Comparative Proteomic Analysis of Differentially Expressed Proteins between Injured Sensory and Motor Nerves after Peripheral Nerve Transection.

Peripheral nerve repair and functional recovery depend on the rate of nerve regeneration and the quality of target reinnervation. It is important to fully understand the cellular and molecular basis underlying the specificity of peripheral nerve regeneration, which means achieving corresponding correct pathfinding and accurate target reinnervation for regrowing motor and sensory axons. In this study, a quantitative proteomic technique, based on isobaric tags for relative and absolute quantitation (iTRAQ), was used to profile the protein expression pattern between single motor and sensory nerves at 14 days after peripheral nerve transection. Among a total of 1259 proteins identified, 176 proteins showed the differential expressions between injured motor and sensory nerves. Quantitative RT-PCR and western blot analysis were applied to validate the proteomic data on representative differentially expressed proteins. Functional categorization indicated that differentially expressed proteins were linked to a diverse array of molecular functions, including axonogenesis, response to axon injury, tissue remodeling, axon ensheathment, cell proliferation and adhesion, vesicle-mediated transport, response to oxidative stress, internal signal cascade, and macromolecular complex assembly, which might play an essential role in peripheral motor and sensory nerve regeneration. Overall, we hope that the proteomic database obtained in this study could serve as a solid foundation for the comprehensive investigation of differentially expressed proteins between injured motor and sensory nerves and for the mechanism elucidation of the specificity of peripheral nerve regeneration. Data are available via ProteomeXchange with identifier PXD022097.

[Antidepressant mechanism of Shugan Lipi recipe in regulating tryptophan metabolism and its common pharmacodynamic substances].

This article is to explore the antidepressant mechanism of Shugan Lipi recipe in regulating tryptophan metabolism,and to find out their common pharmacodynamic substances. UPLC-Q-TOF-MS technology was used to establish fingerprints of Shugan Lipi recipe,and 124 components were identified. The depressed mouse model was replicated by triple-one multiple stress method. Chaihu Shugan Powder,Sini Powder and Xiaoyao Powder were administered in groups to observe the changes in body weight and behavior of the mice. The results showed that compared with the model group,the body weight,sucrose preference percentage and autonomous activity behavior of each administration group were improved. Among them,the effect of Chaihu Shugan Powder was better than that of Sini Powder and Xiaoyao Powder. LC-MS/MS method was used to determine the contents of 5-hydroxytryptamine( 5-HT),kynurenine( KYN) and tryptophan( TPP) in blood,liver,brain,colon and other tissues,as well as TDO enzyme activity in liver. Western blot and RT-PCR were used to detect the protein and gene expression of TDO enzyme,respectively. It was found that the three prescriptions increased the ratio of 5-HT/KYN in different degrees,decreased the ratio of KYN/TRP in liver,colon and brain,and decreased the expression level and activity of TDO enzyme in liver. The order of their ability to regulate tryptophan metabolism was Chaihu Shugan Powder>Sini Powder>Xiaoyao Powder. In addition,the correlation between the chromatographic peaks in the fingerprints of Shugan Lipi recipes and the pharmacodynamic indexes of tryptophan metabolism was analyzed by the grey relation analysis. The grey relation analysis found that the chemical components with the highest correlation with tryptophan metabolism were mainly from Paeoniae Radix Alba,Citri Reticulatae Pericarpium,Aurantii Fructus Immaturus and Aurantii Fructus. UPLC-Q-TOF-MS was used to analyze the migration components in the plasma of mice after administration of Shugan Lipi recipe,and to verify the common pharmacodynamic substances of Shugan Lipi recipe. The migration of these detected components in plasma was studied,and a total of 18 prototype components and 36 metabolites were identified. Therefore,it was believed that Chaihu Shugan Powder,Sini Powder and Xiaoyao Powder could play an antidepressant role by reducing the expression of TDO enzyme in the liver and regulating the metabolism of tryptophan.The components contained in Paeoniae Radix Alba,Citri Reticulatae Pericarpium,Aurantii Fructus Immaturus and Aurantii Fructus were the common pharmacodynamic substances of Shugan Lipi recipe,which played an important role in regulating tryptophan metabolism.

Indole-3-Carbinol Induces Apoptosis of Hepatic Stellate Cells through K63 De-Ubiquitination of RIP1 in Rats.

BACKGROUND/AIMS: The apoptosis of activated hepatic stellate cells (HSCs) is the central event in the reversal of liver fibrosis. K63 de-ubiquitinated receptor-interacting protein (RIP)1 promotes apoptosis in tumor necrosis factor (TNF)-α signaling pathway. In the previous study, we have proved that indole-3-carbinol (I3C) could reverse different models of liver fibrosis in rats, but the mechanism is still unclear. Thus, the present research aimed to demonstrate the induction of I3C on apoptosis of HSCs and the underlying molecular mechanism. METHODS: HSC-T6, an immortalized rat liver stellate cell line, was treated for 24 hours with 25, 50 and 100 µM of I3C. The apoptosis of HSC-T6 was analyzed by flow cytometric analysis, acridine orange staining and RT-PCR, respectively. K63 de-ubiquitination of RIP1 and the expression of ubiquitin ligases and deubiquitinases were analyzed by Co-IP assay and western blot. Knockdown of deubiquitinases was undertaken by small interfering RNA (siRNA). RESULTS: The results of flow cytometric analysis indicated that the apoptotic rate of HSC-T6 was induced by I3C in a dose-dependent manner. Observation by acridine orange staining exhibited nuclear condensation and apoptotic bodies in I3C treated cells. Consistently, the expression ratio of Bax/bcl-2 was markedly increased by I3C. These results indicated that I3C could significantly induce apoptosis of HSC-T6 cells. Furthermore, Co-IP assay revealed K63 de-ubiquitination of RIP1 by I3C, associated with the induction of caspase 8. Although I3C had no effect on the expression of ubiquitin ligases cellular inhibitor of apoptosis 1/2 (cIAP1/2), the protein level of deubiquitinase cylindromatosis (CYLD) was significantly induced by I3C. Moreover, CYLD silencing reversed the pro-apoptosis induction effect of I3C and reduced the expression ratio of Bax/bcl-2 in HSC-T6 cells. CONCLUSION: These results demonstrated that I3C could induce apoptosis of HSC through RIP1 K63 de-ubiquitination by upregulating deubiquitinase CYLD.

The Relationship Between MMP-2 -1306C>T and MMP-9 -1562C>T Polymorphisms and the Risk and Prognosis of T-Cell Acute Lymphoblastic Leukemia in a Chinese Population: A Case-Control Study.

BACKGROUND: T-cell acute lymphoblastic leukemia (T-ALL) is a malignant hematological disease and is often accompanied by a variety of genetic abnormalities. Hence, our study aims to investigate the relationship between MMP-2 -1306C>T and MMP-9 -1562C>T polymorphisms and the risk and prognosis of T-ALL. METHODS: From April 2009 to February 2011, a total of 376 T-ALL patients were chosen as the case group. Meanwhile, 352 healthy people who passed routine health examinations were selected as the control group. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was used to detect the frequency of MMP-2 -1306C>T (rs243865) and MMP-9 -1562C>T (rs3918242) polymorphisms in the study subjects. The serum levels of MMP-2 and MMP-9 were detected using enzyme-linked immunosorbent assay (ELISA). A Kaplan-Meier analysis was employed to analyze the event-free survival (EFS) rates of the T-All patients with different MMP-2 and MMP-9 genotypes. A multivariate COX model was applied to analyze the relationship between MMP-2 and MMP-9 polymorphisms and the prognosis of T-ALL patients. A C-statistic and net reclassification index (NRI) was carried out to evaluate the predictive value of MMP-2 and MMP-9 gene polymorphisms using the Cox model. RESULTS: Compared to the control group, the genotypic frequency of MMP-2 -1306C>T (CT + TT) and MMP-9 -1562C>T (CT + TT) in the case group was significantly higher. The serum level of MMP-9 was markedly elevated in T-ALL patients with the CT + TT genotype compared to patients with the CC genotype. The results of the Kaplan-Meier analysis showed that the median EFS was lower in T-ALL patients with the CT + TT genotype of MMP-9 -1562C>T compared to patients with the CC genotype. The results of a multivariate analysis using the Cox proportional hazard model indicated that the MMP-9 -1562C>T polymorphism was associated with the prognosis of T-ALL patients. CONCLUSION: These results indicated that MMP-2 -1306C/T and MMP-9 -1562C/T polymorphisms might be associated with an increased risk of T-ALL. The MMP-9 -1562C>T polymorphism may also be related to the prognosis of T-ALL patients.

Effects of Blood Purification on Serum Levels of Inflammatory Cytokines and Cardiac Function in a Rat Model of Sepsis.

OBJECTIVE: The study aimed to explore the effects of blood purification (BP) on serum levels of inflammatory cytokines and cardiac function in a rat model of sepsis. METHODS: A rat model of sepsis was established by cecal ligation and puncture. All rats were divided into the normal control, sham operation, model, sham treatment, and BP treatment groups. Cardiac functions, inflammatory cytokines, myocardial enzymes, pathological score of cardiac muscle tissue, and myocardial apoptosis of rats in each group were compared. RESULTS: Sepsis rats had higher serum levels of inflammatory cytokines and lower cardiac function than those in the normal control and sham operation groups. Compared with the model and sham treatment groups, improved cardiac functions, decreased inflammatory cytokines, myocardial enzymes, pathological score, and myocardial apoptosis and mortality were observed in the BP treatment group. CONCLUSION: BP may reduce serum levels of inflammatory cytokines and improve cardiac function of sepsis rats.

Hormetic Effects of Trifloxystrobin on Aggressiveness of Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a devastating ascomycete plant pathogen with an extremely wide host range. Fungicides are still the mainstay for control of this pathogen, and stimulations to mycelial growth and aggressiveness by subtoxic doses of fungicides carbendazim and dimethachlon have been reported. The present study assessed hormetic effects of the quinone outside inhibitor (QoI) fungicide trifloxystrobin on aggressiveness of S. sclerotiorum. Trifloxystrobin at 0.0001, 0.0005, and 0.001 µg/ml exerted significant stimulatory effects on aggressiveness to potted rapeseed plants, and the highest percent stimulation were 20.5 and 24.2% for isolates HB15 and SX11, respectively. At 18 h postinoculation (HPI), initial necrotic lesions were visible to the naked eye on leaves treated with trifloxystrobin, whereas no obvious disease symptoms were discerned for the nontreated control. At 24, 36, and 48 HPI, aggressiveness stimulation was more obvious than at 18 HPI. Scanning electron microscopic observations demonstrated that no mycelia were detected on the nontreated leaves at 4 HPI; by contrast, mycelia were observed on leaves treated with trifloxystrobin at 0.0001 µg/ml. At 8 and 12 HPI, there were more mycelia and infecting hyphae on the treated leaves than on the nontreated control. These results indicated that fungal stimulation had occurred in the first 4 and 8 HPI, suggesting that direct stimulation was likely to be the underlying mechanism for hormetic actions of trifloxystrobin. Pretreatment with trifloxystrobin did not significantly affect subsequent mycelial growth on PDA or aggressiveness to detached rapeseed leaves in the absence of trifloxystrobin. However, in the presence of trifloxystrobin, mycelial growth and aggressiveness were significantly (P < 0.05) greater for the pretreatment with trifloxystrobin at 0.003 and 0.03 µg/ml compared with the nonpretreatment control, indicating that a prior exposure to the fungicide may undermine its subsequent effectiveness. These studies will raise our awareness of fungicide hormesis and have important implications for judicious application of fungicides.

Anisotropic microtopography surface of chitosan scaffold regulating skin precursor-derived Schwann cells towards repair phenotype promotes neural regeneration.

For repairing peripheral nerve and spinal cord defects, biomaterial scaffold-based cell-therapy was emerged as an effective strategy, requiring the positive response of seed cells to biomaterial substrate and environment signals. Previous work highlighted that the imposed surface properties of scaffold could provide important guidance cues to adhered cells for polarization. However, the insufficiency of native Schwann cells and unclear cellular response mechanisms remained to be addressed. Given that, this study aimed to illuminate the micropatterned chitosan-film action on the rat skin precursor-derived Schwann cells (SKP-SCs). Chitosan-film with different ridge/groove size was fabricated and applied for the SKP-SCs induction. Results indicated that SKP-SCs cultured on 30 µm size microgroove surface showed better oriented alignment phenotype. Induced SKP-SCs presented similar genic phenotype as repair Schwann cells, increasing expression of c-Jun, neural cell adhesion molecule, and neurotrophic receptor p75. Moreover, SKP-SC-secretome was subjected to cytokine array GS67 assay, data indicated the regulation of paracrine phenotype, a panel of cytokines was verified up-regulated at secreted level and gene expression level in induced SKP-SCs. These up-regulated cytokines exhibit a series of promotive neural regeneration functions, including cell survival, cell migration, cell proliferation, angiogenesis, axon growth, and cellular organization etc. through bioinformatics analysis. Furthermore, the effectively polarized SKP-SCs-sourced secretome, promoted the proliferation and migration capacity of the primarily cultured native rat Schwann cells, and augmented neurites growth of the cultured motoneurons, as well as boosted axonal regrowth of the axotomy-injured motoneurons. Taken together, SKP-SCs obtained pro-neuroregeneration phenotype in adaptive response to the anisotropic topography surface of chitosan-film, displayed the oriented parallel growth, the transition towards repair Schwann cell genic phenotype, and the enhanced paracrine effect on neural regeneration. This study provided novel insights into the potency of anisotropic microtopography surface to Schwann-like cells phenotype regulation, that facilitating to provide promising engineered cell-scaffold in neural injury therapies.

IKVAV functionalized oriented PCL/Fe3O4 scaffolds for magnetically modulating DRG growth behavior.

The re-bridging of the deficient nerve is the main problem to be solved after the functional impairment of the peripheral nerve. In this study, a directionally aligned polycaprolactone/triiron tetraoxide (PCL/Fe3O4) fiber scaffolds were firstly prepared by electrospinning technique, and further then grafted with IKVAV peptide for regulating DRG growth and axon extension in peripheral nerve regeneration. The results showed that oriented aligned magnetic PCL/Fe3O4 composite scaffolds were successfully prepared by electrospinning technique and possessed good mechanical properties and magnetic responsiveness. The PCL/Fe3O4 scaffolds containing different Fe3O4 concentrations were free of cytotoxicity, indicating the good biocompatibility and low cytotoxicity of the scaffolds. The IKVAV-functionalized PCL/Fe3O4 scaffolds were able to guide and promote the directional extension of axons, the application of external magnetic field and the grafting of IKVAV peptides significantly further promoted the growth of DRGs and axons. The ELISA test results showed that the AP-10 F group scaffolds promoted the secretion of nerve growth factor (NGF) from DRG under a static magnetic field (SMF), thus promoting the growth and extension of axons. Importantly, the IKVAV-functionalized PCL/Fe3O4 scaffolds could significantly up-regulate the expression of Cntn2, PCNA, Sox10 and Isca1 genes related to adhesion, proliferation and magnetic receptor function under the stimulation of SMF. Therefore, IKVAV-functionalized PCL/Fe3O4 composite oriented scaffolds have potential applications in neural tissue engineering.

Chitosan/PLGA-based tissue engineered nerve grafts with SKP-SC-EVs enhance sciatic nerve regeneration in dogs through miR-30b-5p-mediated regulation of axon growth.

Extracellular vesicles from skin-derived precursor Schwann cells (SKP-SC-EVs) promote neurite outgrowth in culture and enhance peripheral nerve regeneration in rats. This study aimed at expanding the application of SKP-SC-EVs in nerve grafting by creating a chitosan/PLGA-based, SKP-SC-EVs-containing tissue engineered nerve graft (TENG) to bridge a 40-mm long sciatic nerve defect in dogs. SKP-SC-EVs contained in TENGs significantly accelerated the recovery of hind limb motor and electrophysiological functions, supported the outgrowth and myelination of regenerated axons, and alleviated the denervation-induced atrophy of target muscles in dogs. To clarify the underlying molecular mechanism, we observed that SKP-SC-EVs were rich in a variety of miRNAs linked to the axon growth of neurons, and miR-30b-5p was the most important among others. We further noted that miR-30b-5p contained within SKP-SC-EVs exerted nerve regeneration-promoting effects by targeting the Sin3a/HDAC complex and activating the phosphorylation of ERK, STAT3 or CREB. Our findings suggested that SKP-SC-EVs-incorporating TENGs represent a novel type of bioactive material with potential application for peripheral nerve repair in the clinic.

Promotive effect of skin precursor-derived Schwann cells on brachial plexus neurotomy and motor neuron damage repair through milieu-regulating secretome.

Brachial plexus injury (BPI) with motor neurons (MNs) damage still remain poor recovery in preclinical research and clinical therapy, while cell-based therapy approaches emerged as novel strategies. Previous work of rat skin precursor-derived Schwann cells (SKP-SCs) provided substantial foundation for repairing peripheral nerve injury (PNI). Given that, our present work focused on exploring the repair efficacy and possible mechanisms of SKP-SCs implantation on rat BPI combined with neurorrhaphy post-neurotomy. Results indicated the significant locomotive and sensory function recovery, with improved morphological remodeling of regenerated nerves and angiogenesis, as well as amelioration of target muscles atrophy and motor endplate degeneration. Besides, MNs could restore from oxygen-glucose-deprivation (OGD) injury upon SKP-SCs-sourced secretome treatment, implying the underlying paracrine mechanisms. Moreover, rat cytokine array assay detected 67 cytokines from SKP-SC-secretome, and bioinformatic analyses of screened 32 cytokines presented multiple functional clusters covering diverse cell types, including inflammatory cells, Schwann cells, vascular endothelial cells (VECs), neurons, and SKP-SCs themselves, relating distinct biological processes to nerve regeneration. Especially, a panel of hypoxia-responsive cytokines (HRCK), can participate into multicellular biological process regulation for permissive regeneration milieu, which underscored the benefits of SKP-SCs and sourced secretome, facilitating the chorus of nerve regenerative microenvironment. Furthermore, platelet-derived growth factor-AA (PDGF-AA) and vascular endothelial growth factor-A (VEGF-A) were outstanding cytokines involved with nerve regenerative microenvironment regulating, with significantly elevated mRNA expression level in hypoxia-responsive SKP-SCs. Altogether, through recapitulating the implanted SKP-SCs and derived secretome as niche sensor and paracrine transmitters respectively, HRCK would be further excavated as molecular underpinning of the neural recuperative mechanizations for efficient cell therapy; meanwhile, the analysis paradigm in this study validated and anticipated the actions and mechanisms of SKP-SCs on traumatic BPI repair, and was beneficial to identify promising bioactive molecule cocktail and signaling targets for cell-free therapy strategy on neural repair and regeneration.

MircoRNA-25-3p in skin precursor cell-induced Schwann cell-derived extracellular vesicles promotes axon regeneration by targeting Tgif1.

Nerve injury often leads to severe dysfunction because of the lack of axon regeneration in adult mammal. Intriguingly a series of extracellular vesicles (EVs) have the obvious ability to accelerate the nerve repair. However, the detailed molecular mechanisms to describe that EVs switch neuron from a transmitter to a regenerative state have not been elucidated. This study elucidated the microRNA (miRNA) expression profiles of two types of EVs that promote nerve regeneration. The functions of these miRNAs were screened in vitro. Among the 12 overlapping miRNAs, miR-25-3p was selected for further analysis as it markedly promoted axon regeneration both in vivo and in vitro. Furthermore, knockdown experiments confirmed that PTEN and Klf4, which are the major inhibitors of axon regeneration, were the direct targets of miR-25-3p in dorsal root ganglion (DRG) neurons. The utilization of luciferase reporter assays and functional tests provided evidence that miR-25-3p enhances axon regeneration by targeting Tgif1. Additionally, miR-25-3p upregulated the phosphorylation of Erk. Furthermore, Rapamycin modulated the expression of miR-25-3p in DRG neurons. Finally, the pro-axon regeneration effects of EVs were confirmed by overexpressing miR-25-3p and Tgif1 knockdown in the optic nerve crush model. Thus, the enrichment of miR-25-3p in EVs suggests that it regulates axon regeneration, proving a potential cell-free treatment strategy for nerve injury.

Cytotoxic phenazine and antiallergic phenoxazine alkaloids from an arctic Nocardiopsis dassonvillei SCSIO 502F.

Microbes well-adapted to the Arctic Ocean are promising for producing novel compounds, due to their fancy strategies for adaptation and being under-investigated. Two new phenazine alkaloids (1 and 2) and one new phenoxazine (3) were isolated from Nocardiopsis dassonvillei 502F, a strain originally isolated from Arctic deep-sea sediments. AntiSMASH analysis of the genome of Nocardiopsis dassonvillei 502F revealed the presence of 16 putative biosynthetic gene clusters (BGCs), including a phenazine BGC. Most of the isolated compounds were evaluated for their antibacterial, antiallergic, and cytotoxic activities. Among them, compounds 4 and 5 exhibited potent in vitro cytotoxic activities against osteosarcoma cell line 143B with IC50 values 0.16 and 20.0 µM, respectively. Besides, the results of antiallergic activities of compounds 6-8 exhibited inhibitory activities with IC50 values of 10.88 ± 3.05, 38.88 ± 3.29, and 2.44 ± 0.17 µg/mL, respectively (IC50 91.6 µM for the positive control loratadine).

Nanoscale Treatment of Intervertebral Disc Degeneration: Mesenchymal Stem Cell Exosome Transplantation.

A common surgical disease, intervertebral disc degeneration (IVDD), is increasing at an alarming rate in younger individuals. Repairing damaged intervertebral discs (IVDs) and promoting IVD tissue regeneration at the molecular level are important research goals.Exosomes are extracellular vesicles (EVs) secreted by cells and can be derived from most body fluids. Mesenchymal stem cell-derived exosomes (MSC-exos) have characteristics similar to those of the parental MSCs. These EVs can shuttle various macromolecular substances, such as proteins, messenger RNAs (mRNAs), and microRNAs (miRNAs) and regulate the activity of recipient cells through intercellular communication. Reducing inflammation and apoptosis can significantly promote IVD regeneration to facilitate the repair of the IVD. Compared with MSCs, exosomes are more convenient to store and transport, and the use of exosomes can prevent the risk of rejection with cell transplantation. Furthermore, MSC-exo-mediated treatment may be safer and more effective than MSC transplantation. In this review, we summarize the use of bone marrow mesenchymal stem cells (BMSCs), adipose-derived mesenchymal stem cells (AMSCs), nucleus pulposus mesenchymal stem cells (NPMSCs), and stem cells from other sources for tissue engineering and use in IVDD. Here, we aim to describe the role of exosomes in inhibiting IVDD, their potential therapeutic effects, the results of the most recent research, and their clinical application prospects to provide an overview for researchers seeking to explore new treatment strategies and improve the efficacy of IVDD treatment.

[Effect of Nucleolin on Lymphoma Proliferation by Regulating Thymidine Kinase 1].

OBJECTIVE: To investigate the mechanism of nucleolin (NCL) involved in lymphoma proliferation by regulating thymidine kinase 1 (TK1). METHODS: Twenty-three patients with diffuse large B-cell lymphoma (DLBCL) were selected and divided into initial treatment group (14 cases) and relapsed/refractory group (9 cases). Serum TK1 and C23 protein in peripheral blood mononuclear cells were detected. Cell models of CA46-NCL-KD (CA46-NCL-knockdown) and CA46-NCL-KNC (CA46-NCL-knockdown negative control) were established by lentivirus vector mediated transfection in Burkitt lymphoma cell line CA46. The half maximal inhibitory concentration (IC50) of CA46-NCL-KD, CA46-NCL-KNC, and CA46 to adriamycin were detected by cell proliferation assay (MTS). The expression of NCL mRNA and protein in CA46-NCL-KD and CA46-NCL-KNC cells were dectected by Q-PCR and Western blot, respectively. The cell cycle of CA46-NCL-KD, CA46-NCL-KNC, and CA46 cells were detected by flow cytometry. The expression of TK1 protein in CA46-NCL-KD and CA46-NCL-KNC cells was detected by an enhanced chemiluminescence (ECL) dot blot assay. RESULTS: The level of serum TK1 in the initial treatment group was 0.43(0-30-1.01) pmol/L, which was lower than 10.56(2.19-14.99) pmol/L in the relapsed/refractory group (P<0-01), and the relative expression level of NCL protein in peripheral blood was also significantly lower. The IC50 of CA46-C23-KD cells to adriamycin was (0.147±0.02) µg/ml, which was significantly lower than (0.301±0.04) µg/ml of CA46-C23-KNC cells and (0.338±0.05) µg/ml of CA46 cells (P<0.05). Compared with CA46-NCL-KNC cells, the expression of NCL mRNA and protein, TK1 protein decreased in CA46-NCL-KD cells, and the proportion of S phase and G2/M phase also decreased, while G0/G1 phase increased in cell cycle. CONCLUSION: The increased expression of NCL in DLBCL and CA46 cells indicates low sensitivity to drug. NCL may participate in regulation of lymphoma proliferation by affecting TK1 expression, thereby affecting the drug sensitivity.

Spatio-temporally deciphering peripheral nerve regeneration in vivo after extracellular vesicle therapy under NIR-II fluorescence imaging.

Extracellular vesicles (EVs) show potential as a therapeutic tool for peripheral nerve injury (PNI), promoting neurological regeneration. However, there are limited data on the in vivo spatio-temporal trafficking and biodistribution of EVs. In this study, we introduce a new non-invasive near-infrared fluorescence imaging strategy based on glucose-conjugated quantum dot (QDs-Glu) labeling to target and track EVs in a sciatic nerve injury rat model in real-time. Our results demonstrate that the injected EVs migrated from the uninjured site to the injured site of the nerve, with an increase in fluorescence signals detected from 4 to 7 days post-injection, indicating the release of contents from the EVs with therapeutic effects. Immunofluorescence and behavioral tests revealed that the EV therapy promoted nerve regeneration and functional recovery at 28 days post-injection. We also found a relationship between functional recovery and the NIR-II fluorescence intensity change pattern, providing novel evidence for the therapeutic effects of EV therapy using real-time NIR-II imaging at the live animal level. This approach initiates a new path for monitoring EVs in treating PNI under in vivo NIR-II imaging, enhancing our understanding of the efficacy of EV therapy on peripheral nerve regeneration and its mechanisms.

The Effect of Human Bone Marrow Mesenchymal Stem Cell-Derived Exosomes on Cartilage Repair in Rabbits.

Mesenchymal stem cells (MSCs) have shown chondroprotective effects in cartilage repair. However, side effects caused by MSC treatment limit their application in clinic. As a cell-free therapy, MSC-derived exosomes (EXOs) have attracted much more attention in recent years. In the present study, we prepared EXOs from human bone marrow mesenchymal stem cells (hBMSCs) and examined their therapeutic potentials in cartilage repair. Our results showed that the prepared extracellular vesicles exhibit classical features of EXOs, such as cup-like shape, around 100 nm diameter, positive protein markers (CD81, TSG101, and Flotillin 1), and ability of internalization. In primary chondrocytes, the treatment of hBMSC-EXOs markedly increases cell viability and proliferation in a dose-dependent manner. Moreover, wound healing assay showed that hBMSC-EXOs accelerate cell migration in primary chondrocytes. JC-1 staining revealed that the mitochondrial membrane potential was enhanced by hBMSC-EXOs, indicating cell apoptosis was decreased in the presence of hBMSC-EXOs. In rabbits with articular cartilage defects, local administration with hBMSC-EXOs facilitates cartilage regeneration as evidenced by gross view and hematoxylin-eosin (H&E) and Saf-O/Fast Green staining. In addition, the International Cartilage Repair Society (ICRS) score was increased by the application of hBMSC-EXOs. Overall, our data indicate that the treatment with hBMSC-EXOs is a suitable cell-free therapy for treating cartilage defects, and these benefits are likely due to improved cell proliferation and migration in chondrocytes.

Peripheral nerve fibroblasts secrete neurotrophic factors to promote axon growth of motoneurons.

Peripheral nerve fibroblasts play a critical role in nerve development and regeneration. Our previous study found that peripheral nerve fibroblasts have different sensory and motor phenotypes. Fibroblasts of different phenotypes can guide the migration of Schwann cells to the same sensory or motor phenotype. In this study, we analyzed the different effects of peripheral nerve-derived fibroblasts and cardiac fibroblasts on motoneurons. Compared with cardiac fibroblasts, peripheral nerve fibroblasts greatly promoted motoneuron neurite outgrowth. Transcriptome analysis results identified 491 genes that were differentially expressed in peripheral nerve fibroblasts and cardiac fibroblasts. Among these, 130 were significantly upregulated in peripheral nerve fibroblasts compared with cardiac fibroblasts. These genes may be involved in axon guidance and neuron projection. Three days after sciatic nerve transection in rats, peripheral nerve fibroblasts accumulated in the proximal and distal nerve stumps, and most expressed brain-derived neurotrophic factor. In vitro, brain-derived neurotrophic factor secreted from peripheral nerve fibroblasts increased the expression of ß-actin and F-actin through the extracellular regulated protein kinase and serine/threonine kinase pathways, and enhanced motoneuron neurite outgrowth. These findings suggest that peripheral nerve fibroblasts and cardiac fibroblasts exhibit different patterns of gene expression. Peripheral nerve fibroblasts can promote motoneuron neurite outgrowth.

Improvement of sensory neuron growth and survival via negatively regulating PTEN by miR-21-5p-contained small extracellular vesicles from skin precursor-derived Schwann cells.

BACKGROUND: Patients with peripheral nerve injury (PNI) often suffer from hypoxic ischemic impairments, in particular when combined with vascular damage, causing neuronal dysfunction and death. Increasing attention has been paid on skin precursor-derived Schwann cells (SKP-SCs), and previous study has shown that SKP-SCs could promote sensory recovery after cell therapy for PNI, resembling the effect of naive SCs, and SKP-SC-derived extracellular vesicles (SKP-SC-EVs) are putatively supposed to be promising therapeutic agents for neural regeneration. METHODS: SKPs were induced to differentiate towards SCs with cocktail factors (N2, neuregulin-1ß, and forskolin) in vitro. SKP-SC-EVs were isolated by exoEasy Maxi Kit and characterized by morphology and phenotypic markers of EVs. Rat sensory neurons from dorsal root ganglions (DRGs) were primarily cultured in regular condition or exposed to oxygen-glucose-deprivation (OGD) condition. SKP-SC-EVs were applied to DRGs or sensory neurons, with LY294002 (a PI3K inhibitor) added; the effect on neurite outgrowth and cell survival was observed. Moreover, microRNA (miR) candidate contained in SKP-SC-EVs was screened out, and miR-mimics were transfected into DRG neurons; meanwhile, the negative regulation of PTEN/PI3K/Akt axis and downstream signaling molecules were determined. RESULTS: It was shown that SKP-SC-EVs could improve the neurite outgrowth of DRGs and sensory neurons. Furthermore, SKP-SC-EVs enhanced the survival of sensory neurons after OGD exposure by alleviating neuronal apoptosis and strengthening cell viability, and the expression of GAP43 (a neuron functional protein) in neurons was upregulated. Moreover, the neuro-reparative role of SKP-SC-EVs was implicated in the activation of PI3K/Akt, mTOR, and p70S6k, as well as the reduction of Bax/Bcl-2 ratio, that was compromised by LY294002 to some extent. In addition, transferring miR-21-5p mimics into sensory neurons could partly protect them from OGD-induced impairment. CONCLUSIONS: Sum up, SKP-SC-EVs could improve neurite outgrowth of DRG sensory neurons in physiological and pathological condition. Moreover, the in vitro therapeutic potential of SKP-SC-EVs on the survival and restoration of OGD-injured sensory neurons was evidenced to be associated with miR-21-5p contained in the small EVs and miR-21-5p/PTEN/PI3K/Akt axis.

Repair of peripheral nerve defects by nerve grafts incorporated with extracellular vesicles from skin-derived precursor Schwann cells.

Our previous studies have shown that extracellular vesicles from skin-derived precursor Schwann cells (SKP-SC-EVs) promote neurite outgrowth of sensory and motor neurons in vitro. This study was aimed at generating an artificial nerve graft incorporated with SKP-SC-EVs to examine in vivo effects of SKP-SC-EVs on peripheral nerve regeneration. Here SKP-SC-EVs were isolated and then identified by morphological observation and phenotypic marker expression. Following co-culture with SCs or motoneurons, SKP-SC-EVs were internalized, showing the capability to enhance SC viability or motoneuron neurite outgrowth. In vitro, SKP-SC-EVs released from Matrigel could maintain cellular uptake property and neural activity. Nerve grafts were developed by incorporating Matrigel-encapsulated SKP-SC-EVs into silicone conduits. Functional evaluation, histological investigation, and morphometric analysis were performed to compare the nerve regenerative outcome after bridging the 10-mm long sciatic nerve defect in rats with our developed nerve grafts, silicone conduits (filled with vehicle), and autografts respectively. Our developed nerve grafts significantly accelerated the recovery of motor, sensory, and electrophysiological functions of rats, facilitated outgrowth and myelination of regenerated axons, and alleviated denervation-induced atrophy of target muscles. Collectively, our findings suggested that incorporation of SKP-SC-EVs into nerve grafts might represent a promising paradigm for peripheral nerve injury repair. STATEMENT OF SIGNIFICANCE: Nerve grafts have been progressively developed to meet the increasing requirements for peripheral nerve injury repair. Here we reported a design of nerve grafts featured by incorporation of Matrigel-encapsulated extracellular vesicles from skin-derived precursor Schwann cells (SKP-SC-EVs), because SKP-SC-EVs were found to possess in vitro neural activity, thus raising the possibility of cell-free therapy. Our developed nerve grafts yielded the satisfactory outcome of nerve grafting in rats with a 10-mm long sciatic nerve defect, as evaluated by functional and morphological assessments. The promoting effects of SKP-SC-EVs-incorporating nerve grafts on peripheral nerve regeneration might benefit from in vivo biological cues afforded by SKP-SC-EVs, which had been released from Matrigel and then internalized by residual neural cells in sciatic nerve stumps.

Extracellular vesicles from skin precursor-derived Schwann cells promote axonal outgrowth and regeneration of motoneurons via Akt/mTOR/p70S6K pathway.

BACKGROUND: Skin precursor-derived Schwann cells (SKP-SCs) have been shown to benefit the recovery of spinal cord injury (SCI) and peripheral nerve injury (PNI) with motor dysfunction. However, the effect of extracellular vesicles (EVs) from SKP-SCs responsible for neuroregeneration remains unknown. METHODS: Based on the obtainment and identification of rat SKP-SCs and their derived EVs, the primary rat injury model of motoneurons resulting from axotomy in vitro or nerve crush in vivo, as well as the secondary rat ischemic hypoxic injury model of motoneuron exposure to oxygen-glucose-deprivation (OGD) in vitro, were treated with EVs from skin precursor-derived Schwann cells (SKP-SC-EVs), respectively. Then, the axonal outgrowth and regrowth was observed and compared, and cell viability as well as the protein kinase B/mammalian target of rapamycin/p70 S6 kinase (Akt/mTOR/p70S6K) signaling pathway was detected, moreover, rapamycin (an mTOR inhibitor) was used to further reveal the underlying molecular mechanism. RESULTS: The internalization of SKP-SC-EVs by neuronal cells was identified in vitro and in vivo. Besides the pro-axonal outgrowth effect of SKP-SC-EVs, prospectively, the treatment of OGD-injured motoneurons with SKP-SC-EVs potentiated the restoration of neuronal viability and axonal regrowth. Furthermore, the axotomizing injury could be improved with SKP-SC-EVs treatment in vitro and in vivo. Finally, it was shown that the application of SKP-SC-EVs could activate the Akt/mTOR/p70S6K signaling pathway that can be abolished by rapamycin. CONCLUSIONS: In summary, the addition of SKP-SC-EVs could regulate the cell growth and death signaling pathway mediated by Akt/mTOR/p70S6K, owing to the transmission of cargos in EVs to damaged motoneurons, which leads to axonal regrowth and neuronal resurrection. Thus, SKP-SC-EVs treatment could be a novel promising strategy for improving the axonal outgrowth and regeneration of motoneurons.