeks after surgery, three-dimensional blood vessel reconstructions were made through MICROFIL perfusion and micro-CT scanning, and parameter analysis of the tissue-engineered nerves was performed. New blood vessels grew into the tissue-engineered nerves from three main directions: the proximal end, the distal end, and the middle. The parameter analysis of the three-dimensional blood vessel images yielded several parameters, including the number, diameter, connection, and spatial distribution of blood vessels. The new blood vessels were mainly capillaries and microvessels, with diameters ranging from 9 to 301 µm. The blood vessels with diameters from 27 to 155 µm accounted for 82.84% of the new vessels. The microvessels in the tissue-engineered nerves implanted in vivo were relatively well-identified using the MICROFIL perfusion and micro-CT scanning method, which allows the evaluation and comparison of differences and changes of angiogenesis in tissue-engineered nerves implanted in vivo.

Mucoepidermoid carcinoma of esophagus combined with squamous carcinoma of lung: A case report and literature review.

Mucoepidermoid carcinoma (MEC) is typically located in the salivary, lacrimal, and tracheobronchial glands and rarely presents in the esophagus. MEC is commonly characterized by squamous cells, mucus-secreting cells, and intermediate cells. This report presents the case of a 57-years-old male with a three months history of cough and shortness of breath. Computer tomography (CT) scans revealed a tumor locating in the left hilar. The histological report was squamous carcinoma. After three circles of chemotherapy, the patient complained of dysphagia. The electronic gastroscope showed a protrusion which 30-34 cm from the incisors. The tumor was histopathologically determined to be MEC of esophagus. The patient refused to surgery and concurrent chemoradiotheray; so, radiotherapy and sequential chemotherapy were performed, and after one year of follow up, the disease of esophagus recurrence; the patient was died of hemorrhage of esophagus for tumor progression. The literatures of MEC are also reviewed in this study.

Synthesis and neuroprotective effects of the fluorine substituted salidroside analogues in the PC12 cell model exposed to hypoglycemia and serum limitation.

Salidroside (Sal) is a natural antioxidant extracted from the root of Rhodiola rosea L., a traditional Chinese medicinal plant, which elicits neuroprotective effects in the treatment of ischemic stroke. In an attempt to improve its neuroprotective effects, fluorine substituted Sal analogues were synthesized and their neuroprotective activities against the hypoglycemia and serum limitation induced cell injury in differentiated PC12 cells were evaluated. The target compounds displayed strong protective effects on the cell viability against the damage caused by hypoglycemia and serum limitation, especially for D1, which had a great potency superior to Sal and efficiently inhibited hypoglycemia and serum limitation induced cell nuclear morphologic changes and the increased apoptotic rate in a dose-dependent manner. These new findings may provide potentially important information for further development of Sal analogues and lay the basis for further studies on the cerebral ischemic stroke and neurodegenerative diseases for human clinical treatment.

Synthesis and protective effects of novel salidroside analogues on glucose and serum depletion induced apoptosis in PC12 cells.

Salidroside is a natural product isolated from Rhodiola rosea L. which possesses a wide range of biological activities, especially neuroprotective effects in the treatment of ischemic stroke. In an attempt to improve its neuroprotective effects, a series of novel salidroside analogues were synthesized and their neuroprotective activities were evaluated against the glucose and serum depletion-induced cell death in differentiated PC12 cells. Most target compounds displayed protective effects on the cell viability, especially for compound 6, which had a great potency superior to salidroside. MTT assay and Hoechst 33342 staining collectively showed that pretreatment with 6 attenuated cell viability loss and reduced apoptotic death in cultured PC12 cells with glucose and serum depletion. And its neuroprotective effects might be associated with the increase of the apoptosis-related protein Bcl-2/Bax expression ratio, and also with the inhibition of caspase-3 activation. Therefore, our new findings may provide potentially important information for further development of salidroside analogues and lay the basis for further studies on the cerebral ischemic stroke and neurodegenerative diseases for human clinical treatment.

Preparation of uniaxial multichannel silk fibroin scaffolds for guiding primary neurons.

Physical guidance cues have been exploited to stimulate neuron adhesion and neurite outgrowth. In the present study, three-dimensional (3-D) silk fibroin scaffolds with uniaxial multichannels (42-142 µm in diameter) were prepared by a directional temperature field freezing technique, followed by lyophilization. By varying the initial silk fibroin concentration, the chemical potential and quantity of free water around cylindrical ice crystals could be controlled to control the cross-section morphology of the scaffold channels. Aligned ridges also formed on the inner surface of the multichannels in parallel to the direction of the channels. In vitro, primary hippocampal neurons were seeded in these 3-D silk fibroin scaffolds with uniaxial multichannels of ∼120 µm in diameter. The morphology of the neurons was multipolar and alignment along the scaffold channels was observed. Cell-cell networks and cell-matrix interactions established by newly formed axons were observed after 7 days in culture. These neurons expressed ß-III-tubulin, nerve filament and microtubule-associated protein, while glial fibrillary acidic protein immunofluorescence was barely above background. The ridges on the inner surface of the channels played a critical role in the adhesion and extension of neurons by providing continuous contact guidance. These new 3-D silk scaffolds with uniaxial multichannels provided a favorable microenvironment for the development of hippocampal neurons by guiding axonal elongation and cell migration.