CD206+ M2-like macrophages protect against intervertebral disc degeneration partially by targeting R-spondin-2.

OBJECTIVE: This study aimed to explore the specific function of M2 macrophages in intervertebral disc degeneration (IDD). METHODS: Intervertebral disc (IVD) samples from normal (n = 4) and IDD (n = 6) patients were collected, and the expression of M2-polarized macrophage marker, CD206, was investigated using immunohistochemical staining. Nucleus pulposus cells (NPCs) in a TNF-α environment were obtained, and a mouse caudal IVD puncture model was established. Mice with Rheb deletions, specifically in the myeloid lineage, were generated and subjected to surgery-induced IDD. IDD-induced damage and cell apoptosis were measured using histological scoring, X-ray imaging, immunohistochemical staining, and TdT-mediated dUTP nick end labeling (TUNEL) assay. Finally, mice and NPCs were treated with R-spondin-2 (Rspo2) or anti-Rspo2 to investigate the role of Rspo2 in IDD. RESULTS: Accumulation of CD206 in human and mouse IDD tissues was detected. Rheb deletion in the myeloid lineage (RheBcKO) increased the number of CD206+ M2-like macrophages (mean difference 18.6% [15.7-21.6%], P < 0.001), decreased cell apoptosis (mean difference -15.6% [-8.9 to 22.2%], P = 0.001) and attenuated the IDD process in the mouse IDD model. NPCs treated with Rspo2 displayed increased extracellular matrix catabolism and apoptosis; co-culture with a conditioned medium derived from RheBcKO mice inhibited these changes. Anti-Rspo2 treatment in the mouse caudal IVD puncture model exerted protective effects against IDD. CONCLUSIONS: Promoting CD206+ M2-like macrophages could reduce Rspo2 secretion, thereby alleviating experimental IDD. Rheb deletion may help M2-polarized macrophages accumulate and attenuate experimental IDD partially by inhibiting Rspo2 production. Hence, M2-polarized macrophages and Rspo2 may serve as therapeutic targets for IDD.

lncRNA MEG3 Promotes PDK4/GSK-3ß/ß-Catenin Axis in MEFs by Targeting miR-532-5p.

Studies reported the positive and negative osteogenic effects of MEG3 in mesenchymal stem cells (MSCs). This study aims at clarifying the osteogenic potential of MEG3 and the underlying mechanism. Bone morphogenetic protein 9- (BMP9-) transfected MSCs were recruited as an osteogenic model in vitro, and ectopic bone formation were used in vivo to explore the effect of MEG3 on osteogenesis. We found that overexpression of MEG3 facilitated BMP9-induced osteogenic markers, ALP activities, and matrix mineralization. However, knockdown of MEG3 attenuated BMP9-induced osteogenic markers. MEG3 increased the phosphorylation of GSK-3ß and the protein level of ß-catenin. Pyruvate dehydrogenase kinase 4 (PDK4) can also combine with GSK-3ß and increase the latter phosphorylation. Moreover, MEG3 increased the mRNA level of PDK4. The ceRNA analysis showed that MEG3 may regulate the expression of PDK4 via microRNA 532-5p (miR-532-5p). The MEG3-enhanced GSK-3ß/ß-catenin axis can be attenuated by miR-532-5p, and miR-532-5p inhibitor partly rescued endogenous PDK4 and MEG3-mediated expression of PDK4. MEG3 may potentiate PDK4 and GSK-3ß/ß-catenin by inhibiting miR-532-5p.

The role of aquaporin 4 (AQP4) in spinal cord injury.

Aquaporin-4 (AQP-4) is an aquaporin composed of six helical transmembrane domains and two highly conserved ASN-pro-ALA (NPA) motifs. It is strongly expressed in rodent and human spinal cord tissues and plays a key role in the pathological process after SCI. After SCI, edema, glial scarring, and inflammation can accelerate the progression of injury and lead to deterioration of function. Many studies have reported that AQP-4 plays an important role in SCI. In particular, it plays an important role in secondary pathological processes (spinal cord edema, glial scar formation, and inflammatory response) after SCI. Loss of AQP-4 has been associated with reduced spinal edema and improved prognosis after SCI in mice. In addition, downregulation of AQP-4 reduces glial scar formation and the inflammatory response after SCI. There is a consensus from numerous studies that AQP-4 may be a potential target for SCI therapy, which guides the ongoing investigation for molecular therapy of SCI. Here, we review the structure of AQP-4, its expression in normal and damaged spinal cord, and its role in SCI, as well as discuss the theoretical basis for the treatment of SCI.

Connexin 43 Modulates Osteogenic Differentiation of Bone Marrow Stromal Cells Through GSK-3beta/Beta-Catenin Signaling Pathways.

BACKGROUND/AIMS: Bone marrow stromal cells (BMSCs) are multipotent precursors that give rise to osteoblasts, and contribute directly to bone formation. Connexin 43 (Cx43) is the most ubiquitous gap junction protein expressed in bone cell types, and plays crucial roles in regulating intercellular signal transmission for bone development, differentiation and pathology. However, the precise role and mechanism of Cx43 in BMSCs are less known. Here, we investigate the function of Cx43 in osteogenic differentiation of BMSCs in vitro. METHODS: BMSCs were isolated by whole bone marrow adherent culture. Knock down of Cx43 was performed by using lentiviral transduction of Cx43 shRNA. BMSCs were induced to differentiate by culturing in a-MEM, 10% FBS, 50 µM ascorbic acid, 10 mM beta-glycerophosphate, and 100 nM dexamethasone. Alkaline phosphatase (ALP) activity and alizarin red S staining were used to evaluate osteogenic differentiation in calcium nodules. Target mRNAs and proteins were analyzed by using real-time quantitative PCR (qPCR) and western blotting. RESULTS: Cx43 expression markedly increased during osteogenic differentiation. Osteogenic differentiation was suppressed following lentiviral-mediated knockdown of Cx43 expression, as judged by decreased levels of Runt-related transcription factor 2 (Runx2), bone sialoprotein (BSP), osteocalcin (Bglap), Osterix (Osx), alkaline phosphatase (ALP) activity and the number of calcium nodules in response to osteogenic differentiation stimuli. Knock down of Cx43 reduced the level of phosphorylation of GSK-3beta at Ser9 (p-GSK-3beta), resulting in decreased beta-catenin expression and activation. Furthermore, treatment of Cx43-knockdown cells with lithium chloride (LiCl), a GSK-3beta inhibitor, reduced osteogenic differentiation and decreased GSK-3beta levels, as well as partially rescued levels of both total and activated beta-catenin. CONCLUSION: These findings indicate that Cx43 positively modulates osteogenic differentiation of BMSCs by up-regulating GSK-3beta/beta-catenin signaling pathways, suggesting a potential role for Cx43 in determining bone mass and bone mineral density by modulating osteogenesis.

[Case-control study of therapeutic effects between extreme lateral interbody fusion and conventional posterior operation for the treatment of upper lumbar disc herniation].

OBJECTIVE: To evaluate the clinical outcomes between extreme lateral interbody fusion and conventional posterior operation in the treatment of upper lumbar disc herniation. METHODS: Among 60 patients with upper lumbar disc herniation were treated with extreme lateral interbody fusion(XLIF) or conventional posterior operation from June 2010 to December 2014, 30 patients(19 males and 11 females) were treated with XLIF (XLIF group); and the other 30 patients(17 males and 13 females) were treated with conventional posterior operation (conventional group). In XLIF group, the lesions occurred at T12L1 segments in 2 patients, at L1,2 segments in 6 patients, at L2,3 segments in 10 patients, and at L3,4 segment in 12 patients. In conventional group, the lesions occurred at T12L1 segments in 1 patient, at L1,2 segments in 6 patients, at L2,3 segments in 8 patients, and at L3,4 segment in 15 patients. Operative incision lengths, time, blood loss, postoperative draining volume, hospital stays were recorded. Pre-and post-operative visual analogue score(VAS) and Japanese Orthopedic Association(JOA) were compared between two groups. According to the image data, the intervertebral fusion device was observed to be displaced and the rate of interbody fusion was analyzed. RESULTS: All the patients were followed up, and the duration ranged from 12 to 48 months, with an average of 29 months. The complications included 2 femoral nerve damage in XLIF group (postoperative recovery within 3 months) and superficial incision infection in conventional group(cured by anti-infection). There were no patients with cerebrospinal fluid leakage(CSFL), cauda equina injuries or functional deterioration in the nerve root of lower limbs. In the XLIF group: the operative time was (65.6±20.5) minutes, blood loss was (48.8±15.3) ml, postoperative draining volume was 0 ml. In the conventional group: the operative time was (135.2±33.9) minutes, blood loss was (260.3±125.7) ml, postoperative draining volume was (207.1±50.2) ml. The operative time, blood loss, postoperative draining volume in XLIF group were less than those in the conventional group(P<0.05). The JOA and VAS score were significantly improved in both groups during the follow-up period compared with those before operation(P<0.05). But the difference of the JOA and VAS score between the two groups 1, 6, and 24 months after surgery had not significant differences(P>0.05). There were no significant differences in the fusion rate between the two groups 6 and 12 months after operation(P>0.05). CONCLUSIONS: The XLIF fusion for the treatment of upper lumbar disc herniation has several advantages such as minimal invasive, stable vertebral plate, less complications and postoperative fusion rate, which has a better clinical effect.

Determination of phosphoserine/threonine by nano ultra-performance liquid chromatography-tandem mass spectrometry coupled with microscale labeling.

Protein phosphorylation is an important regulatory post-translational modification in many biochemical processes. The phosphopeptide analysis strategies developed in this study were all at microscale. After using a standard microwave oven to assist protein digestion, phosphoserine and phosphothreonine were tagged with chemical analogues, such as 2-mercaptoethanol and 3-mercapto-1-propanol, to enable simultaneously relative quantitation and identification. This method enabled the use of thio alcohols for direct labeling of phosphorylated sites (not labeled at the mercapto, amino, hydroxyl, or carboxyl groups) of phosphopeptides. Various digestion parameters (e.g., microwave power, reaction time, NH4HCO3 concentration) and derivatization efficiency parameters (e.g., reaction time, labeling tag concentration) were studied and optimized. In both control and experimental samples, microwave-assisted digestion coupled with relative quantitation using analogue tags enabled calculation of phosphopeptide ratios in the same sequence. A non-labeling method was also established for quantifying phosphopeptides in human plasma by using the abundant protein albumin as an internal control for normalizing relative quantities of phosphopeptides. Nano ultra-performance liquid chromatography (nanoUPLC) was combined with LTQ Orbitrap to enable simultaneous protein relative quantitation and identification. These strategies proved to be effective for quantifying phosphopeptides in biological samples.

Sequence-specific detection of trace DNA via a junction-probe electrochemical sensor employed template-enhanced hybridization strategy.

A novel junction-probe electrochemical biosensor for the sequence-specific detection of DNA with higher sensitivity and higher discrimination ability was described in here. This DNA biosensor is based on "junction-probe" detection strategy, which operates via a concept called template-enhanced hybridization processes (TeHyP). TeHyP encompasses a design strategy whereby two probes that do not hybridize to each other at a specific temperature can be made to anneal to each other in the presence of a template (target) via the formation of a ternary complex ("Y" junction structure). The resulting structure that forms after the template-enhanced hybridization then was detected by electrochemical method with [Ru(NH(3))(6)](3+) as signal molecule. We demonstrated that the formation of "Y" junction structure brings more [Ru(NH(3))(6)](3+) to the electrode surface via electrostatic interaction and results in an increasing electrochemical signal. The increasing electrochemical signal sensitively reflects the concentration of target DNA and shows a good linear relationship with the concentration of target DNA. By employing above strategy, this DNA biosensor could detect as low as 7.6 x10(-13)M target DNA and exhibited high discrimination ability even against single-base mismatch. In addition, this novel DNA biosensor is easy to fabricate and convenient to operate, and shows good stability, reproducibility and reusability.

[Effects of enteral supplementation with glutamine on mitochondria respiratory function of intestinal epithelium in burned rats].

OBJECTIVE: To investigate the effects of enteral supplementation with glutamine on mitochondria respiratory function of intestinal epithelium in burned rats. METHODS: Wistar rats inflicted with 30% total body surface area (TBSA) full thickness thermal injury were randomly divided into three groups, i.e. burn with enteral nutrition (EN), burn with glutamine treatment (GLN), and normal control (C) groups. Burned rats were infused 732.2 kJ.kg-1.d-1 solution for intravenous nutrition and oral administration, in which the supply energy ratio of glucose, fat and protein was 55:30:15 respectively, glucose was 15.3% and the proportion of calorie to nitrogen was 183:1. The following indices including respiratory control rate (RCR), oxygen extraction (Oext), P/O ratio and intestine mucosal blood flow (IMBF) were measured on postburn days 1, 3, 5, 7, 10. RESULTS: After burn injury, the RCR, Oext, P/O ratio, and IMBF were significant decreased in both EN and GLN groups, but all above indices were markedly increased in GLN group compared to those in EN group. CONCLUSION: After burn injury, the IMBF and Oext were declined, resulting in mitochondria respiratory oxidative dysfunction and phosphorylation discoupling in intestinal epithelium. GLN supplementation appears to be beneficial to improving IMBF, increasing Oext, abating the extent of mitochondria respiration dysfunction, and promoting oxidative phosphorylation.