Jian-Hua Lin

Effect of neuroglobin genetically modified bone marrow mesenchymal stem cells transplantation on spinal cord injury in rabbits.

Objective This study aims to investigate the potentially protective effect of neuroglobin (Ngb) gene-modified bone marrow mesenchymal stem cells (BMSCs) on traumatic spinal cord injury (SCI) in rabbits. Methods A lentiviral vector containing an Ngb gene was constructed and used to deliver Ngb to BMSCs. Ngb gene-modified BMSCs were then injected at the SCI sites 24 hours after SCI. The motor functions of the rabbits were evaluated by the Basso–Beattie–Bresnahan rating scale. Fluorescence microscopy, quantitative real-time PCRs, Western blots, malondialdehyde (MDA) tests, and terminal deoxynucleotidyltransferase-mediated UTP end labeling assays were also performed. Results Ngb expression in the Ngb-BMSC group increased significantly. A more significant functional improvement was observed in the Ngb-BMSC group compared with those in the other groups. Traumatic SCI seemingly led to an increase in MDA level and number of apoptotic cells, which can be prevented by Ngb-BMSC treatment. Conclusion This study demonstrates that Ngb gene-modified BMSCs can strengthen the therapeutic benefits of BMSCs in reducing secondary damage and improving the neurological outcome after traumatic SCI. Therefore, the combined strategy of BMSC transplantation and Ngb gene therapy can be used to treat traumatic SCI.

Overexpressing neuroglobin improves functional recovery by inhibiting neuronal apoptosis after spinal cord injury

The current study was performed to evaluate the mechanisms and therapeutic effects of overexpressing neuroglobin (Ngb) on spinal cord injury (SCI). Adeno-associated virus (AAV) was injected in the T12 section 7 days before SCI. Animals were randomly divided into four groups: a sham group, a vehicle group, an AAV-EGFP group and an AAV-Ngb group. Recovery of hind limb locomotor function was determined during the 3-week post operation period by the Basso, Beattie and Bresnahan locomotor rating scale. At 24 h after SCI and at the end of the study, the segments of spinal cord, centered with the lesion site were harvested for histopathological analysis. Immunofluorescence was performed using antibodies to recognize neuN in the lesion sections. At 24 h after SCI, the spinal cord tissue samples were removed to analyze tissue concentrations of superoxide dismutase (SOD) and malondialdehyde (MDA). Apoptotic cells were assessed using a terminal deoxynucleotidyl transferase, dUTP nick end labeling (TUNEL) kit. The expression of bcl-2, bax, cytochrome c, and cleaved caspase-3, were determined by Western blot assay and immunostaining analysis. The results showed that animals overexpressing Ngb had significantly greater recovery of locomotor function, less neuronal loss and fewer apoptotic cells. In addition, overexpressing Ngb significantly increased bcl-2 expression and SOD level, decreased bax expression, attenuated the release of cytochrome c from mitochondria to the cytosol fraction, and reduced the activity of caspase-3 and MDA level after SCI. These findings suggest, that overexpressing Ngb can significantly improve the recovery of locomotor function. This neuroprotective effect may be associated with the inhibition of neural apoptosis via the mitochondrial pathway.

Heme oxygenase-1 promotes neuron survival through down-regulation of neuronal NLRP1 expression after spinal cord injury

BackgroundUnderstanding the mechanisms underlying neuronal death in spinal cord injury (SCI) and developing novel therapeutic approaches for SCI-induced damage are critical for functional recovery. Here we investigated the role of heme oxygenase-1 (HO-1) in neuroprotection after SCI.MethodsAdeno-associated virus expressing HO-1 was prepared and injected into rat spinal cords before SCI model was performed. HO-1 expression, inflammasome activation, and the presence of inflammatory cytokines were determined by quantitative polymerase chain reaction, immunohistological staining, immunoblot, and immunoprecipitation. Neuronal apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling. The hindlimb locomotor function was evaluated for extent of neurologic damage. In an in vitro model, hydrogen peroxide was used to induce similar inflammasome activation in cultured primary spinal cord neurons, followed by evaluation of above parameters with or without transduction of HO-1-expressing adeno-associated virus.ResultsEndogenous HO-1 expression was found in spinal cord neurons after SCI in vivo, in association with the expression of Nod-like receptor protein 1 (NLRP1) and the formation of NLRP1 inflammasomes. Administration of HO-1-expressing adeno-associated virus effectively decreased expression of NLRP1, therefore alleviating NLRP1 inflammasome-induced neuronal death and improving functional recovery. In the in vitro model, exogenous HO-1 expression protected neurons from hydrogen peroxide-induced neuronal death by inhibiting NLRP1 expression. In addition, HO-1 inhibited expression of activating transcription factor 4 (ATF4), which is a transcription factor regulating NLRP1 expression.ConclusionsHO-1 protects spinal cord neurons after SCI through inhibiting NLRP1 inflammasome formation.

Polymorphism in the Hypoxia-Inducible Factor 1alpha Gene May Confer Susceptibility to LDD in Chinese Cohort

Objective This study aimed to investigate whether or not hypoxia-inducible factor-1α (HIF-1α) gene variants are associated with the susceptibility and clinical characteristics of lumbar disc degeneration (LDD). Methods We examined 320 patients with LDD and 447 gender- and age-matched control subjects. We also determined the HIF-1α gene variants, including C1772T (P582S) and G1790A (A588T) polymorphisms. Results Significant differences were observed in allelic and genotypic distributions of 1790 A > G polymorphisms between LDD cases and control subjects. Logistic regression revealed that 1790 AA genotypes indicated a protective effect against the development of LDD. The HIF-1α 1790 A > G polymorphisms also affected the severity of LDD as evaluated based on the modified Japanese Orthopedic Association (mJOA) scores. The 1790 AA genotype carriers exhibited significantly lower mJOA scores than AG and GG carriers. C1772T did not show any association with the risk and severity of LDD. Conclusion Our study suggested that HIF-1α 1790 A > G polymorphisms may be used as a molecular marker to determine the susceptibility and severity of LDD.

Heme oxygenase-1 protects spinal cord neurons from hydrogen peroxide-induced apoptosis via suppression of Cdc42/MLK3/MKK7/JNK3 signaling

The mechanisms by which oxidative stress induces spinal cord neuron death has not been completely understood. Investigation on the molecular signal pathways involved in oxidative stress-mediated neuronal death is important for development of new therapeutics for oxidative stress-associated spinal cord disorders. In current study we examined the role of heme oxygenase-1 (HO-1) in the modulation of MLK3/MKK7/JNK3 signaling, which is a pro-apoptotic pathway, after treating primary spinal cord neurons with H2O2. We found that MLK3/MKK7/JNK3 signaling was substantially activated by H2O2 in a time-dependent manner, demonstrated by increase of activating phosphorylation of MLK3, MKK7 and JNK3. H2O2 also induced expression of HO-1. Transduction of neurons with HO-1-expressing adeno-associated virus before H2O2 treatment introduced expression of exogenous HO-1 in neurons. Exogenous HO-1 reduced phosphorylation of MLK3, MKK7 and JNK3. Consistent with its inhibitory effect on MLK3/MKK7/JNK3 signaling, exogenous HO-1 decreased H2O2-induced neuronal apoptosis and necrosis. Furthermore, we found that exogenous HO-1 inhibited expression of Cdc42, which is crucial for MLK3 activation. In addition, HO-1-induced down-regulation of MLK3/MKK7/JNK3 signaling might be related to up-regulation of microRNA-137 (mir-137). A mir-137 inhibitor alleviated the inhibitory effect of HO-1 on JNK3 activation. This inhibitor also increased neuronal death even when exogenous HO-1 was expressed. Therefore, our study suggests a novel mechanism by which HO-1 exerted its neuroprotective efficacy on oxidative stress.

FIM-A, a phosphorus-containing sirolimus, inhibits the angiogenesis and proliferation of osteosarcomas.

The mTOR pathway is a central control of cell growth, proliferation, metabolism, and survival, and is deregulated in most cancers. Cancer cells are addicted to increased activity of mTOR kinase-mediated signaling pathways, leading to numerous inhibitors of mTOR signaling in preclinic and clinical trials for cancer therapy. Phosphorus-containing sirolimus (FIM-A), which targets mTOR signaling, inhibits cancer cell growth in vitro. Here we report that FIM-A reduces the angiogenesis and proliferation of osteosarcoma both in vitro and in vivo. In cultured osteosarcoma cell lines, FIM-A inhibited cell proliferation and arrested cells in the G1 phase of the cell cycle, accompanied with reduction of VEGF and HIF-1alpha. With in vivo mouse osteosarcoma xenografts, FIM-A treatment resulted in the inhibition of mTORC1 signaling as demonstrated by the decreased phosphorylation of p70S6K1 and 4E-BP1. Consistent with this finding, FIM-A significantly decreased the average tumor volume, nuclei staining of PCNA, and the number of intratumoral microvessels. Our data demonstrated that targeting mTORC1 by FIM-A inhibited the growth of osteosarcoma in vitro and in vivo, providing the basis for further development of FIM-A as a therapy for osteosarcoma patients.

Comparison of culture and broad-range polymerase chain reaction methods for diagnosing periprosthetic joint infection: analysis of joint fluid, periprosthetic tissue, and sonicated fluid

PurposeThis study compared the diagnostic capabilities of culture and broad-range polymerase chain reaction (PCR) using joint fluid (JF), periprosthetic tissue (PT), and sonicated fluid (SF) for the diagnosis of periprosthetic joint infection (PJI).MethodsSixty-seven subjects underwent knee or hip revision surgery, with 53 PJI and 14 aseptic failure (AF) cases included retrospectively. JF, PT, and SF samples were collected after a suspension of antibiotics more than two weeks, and culture and broad-range PCR were performed for all samples.ResultsThe sensitivities of SF culture (83.0%), JF-PCR (83.0%), and SF-PCR (84.9%) were similar (P > 0.05), but each was significantly more sensitive than JF culture (69.8%), PT culture (71.7%), and PT-PCR (34.0%) (P < 0.05). The specificities of JF culture, PT culture, SF culture, JF-PCR, PT-PCR, and SF-PCR were similar (100, 100, 85.7, 85.7, 100, and 78.6%, respectively) (P > 0.05). PCR was unable to accurately detect six polymicrobial infections and two fungal infections.ConclusionsSF culture, JF-PCR, and SF-PCR were more sensitive than JF culture, PT culture, and PT-PCR for diagnosing PJI among patients who have stopped taking antibiotics for two weeks or more. Compared with PCR methods, SF culture has the advantage of detecting polymicrobial or fungal infections. PT-PCR proved to be insufficiently sensitive for providing correct diagnoses.

Transplantation of a Peripheral Nerve with Neural Stem Cells Plus Lithium Chloride Injection Promote the Recovery of Rat Spinal Cord Injury

Transplantation of neural stem cells (NSCs) holds great potential for the treatment of spinal cord injury (SCI). However, transplanted NSCs poorly survive in the SCI environment. We injected NSCs into tibial nerve and transplanted tibial nerve into a hemisected spinal cord and investigated the effects of lithium chloride (LiCl) on the survival of spinal neurons, axonal regeneration, and functional recovery. Our results show that most of the transplanted NSCs expressed glial fibrillary acidic protein, while there was no obvious expression of nestin, neuronal nuclei, or acetyltransferase found in NSCs. LiCl treatment produced less macrosialin (ED1) expression and axonal degeneration in tibial nerve after NSC injection. Our results also show that a regimen of LiCl treatment promoted NSC differentiation into NF200-positive neurons with neurite extension into the host spinal cord. The combination of tibial nerve transplantation with NSCs and LiCl injection resulted in more host motoneurons surviving in the spinal cord, more regenerated axons in tibial nerve, less glial scar area, and decreased ED1 expression. We conclude that lithium may have therapeutic potential in cell replacement strategies for central nervous system injury due to its ability to promote survival and neuronal generation of grafted NSCs and reduced host immune reaction.