Schwann Cell-Derived Exosomes and Methylprednisolone Composite Patch for Spinal Cord Injury Repair.

Spinal cord injury (SCI) can cause permanent loss of sensory and motor function, and there is no effective clinical treatment, to date. Due to the complex pathological process involved after injury, synergistic treatments are very urgently needed in clinical practice. We designed a nanofiber scaffold hyaluronic acid hydrogel patch to release both exosomes and methylprednisolone to the injured spinal cord in a non-invasive manner. This composite patch showed good biocompatibility in the stabilization of exosome morphology and toxicity to nerve cells. Meanwhile, the composite patch increased the proportion of M2-type macrophages and reduced neuronal apoptosis in an in vitro study. In vivo, the functional and electrophysiological performance of rats with SCI was significantly improved when the composite patch covered the surface of the hematoma. The composite patch inhibited the inflammatory response through macrophage polarization from M1 type to M2 type and increased the survival of neurons by inhibition neuronal of apoptosis after SCI. The therapeutic effects of this composite patch can be attributed to TLR4/NF-κB, MAPK, and Akt/mTOR pathways. Thus, the composite patch provides a medicine-exosomes dual-release system and may provide a non-invasive method for clinical treatment for individuals with SCI.

The discovery of the new mechanism: Celastrol improves spinal cord injury by increasing cAMP through VIP-ADCYAP1R1-GNAS pathway.

Spinal cord injury (SCI) is a debilitating condition that results in significant impairment of motor function and sensation. Despite the ongoing efforts to develop effective treatments, there are currently very limited options available for patients with SCI. Celastrol, a natural anti-inflammatory compound extracted from Tripterygium wilfordii, has been shown to exhibit anti-inflammatory and anti-apoptotic properties. In this study, we aimed to explore the therapeutic potential of celastrol for SCI and elucidate the underlying molecular mechanisms involved. We found that local tissue often experiences a significant decrease in cAMP content and occurrs apoptosis after SCI. However, the treatment of celastrol could promote the production of cAMP by up-regulating the VIP-ADCYAP1R1-GNAS pathway. This could effectively inhibit the phosphorylation of JNK and prevent apoptosis, ultimately improving the exercise ability after SCI. Together, our results reveal celastrol may be a promising therapeutic agent for the treatment of SCI.

Growth hormone treatment improves the development of follicles and oocytes in prepubertal lambs.

BACKGROUND: When prepubertal lambs are superovulated, the ovarian response to gonadotropin stimulation has great individual difference and the collected oocytes have lower developmental ability than that of adult ewes. Over the years, growth hormone (GH) has been used in assisted reproduction because it can improve the reproductive performance in humans and animals. However, the effect of GH on ovaries and oocytes of prepubertal lambs remains unclear. METHODS: Before and during follicle-stimulating hormone (FSH) superovulation of prepubertal lambs (4‒6-week-old), the lambs were treated with high (50 mg) or low dose (25 mg) of ovine GH in a long (5 days) or short (2 days) period. The recovered oocytes were used for in vitro maturation and fertilization, and several parameters of oocyte quality and development capacity were evaluated. The possible underlying mechanisms of GH action were explored by analysis of granulosa cell (GC) transcriptome, ovarian proteome and follicular fluid metabolome. RESULTS: Treatment of lambs with 50 mg GH over 5 days (long treatment) potentially promoted the response of lambs to superovulation and improved the development capacity of retrieved oocytes, consequently increasing the high quality embryo yield from lambs. A number of differently expressed genes or proteins were found in ovaries between GH-treated and untreated lambs. Cellular experiments revealed that GH reduced the oxidative stress of GCs and promoted the GC proliferation probably through activation of the PI3K/Akt signaling pathway. Finally, analysis of follicular fluid metabolome indicated that GH treatment altered the abundance of many metabolites in follicular fluid, such as antioxidants and fatty acids. CONCLUSIONS: GH treatment has a beneficial role on function of lamb ovaries, which supports the development of follicles and oocytes and improves the efficiency of embryo production from prepubertal lambs.

Ang-(1-7)/MasR axis promotes functional recovery after spinal cord injury by regulating microglia/macrophage polarization.

BACKGROUND: Inflammatory response is an essential part of secondary injury after spinal cord injury (SCI). During this period, the injury may be exacerbated through the release of a large number of inflammatory factors and the polarization of infiltrating macrophages and microglia towards M1. Ang-(1-7), mainly generated by Ang II via angiotensin-converting enzyme 2 (ACE2), can specifically bind to the G protein-coupled receptor Mas (MasR) and plays an important role in regulating inflammation and alleviating oxidative stress. METHODS: We aimed to investigate whether activating the Ang-(1-7)/MasR axis in rats after SCI can regulate local neuroinflammation to achieve functional recovery and obtain its potential mechanism. MasR expression of bone marrow-derived macrophages was determined by Western blot. Immunofluorescence, Western blot, Flow cytometry, and RT-qPCR were applied to evaluate the polarization of Ang-(1-7) on macrophages and the regulation of inflammatory cytokines. Previous evaluation of the spinal cord and bladder after SCI was conducted by hematoxylin-eosin staining, Basso, Beattie, and Bresnahan (BBB) score, inclined plate test, electrophysiology, and catwalk were used to evaluate the functional recovery of rats. RESULTS: MasR expression increased in macrophages under inflammatory conditions and further elevated after Ang-(1-7) treatment. Both in vivo and in vitro results confirmed that Ang-(1-7) could regulate the expression of inflammatory cytokines by down-regulating proinflammatory cytokines and up-regulating anti-inflammatory cytokines, and bias the polarization direction of microglia/macrophages to M2 phenotypic. After SCI, Ang-(1-7) administration in situ led to better histological and functional recovery in rats, and this recovery at least partly involved the TLR4/NF-κB signaling pathway. CONCLUSION: As shown in our data, activating Ang-(1-7)/MasR axis can effectively improve the inflammatory microenvironment after spinal cord injury, promote the polarization of microglia/macrophages towards the M2 phenotype, and finally support the recovery of motor function. Therefore, we suggest using Ang-(1-7) as a feasible treatment strategy for spinal cord injury to minimize the negative consequences of the inflammatory microenvironment after spinal cord injury.

Schwann cell-derived exosomes containing MFG-E8 modify macrophage/microglial polarization for attenuating inflammation via the SOCS3/STAT3 pathway after spinal cord injury.

Macrophage/microglia polarization acts as an important part in regulating inflammatory responses in spinal cord injury (SCI). However, the regulation of inflammation of Schwann cell-derived exosomes (SCDEs) for SCI repair is still unclear. Therefore, we intend to find out the effect of SCDEs on regulating the inflammation related to macrophage polarization during the recovery of SCI. Firstly, the thesis demonstrated that SCDEs could attenuate the LPS- inflammation in BMDMs by suppressing M1 polarization and stimulating M2 polarization. Similarly, SCDEs improved functional recovery of female Wistar rats of the SCI contusion model according to BBB (Basso, Beattie, and Bresnahan) score, electrophysiological assay, and the gait analysis system of CatWalk XT. Moreover, MFG-E8 was verified as the main component of SCDEs to improve the inflammatory response by proteomic sequencing and lentiviral transfection. Improvement of the inflammatory microenvironment also inhibited neuronal apoptosis. The knockout of MFG-E8 in SCs can reverse the anti-inflammatory effects of SCDEs treatment. The SOCS3/STAT3 signaling pathway was identified to participate in upregulating M2 polarization induced by MFG-E8. In conclusion, our findings will enrich the mechanism of SCDEs in repairing SCI and provide potential applications and new insights for the clinical translation of SCDEs treatment for SCI.

A Conformational Restriction Strategy for the Control of CRISPR/Cas Gene Editing with Photoactivatable Guide RNAs.

The CRISPR/Cas system is one of the most powerful tools for gene editing. However, approaches for precise control of genome editing and regulatory events are still desirable. Here, we report the spatiotemporal and efficient control of CRISPR/Cas9- and Cas12a-mediated editing with conformationally restricted guide RNAs (gRNAs). This approach relied on only two or three pre-installed photo-labile substituents followed by an intramolecular cyclization, representing a robust synthetic method in comparison to the heavily modified linear gRNAs that often require extensive screening and time-consuming optimization. This tactic could direct the precise cleavage of the genes encoding green fluorescent protein (GFP) and the vascular endothelial growth factor A (VEGFA) protein within a predefined cutting region without notable editing leakage in live cells. We also achieved light-mediated myostatin (MSTN) gene editing in embryos, wherein a new bow-knot-type gRNA was constructed with excellent OFF/ON switch efficiency. Overall, our work provides a significant new strategy in CRISPR/Cas editing with modified circular gRNAs to precisely manipulate where and when genes are edited.

Bioinformatics analysis of ferroptosis in spinal cord injury.

Ferroptosis plays a key role in aggravating the progression of spinal cord injury (SCI), but the specific mechanism remains unknown. In this study, we constructed a rat model of T10 SCI using a modified Allen method. We identified 48, 44, and 27 ferroptosis genes that were differentially expressed at 1, 3, and 7 days after SCI induction. Compared with the sham group and other SCI subgroups, the subgroup at 1 day after SCI showed increased expression of the ferroptosis marker acyl-CoA synthetase long-chain family member 4 and the oxidative stress marker malondialdehyde in the injured spinal cord while glutathione in the injured spinal cord was lower. These findings with our bioinformatics results suggested that 1 day after SCI was the important period of ferroptosis progression. Bioinformatics analysis identified the following top ten hub ferroptosis genes in the subgroup at 1 day after SCI: STAT3, JUN, TLR4, ATF3, HMOX1, MAPK1, MAPK9, PTGS2, VEGFA, and RELA. Real-time polymerase chain reaction on rat spinal cord tissue confirmed that STAT3, JUN, TLR4, ATF3, HMOX1, PTGS2, and RELA mRNA levels were up-regulated and VEGFA, MAPK1 and MAPK9 mRNA levels were down-regulated. Ten potential compounds were predicted using the DSigDB database as potential drugs or molecules targeting ferroptosis to repair SCI. We also constructed a ferroptosis-related mRNA-miRNA-lncRNA network in SCI that included 66 lncRNAs, 10 miRNAs, and 12 genes. Our results help further the understanding of the mechanism underlying ferroptosis in SCI.

Quantitative iTRAQ proteomics reveal the proteome profiles of bone marrow mesenchymal stem cells after cocultures with Schwann cells in vitro.

Background: Bone marrow mesenchymal stem cells (BMSCs) combined with Schwann cells (SCs) represent a better therapeutic cell transplantation strategy for treating spinal cord injury (SCI) than transplantation with BMSCs or SCs alone. In previous studies, we demonstrated that BMSCs are able to differentiate in neuron-like cells when cocultured with SCs. The detailed mechanism underlying SCI repair that occurs during the combined transplantation of BMSCs and SCs has not yet been studied. In this study, we adopted an isobaric tag for relative and absolute quantitation (iTRAQ)-based protein identification/quantification approach to examine the effects of the SC and BMSC coculture process on the BMSCs and then obtained and analyzed the differentially expressed proteins (DEPs) and their possible related pathways. Methods: This study included three groups based on the number of coculture days (i.e., 0, 3, and 7 days). Changes in BMSC protein expression levels were measured using the iTRAQ technique. A bioinformatics analysis of all the data was performed. Results: In total, 6,760 types of proteins were detected, corresponding to 5,181 data points with quantitative information. Of these, a total of 243 DEPs were identified, of which 169 proteins were upregulated and 74 proteins were downregulated. These DEPs were identified by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Intercellular adhesion molecule-1 (ICAM-1), integrin, and dioxygenase may play crucial roles in the repair of SCI. The data analysis indicates that the relevant biological processes may be regulated by lysosome function, cell adhesion molecules (CAMs), leukocyte transendothelial migration, and the phosphatidylinositol-3-kinase (PI3K) and peroxisome proliferator-activated receptor (PPAR) signaling pathways. Conclusions: The data provided in this study indicate that several molecular mechanisms and signaling pathways are involved in the BMSC and SC coculture process. This information may be useful for the further identification of specific targets and related mechanisms and guide new directions for SCI treatment.

Microgel reinforced zwitterionic hydrogel coating for blood-contacting biomedical devices.

Zwitterionic hydrogels exhibit eminent nonfouling and hemocompatibility. Several key challenges hinder their application as coating materials for blood-contacting biomedical devices, including weak mechanical strength and low adhesion to the substrate. Here, we report a poly(carboxybetaine) microgel reinforced poly(sulfobetaine) (pCBM/pSB) pure zwitterionic hydrogel with excellent mechanical robustness and anti-swelling properties. The pCBM/pSB hydrogel coating was bonded to the PVC substrate via the entanglement network between the pSB and PVC chain. Moreover, the pCBM/pSB hydrogel coating can maintain favorable stability even after 21 d PBS shearing, 0.5 h strong water flushing, 1000 underwater bends, and 100 sandpaper abrasions. Notably, the pCBM/pSB hydrogel coated PVC tubing can not only mitigate the foreign body response but also prevent thrombus formation ex vivo in rats and rabbits blood circulation without anticoagulants. This work provides new insights to guide the design of pure zwitterionic hydrogel coatings for biomedical devices.

Molecular chaperone GroEL-GroES enhances the soluble expression of biologically active ovine growth hormone in the prokaryotic system.

Growth hormone (GH) plays important roles in growth and development of mammalian animals and is valuable for many applications. This study aimed to express and purify biological active recombinant ovine growth hormone (roGH) through prokaryotic expression system. The roGH coding sequence was ligated into the prokaryotic expression vector and transformed into Escherichia coli (E. coli) for protein expression. Factors that influence the roGH expression were examined and the appropriate culture temperature (20 °C) and inducer (IPTG) concentration (25 µM) were determined. To enhance the soluble expression of the protein, co-expression with the molecular chaperone GroEL-GroES was utilized and eventually achieved a high yield of soluble roGH expressed in E. coli. Further, the fusion tag in expressed target protein could be efficiently removed through thrombin-specific cleavage. The expressed roGH was identified by Western blotting and the LC-MS spectrum confirmed its molecular weight of 22749.22 Da. Finally, the purified roGH had an expected biological activity when assayed in cell models in vitro and experimental mouse in vivo. In conclusion, the present study established an efficient and simple approach to produce recombinant GH, and facilitate relevant research and applications.

Application value of biofluid-based biomarkers for the diagnosis and treatment of spinal cord injury.

Recent studies in patients with spinal cord injuries (SCIs) have confirmed the diagnostic potential of biofluid-based biomarkers, as a topic of increasing interest in relation to SCI diagnosis and treatment. This paper reviews the research progress and application prospects of recently identified SCI-related biomarkers. Many structural proteins, such as glial fibrillary acidic protein, S100-ß, ubiquitin carboxy-terminal hydrolase-L1, neurofilament light, and tau protein were correlated with the diagnosis, American Spinal Injury Association Impairment Scale, and prognosis of SCI to different degrees. Inflammatory factors, including interleukin-6, interleukin-8, and tumor necrosis factor α, are also good biomarkers for the diagnosis of acute and chronic SCI, while non-coding RNAs (microRNAs and long non-coding RNAs) also show diagnostic potential for SCI. Trace elements (Mg, Se, Cu, Zn) have been shown to be related to motor recovery and can predict motor function after SCI, while humoral markers can reflect the pathophysiological changes after SCI. These factors have the advantages of low cost, convenient sampling, and ease of dynamic tracking, but are also associated with disadvantages, including diverse influencing factors and complex level changes. Although various proteins have been verified as potential biomarkers for SCI, more convincing evidence from large clinical and prospective studies is thus required to identify the most valuable diagnostic and prognostic biomarkers for SCI.

Effects of exosome-mediated delivery of myostatin propeptide on functional recovery of mdx mice.

Duchenne muscular dystrophy (DMD) is a devastating disorder caused by loss of functional dystrophin protein, resulting in muscle wasting. Enhancing muscle growth by inhibiting myostatin, a growth factor negatively regulating skeletal muscle mass, is a promising approach to slow disease progression. Direct administration of myostatin propeptide, a natural inhibitor of mature myostatin, has shown limited efficacy probably due to low serum stability. Here, we demonstrate that serum stability, delivery efficiency and efficacy of propeptide can be significantly enhanced by anchoring propeptide to the surface of exosomes by fusing the inhibitory domain of myostatin propeptide into the second extracellular loop of CD63 (EXOpro). Repeated administrations of EXOpro accelerated muscle regeneration and growth, resulting in significantly increased muscle mass and functional rescue without any detectable toxicity in mdx mice. Importantly, EXOpro partially rehabilitated bone structure and promoted bone regeneration in mdx mice. Our findings demonstrate that anchoring to exosomes increased delivery and serum stability of propeptide and augmented the inhibitory efficacy of myostatin propeptide and thus provide a delivery platform for propeptide-based intervention in DMD.