Proteomics and bioinformatics reveal insights into neuroinflammation in the acute to subacute phases in rat models of spinal cord contusion injury.

Neuroinflammation is recognized as a hallmark of spinal cord injury (SCI). Although neuroinflammation is an important pathogenic factor that leads to secondary injuries after SCI, neuroprotective anti-inflammatory treatments remain ineffective in the management of SCI. Moreover, the molecular signatures involved in the pathophysiological changes that occur during the course of SCI remain ambiguous. The current study investigated the proteins and pathways involved in C5 spinal cord hemi-contusion injury using a rat model by means of 4-D label-free proteomic analysis. Furthermore, two Gene Expression Omnibus (GEO) transcriptomic datasets, Western blot assays, and immunofluorescent staining were used to validate the expression levels and localization of dysregulated proteins. The present study observed that the rat models of SCI were associated with the enrichment of proteins related to the complement and coagulation cascades, cholesterol metabolism, and lysosome pathway throughout the acute and subacute phases of injury. Intriguingly, the current study also observed that 75 genes were significantly altered in both the GEO datasets, including ANXA1, C1QC, CTSZ, GM2A, GPNMB, and PYCARD. Further temporal clustering analysis revealed that the continuously upregulated protein cluster was associated with immune response, lipid regulation, lysosome pathway, and myeloid cells. Additionally, five proteins were further validated by means of Western blot assays and the immunofluorescent staining showed that these proteins coexisted with the F4/80+ reactive microglia and infiltrating macrophages. In conclusion, the proteomic data pertaining to the current study indicate the notable proteins and pathways that may be novel therapeutic targets for the treatment of SCI.

Advanced oxidation protein products mediate human keratinocytes apoptosis by inducing cell autophagy through the mTOR-Beclin-1 pathway.

Excessive keratinocyte apoptosis leads to impaired wound healing. Recently, advanced oxidation protein products (AOPP) have been recognized as a marker of oxidative stress and a potent inducer of apoptosis. Previously, we have demonstrated that extracellular AOPP accumulation induced keratinocyte apoptosis, and we discovered that autophagy was involved. To further elucidate the role and mechanism of autophagy in AOPP-induced-apoptosis of keratinocytes, we treated HaCaT cells with increasing concentrations of AOPP-human serum albumin or with AOPP-human serum albumin for increasing durations. Cyto-ID solution staining was used to assess cell autophagy using confocal laser scanning microscopy. Autophagy-related protein interactions were investigated using western blot analysis. Exposure of HaCaT cells to AOPP decreased the expression of mammalian target of rapamycin (mTOR) and increased the expression of autophagy-related proteins Beclin-l and LC3, and eventually led to autophagy. Furthermore, an autophagy agonist significantly decreased the expression of apoptosis-related proteins. Taken together, we showed that accumulation of extracellular AOPP induced autophagy in HaCaT cells via a reactive oxygen species-dependent, mTOR-Beclin-1-mediated pathway, and that excessive autophagy-mediated apoptosis, which resulted in delayed wound healing.

Posterior unilateral exposure and stability reconstruction with pedicle and lamina screw fixation for the cervical dumbbell tumorectomy: a case report and biomechanical study.

PURPOSE: Cervical dumbbell tumor is usually removed via a posterior approach and may require the spinal fixation sometimes. However, the present surgical methods involved either more trauma or a higher risk of instability of the cervical spine. A new technique of unilateral exposure and stability reconstruction with pedicle and lamina screws fixation for posterior cervical dumbbell tumorectomy was described and compared with conventional techniques. METHODS: Posterior unilateral exposure, hemi-laminectomy and facetectomy were performed in one patient with the cervical dumbbell tumor between C3 and C4. The stability was reconstructed by the unilateral pedicle and lamina screws fixation (UPLS), and a strip of shaped allograft bone was also implanted between the superior and inferior lateral mass. Biomechanical stability test of this new technique was investigated using seven fresh-frozen human cervical spine specimens (C4-C7) and compared with unilateral pedicle screw (UPS) and bilateral pedicle screw fixation (BPS) techniques. A continuous pure moment of ± 2.0 Nm was applied to the specimen in flexion, extension, lateral bending and axial rotation. RESULTS: The cervical dumbbell tumor was removed completely, and bone fusion with continuous bone trabecula was maintained in the patient on the final follow-up examination at 18 months postoperatively. Biomechanical stability tests revealed that the range of motion of the UPLS fixation plus graft bone implant was the same as the BPS fixation in flexion (1.8°vs. 1.5°, p = 0.58) and extension (2.3°vs. 2.2°, p = 0.73), but significantly bigger in lateral bending (3.9° vs. 1.0°, p < 0.001) and axial rotation (6.8° vs. 3.8°, p = 0.002), which were significantly smaller than the UPS fixation in all directions (all p < 0.001). CONCLUSIONS: For the treatment of cervical dumbbell tumor, posterior unilateral exposure and stability reconstruction with pedicle and lamina screws fixation following hemi-laminectomy and facetectomy appear to be a more stable and lesser trauma technique. LEVEL OF EVIDENCE: Diagnostic: individual cross-sectional studies with consistently applied reference standard and blinding.

Advanced oxidation protein products induce microglia-mediated neuroinflammation via MAPKs-NF-κB signaling pathway and pyroptosis after secondary spinal cord injury.

BACKGROUND: Inflammatory response mediated by oxidative stress is considered as an important pathogenesis of spinal cord injury (SCI). Advanced oxidation protein products (AOPPs) are novel markers of oxidative stress and their role in inflammatory response after SCI remained unclear. This study aimed to investigate the role of AOPPs in SCI pathogenesis and explore the possible underlying mechanisms. METHODS: A C5 hemi-contusion injury was induced in Sprague-Dawley rats to confirm the involvement of AOPPs after SCI. For in vivo study, apocynin, the NADPH oxidase inhibitor was used to study the neuroprotective effects after SCI. For in vitro study, the BV2 microglia cell lines were pretreated with or without the inhibitor or transfected with or without small interference RNA (siRNA) and then stimulated with AOPPs. A combination of molecular and histological methods was used to clarify the mechanism and explore the signaling pathway both in vivo and in vitro. One-way analysis of variance (ANOVA) was conducted with Bonferroni post hoc tests to examine the differences between groups. RESULTS: The levels of AOPPs in plasma and cerebrospinal fluid as well as the contents in the spinal cord showed significant increase after SCI. Meanwhile, apocynin ameliorated tissue damage in the spinal cord after SCI, improving the functional recovery. Immunofluorescence staining and western blot analysis showed activation of microglia after SCI, which was in turn inhibited by apocynin. Pretreated BV2 cells with AOPPs triggered excessive generation of reactive oxygen species (ROS) by activating NADPH oxidase. Increased ROS induced p38 MAPK and JNK phosphorylation, subsequently triggering nuclear translocation of NF-κB p65 to express pro-inflammatory cytokines. Also, treatment of BV2 cells with AOPPs induced NLRP3 inflammasome activation and cleavage of Gasdermin-d (GSDMD), causing pyroptosis. This was confirmed by cleavage of caspase-1, production of downstream mature interleukin (IL)-1ß and IL-18 as well as rupture of rapid cell membrane. CONCLUSIONS: Collectively, these data indicated AOPPs as biomarkers of oxidative stress, modulating inflammatory response in SCI by multiple signaling pathways, which also included the induction of NADPH oxidase dependent ROS, and NLRP3-mediated pyroptosis, and activation of MAPKs and NF-κB.

Serum cystatin C is increased in acute spinal cord injury: a multicentre retrospective study.

STUDY DESIGN: A multicentre retrospective study. OBJECTIVE: A multicentre retrospective study was performed to observe the changes in serum cystatin C (CysC) levels in patients with acute spinal cord injury (SCI). SETTING: Four hospitals in China. METHODS: Over a 5-year study period, the CysC, creatinine (Cr), and blood urea nitrogen (BUN) levels of people who had incurred SCI in the preceding 7 days were collected and compared with those of people with limb fracture (LF) who were matched for injury time and gender. People with SCI also were grouped by injury duration, ASIA Impairment Scale (AIS) grade and the presence or absence of steroid therapy and compared each day. RESULTS: Three hundred and twenty-three samples from people with SCI were retrospectively collected; their mean serum CysC levels were significantly higher than those of people with LF (p < 0.001); No significant difference was observed in Cr or BUN levels between the two groups (p > 0.14). CysC levels increased on the second day, peaked on day 3, and returned to normal on day 5. The more severely injured individuals had higher CysC levels. Steroid therapy or not had no influence for CysC levels. CONCLUSION: CysC levels are increased in patients with acute SCI, possibly as a direct result of injury. Serum CysC is a potential biomarker of SCI.

Advanced oxidative protein products induced human keratinocyte apoptosis through the NOX-MAPK pathway.

Impaired wound healing is a major diabetes-related complication. Keratinocytes play an important role in wound healing. Multiple factors have been proposed that can induce dysfunction in keratinocytes. The focus of present research is at a more specific molecular level. We investigated the role of advanced oxidative protein products (AOPPs) in inducing human immortalized keratinocyte (HaCaT) cell apoptosis and the cellular mechanism underlying the proapoptotic effect of AOPPs. HaCaT cells were treated with increasing concentrations of AOPP-human serum albumin or for increasing time durations. The cell viability was measured using the thiazolyl blue tetrazolium bromide method, and flow cytometry was used to assess the rate of cell apoptosis. A loss of mitochondrial membrane potential (MMP) and an increase in intracellular reactive oxygen species (ROS) were observed through a confocal laser scanning microscope system, and the level of ROS generation was determined using a microplate reader. Nicotinamide adenine dinucleotide phosphate oxidase (NOX)4, extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), and apoptosis-related downstream protein interactions were investigated using the Western blot analysis. We found that AOPPs triggered HaCaT cell apoptosis and MMP loss. After AOPP treatment, intracellular ROS generation increased in a time- and dose-dependent manner. Proapoptotic proteins, such as Bax, caspase 9/caspase 3, and poly(ADP-ribose) polymerase (PARP)-1 were activated, whereas anti-apoptotic Bcl-2 protein was downregulated. AOPPs also increased NOX4, ERK1/2, and p38 MAPK expression. Taken together, these findings suggest that extracellular AOPP accumulation triggered NOX-dependent ROS production, which activated ERK1/2 and p38 MAPK, and induced HaCaT cell apoptosis by activating caspase 3 and PARP-1.

Advanced oxidation protein products induce chondrocyte apoptosis via receptor for advanced glycation end products-mediated, redox-dependent intrinsic apoptosis pathway.

Pro-inflammatory cytokine-induced chondrocyte apoptosis is a primary cause of cartilage destruction in the progression of rheumatoid arthritis (RA). Advanced oxidation protein products (AOPPs), a novel pro-inflammatory mediator, have been confirmed to accumulate in patients with RA. However, the effect of AOPPs accumulation on chondrocyte apoptosis and the associated cellular mechanisms remains unclear. The present study demonstrated that the plasma formation of AOPPs was enhanced in RA rats compared with normal. Then, chondrocyte were treated with AOPPs-modified rat serum albumin (AOPPs-RSA) in vitro. Exposure of chondrocyte to AOPPs activated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and increased expression of NADPH oxidase subunits, which was mediated by receptor for advanced glycation end products (RAGE), but not scavenger receptor CD36. Moreover, AOPPs challenge triggered NADPH oxidase-dependent ROS generation which induced mitochondrial dysfunction and endoplasmic reticulum stress resulted in activation of caspase family that eventually lead to apoptosis. Lastly, blockade of RAGE, instead of CD36, largely attenuated these signals. Our study demonstrated first time that AOPPs induce chondrocyte apoptosis via RAGE-mediated and redox-dependent intrinsic apoptosis pathway in vitro. These data implicates that AOPPs may represent a novel pathogenic factor that contributes to RA progression. Targeting AOPPs-triggered cellular mechanisms might emerge as a promising therapeutic option for patients with RA.

Preoperative Range of Motion in Extension May Influence Postoperative Cervical Kyphosis After Laminoplasty.

STUDY DESIGN: Retrospective observational study. OBJECTIVE: The objective of this study was to investigate factors associated with cervical kyphosis after laminoplasty. SUMMARY OF BACKGROUND DATA: Many factors are reportedly associated with the deterioration of cervical curvature after laminoplasty, including cervical lordosis angle, cervical spine range of motion (ROM), T1 slope, and C2-C7 sagittal vertical axis. Postlaminoplasty kyphosis or deterioration of cervical curvature is likely caused by multiple factors. There is currently no consensus on these issues. MATERIALS AND METHODS: Data of patients treated with laminoplasty for degenerative cervical myelopathy at our institution during 2008-2018 were reviewed. The following variables were collected for each patient: age and sex; follow-up time; surgery involving C3 (yes or no); surgery involving C7 (yes or no); distribution of segments operated on; number of laminae operated on; flexion, extension, and total ROM; cervical lordotic angle; longitudinal distance index; curvature index; C2-C7 sagittal vertical axis; and T1 slope. Logistic regression analysis was used to assess possible risk factors for postoperative kyphosis. Receiver operating characteristic curves were constructed to determine the cutoff values of risk factors. RESULTS: The study cohort comprised 151 patients. Logistic regression analysis indicated that sex, number of laminae operated on, and preoperative extension ROM were significantly associated with postoperative cervical kyphosis ( P <0.05). There was significantly greater postoperative kyphosis in women than in men; the more segments operated on, the greater the risk of postoperative kyphosis, and the larger the preoperative extension ROM, the lower the risk of postlaminoplasty kyphosis. Receiver operating characteristic curve analysis showed that the cutoff value for preoperative extension ROM is 22.1°. CONCLUSIONS: Preoperative extension ROM may be associated with the development of postoperative kyphosis. The cutoff value of preoperative extension ROM that suggested the prospect of postoperative kyphosis in our sample was 22.1°.

Hesperetin ameliorates spinal cord injury by inhibiting NLRP3 inflammasome activation and pyroptosis through enhancing Nrf2 signaling.

Neuroinflammation is a prominent feature of traumatic spinal cord injuries (SCIs). Hesperetin exhibits anti-inflammatory effects in neurological disorders; however, the potential neuroprotective effects of hesperetin in cases of SCI remain unclear. Sprague-Dawley rats with C5 hemi-contusion injuries were used as an SCI model. Hesperetin was administered to the experimental rats in order to investigate its neuroprotective effects after SCI, and BV2 cells were pretreated with hesperetin or silencing of nuclear factor erythroid 2-related factor 2 (siNrf2), and then stimulated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP). The therapeutic impact and molecular mechanism of hesperetin were elucidated in a series of in vivo and in vitro investigations conducted using a combination of experiments. The results of the present in vivo experiment indicated that hesperetin improved functional recovery and protected spinal cord tissue after SCI. Hesperetin attenuated oxidative stress and microglial activation, lowered malondialdehyde (MDA) levels, and elevated catalase (CAT), glutathione (GSH)-Px, and superoxide dismutase (SOD) levels. Moreover, hesperetin downregulated the expression of advanced oxygenation protein products (AOPPs), ionized calcium-binding adapter molecule 1 (Iba-1), NOD-like receptor protein 3 (NLRP3), and interleukin-1 beta (IL-1ß), but increased the expression of Nrf2. In vitro studies have shown that hesperetin inhibits the generation of reactive oxygen species (ROS), as well as the neuroinflammation associated with the upregulation of Nrf2 and heme oxygenase-1 (HO-1) in BV2 cells. The results of the present study indicated that hesperetin inhibited BV2 cell pyroptosis and significantly blocked the expression of NLRP3 inflammasome proteins (NLRP3 Caspase-1 p10 apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain [ASC]) and pro-inflammatory mediators (IL-18, IL-1ß). Furthermore, the silencing of Nrf2 by small interfering ribonucleic acid (siRNA) partially abolished its antioxidant effect in the aforementioned cell experiments. Collectively, these findings illustrate that through an increase in Nrf2 signaling hesperetin reduces oxidative stress and neuroinflammation by suppressing NLRP3 inflammasome activation and pyroptosis.

Should adjacent asymptomatic lumbar disc herniation be simultaneously rectified? A retrospective cohort study of 371 cases that received an open fusion or endoscopic discectomy only on symptomatic segments.

BACKGROUND CONTEXT: In the treatment of multiple disc herniations, the decision of whether to include the presumed asymptomatic lumbar disc herniation (asLDH) at adjacent segments remains uncertain. On the one hand, the untouched asLDH might soon become symptomatic and require treatment. On the other hand, additional surgery involving more segments will introduce greater risk, complications, and cost. PURPOSE: To investigate the prognosis of untreated asLDH after open fusion or percutaneous endoscopic lumbar discectomy (PELD) on symptomatic lumbar disc herniation (LDHs) in patients. STUDY DESIGN: This is a retrospective cohort study. PATIENT SAMPLE: A total of 371 patients with multiple disc herniations who underwent open discectomy and fusion or PELD only for symptomatic levels from January 2012 to July 2018 were included. OUTCOME MEASURES: The primary outcome of interest was the development of symptomatic LDH at the previous asLDH of both groups that required reoperation. A second analysis was performed to compare the reoperation rate due to deterioration of asLDH among different severity grades of herniation. Reoperation rates of the original surgery at the symptomatic segment in both fusion and PELD groups were also reviewed. METHODS: The patients were divided into two groups based on the surgical procedure, with 264 patients undergoing fusion surgery and 107 patients undergoing PELD. Clinical and imaging follow-ups were performed at routine intervals for more than 3 years. The reoperation rates due to deterioration of previously asLDH and failure of original surgery were investigated and compared between the two groups, as well as among the different severity grades of herniation. RESULTS: The follow-up times were 48.2±24.2 and 41.1±17.5 months for the fusion and the PELD groups, respectively. The overall reoperation rate at the previous adjacent asLDH was 6.7% (25/317). According to the severity of the asLDH, a higher grade of asymptomatic herniation yielded a significantly higher rate of reoperation rate in both groups. If the nerve root was displaced by disc material prominently (nG2), the reoperation rate of asLDHs was 42.9% (3/7) in the fusion group and 20% (3/15) in the PELD group. Twenty out of 264 patients (7.6%) in the fusion group and 5 out of 107 patients (4.7%) in the PELD group required reoperation due to deterioration of asLDH. Reoperation rates due to failure of the original surgery were 7.6% (20/264) in the fusion group and 8.4% (9/107) in the PELD group. CONCLUSIONS: With multilevel LDHs, if the asLDH is left untreated, the reoperation rate is closely related to the degree of herniation. When confronting an asLDH graded as G2, a high possibility of reoperation should be clearly discussed with the patient, regardless of open fusion or PELD techniques. Considering that fusion and minimally invasive nonfusion techniques did not yield significantly different overall reoperation rates, ongoing degeneration seemed to have a greater contribution in terms of the deterioration of asLDH.

Pretreatment with kaempferol attenuates microglia-mediate neuroinflammation by inhibiting MAPKs-NF-κB signaling pathway and pyroptosis after secondary spinal cord injury.

Spinal cord injury (SCI) is a devastating injury that characterized by oxidative stress and inflammatory response. Kaempferol is reported to be an anti-neuroinflammation in neurologic disorders. Nevertheless, the role and mechanism of kaempferol in SCI remains unclear. The present study aims to investigate effects of kaempferol on SCI and its possible underlying mechanisms in in vivo and in vitro models. A C5 hemi-contusion injury was induced in Sprague-Dawley rats to investigate the neuroprotective effects of kaempferol after SCI. For in vitro study, the BV2 microglia cell lines were pretreated with or without kaempferol. A combination of molecular and histological methods was used to clarify the mechanism and explore the signaling pathway both in vivo and in vitro. One-way analysis of variance (ANOVA) was conducted with Bonferroni post hoc tests to examine the differences between groups. The in vivo studies showed that kaempferol could improve the recovery of hindlimb motor function and ameliorate tissue damage in the spinal cord after SCI. Moreover, administration of kaempferol reduced microglia activation and oxidative stress level in the spinal cord. The in vitro studies showed that kaempferol suppressed the microglia activation resulting from the administration of LPS with ATP to BV-2 cells. Pretreated BV2 cells with kaempferol reduced the generation of reactive oxygen species (ROS) by inhibiting NADPH oxidase 4, and then, suppressed the phosphorylation of p38 MAPK and JNK, which subsequently inhibited nuclear translocation of NF-κB p65 to express pro-inflammatory factors. We also observed that kaempferol could inhibite the pyroptosis related proteins (NLRP3 Caspase-1 p10 ASC N-GSDMD) and reduce the release of IL-18 and IL-1ß. In conclusion, kaempferol was able to reduce oxidative stress and inflammatory response through down-regulation of ROS dependent MAPKs- NF-κB and pyroptosis signaling pathway, which suggested that kaempferol might be a novel promising therapeutic agent for SCI.

Association between lumbar sacralization and increased degree of vertebral slippage and disc degeneration in patients with L4 spondylolysis.

OBJECTIVEThe aim of this study was to evaluate the effect of lumbar sacralization on the level of vertebral slip and disc degeneration in patients with L4 spondylolysis.METHODSThe authors analyzed data from 102 cases in which patients underwent surgical treatment for L4 spondylolysis and spondylolisthesis at their institution between March 2007 and September 2016. Lumbar sacralization was characterized by the presence of pseudarthrosis and/or bony fusion between the L5 transverse process and sacrum, and the type of lumbosacral transitional vertebra (LSTV) was evaluated with the Castellvi classification. The amount of vertebral slippage was measured using the Taillard technique and Meyerding grade. Degeneration of the L4-5 segment was quantified using the Pfirrmann and Modic classifications. Patients were divided into 2 groups based on the presence or absence of sacralization, and the amount of vertebral slip and degeneration of the L4-5 segment was compared between groups.RESULTSLumbar sacralization was present in 37 (36%) of 102 patients with L4 spondylolysis. The LSTV was type IIa in 10 cases, type IIb in 7, type IIIa in 2, and type IIIb in 18. The levels of vertebral slip and disc degeneration in the group of patients with sacralization were significantly greater than in the group without sacralization. No significant difference was found between the 2 groups with respect to Modic changes.CONCLUSIONSThe increased stability between a sacralized L5 and the sacrum may predispose the L4-5 segment to greater instability and disc degeneration in patients with L4 spondylolysis.

Advanced Oxidative Protein Products Cause Pain Hypersensitivity in Rats by Inducing Dorsal Root Ganglion Neurons Apoptosis via NADPH Oxidase 4/c-Jun N-terminal Kinase Pathways.

Pain hypersensitivity is the most common category of chronic pain and is difficult to cure. Oxidative stress and certain cells apoptosis, such as dorsal root ganglion (DRG) neurons, play an essential role in the induction and development of pain hypersensitivity. The focus of this study is at a more specific molecular level. We investigated the role of advanced oxidative protein products (AOPPs) in inducing hypersensitivity and the cellular mechanism underlying the proapoptotic effect of AOPPs. Normal rats were injected by AOPPs-Rat serum albumin (AOPPs-RSA) to cause pain hypersensitivity. Primary cultured DRG neurons were treated with increasing concentrations of AOPPs-RSA or for increasing time durations. The MTT, flow cytometry and western blot analyses were performed in the DRG neurons. A loss of mitochondrial membrane potential (MMP) and an increase in intracellular reactive oxygen species (ROS) were observed. We found that AOPPs triggered DRG neurons apoptosis and MMP loss. After AOPPs treatment, intracellular ROS generation increased in a time- and dose-dependent manner, whereas, N-acetyl-L-cysteine (NAC), a specific ROS scavenger could inhibit the ROS generation. Proapoptotic proteins, such as Bax, caspase 9/caspase 3, and PARP-1 were activated, whereas anti-apoptotic Bcl-2 protein was down-regulated. AOPPs also increased Nox4 and JNK expression. Taken together, these findings suggest that AOPPs cause pain hypersensitivity in rats, and extracellular AOPPs accumulation triggered Nox4-dependent ROS production, which activated JNK, and induced DRG neurons apoptosis by activating caspase 3 and PARP-1.

Advanced oxidation protein products sensitized the transient receptor potential vanilloid 1 via NADPH oxidase 1 and 4 to cause mechanical hyperalgesia.

Oxidative stress is a possible pathogenesis of hyperalgesia. Advanced oxidation protein products (AOPPs), a new family of oxidized protein compounds, have been considered as a novel marker of oxidative stress. However, the role of AOPPs in the mechanism of hyperalgesia remains unknown. Our study aims to investigate whether AOPPs have an effect on hyperalgesia and the possible underlying mechanisms. To identify the AOPPs involved, we induced hyperalgesia in rats by injecting complete Freund's adjuvant (CFA) in hindpaw. The level of plasma AOPPs in CFA-induced rats was 1.6-fold in comparison with what in normal rats (P<0.05). After intravenous injection of AOPPs-modified rat serum albumin (AOPPs-RSA) in Sprague-Dawley rats, the paw mechanical thresholds, measured by the electronic von Frey system, significantly declined. Immunofluorescence staining indicated that AOPPs increased expressions of NADPH oxidase 1 (Nox1), NADPH oxidase 4 (Nox4), transient receptor potential vanilloid 1 (TRPV1) and calcitonin gene-related peptide (CGRP) in the dorsal root ganglia (DRG) tissues. In-vitro studies were performed on primary DRG neurons which were obtained from both thoracic and lumbar DRG of rats. Results indicated that AOPPs triggered reactive oxygen species (ROS) production in DRG neurons, which were significantly abolished by ROS scavenger N-acetyl-l-cysteine (NAC) and small-interfering RNA (siRNA) silencing of Nox1 or Nox4. The expressions of Nox1, Nox4, TRPV1 and CGRP were significantly increased in AOPPs-induced DRG neurons. And relevant siRNA or inhibitors notably suppressed the expressions of these proteins and the calcium influxes in AOPPs-induced DRG neurons. In conclusion, AOPPs increased significantly in CFA-induced hyperalgesia rats and they activated Nox1/Nox4-ROS to sensitize TRPV1-dependent Ca2+ influx and CGRP release which led to inducing mechanical hyperalgesia.

Effect of advanced oxidation protein products on articular cartilage and synovium in a rabbit osteoarthritis model.

OBJECTIVE: Advanced oxidation protein products (AOPPs), a marker of oxidative stress, are prevalent in many kinds of disorders. Osteoarthritis (OA), mainly resulting from the regression of cartilage, chronic inflammation of the synovium and the subchondral bone remodeling. Although the inflammatory response of AOPPs on fibroblast-like synoviocytes (FLSs) were reported, the effect of AOPPs on cartilage and synovial in vivo remains unclear. Therefore, our study aims to investigate whether AOPPs have an effect on the articular cartilage and synovial in a rabbit model of OA. METHODS: OA model were created by anterior cruciate ligament transection and medial meniscus resection (ACLT + MMx). Forty-eight male New Zealand rabbits were randomly divided into 3 groups: sham-operated group, AOPPs/ACLT + MMx group, and phosphate buffered saline (PBS)/ACLT + MMx group. In sham-operated group, the anterior cruciate ligament was just exposed without transection, and then the incision was sutured. Then intra-articular injection of AOPPs or PBS was performed in the other two groups. Through four weeks and eight weeks of treatment, rabbits in each group were sacrificed. Both hind legs were removed. India ink staining and Safranin O and fast green staining were used to evaluate the macroscopic and microscopic cartilage morphology. The protein expression of matrix metalloproteinases (MMP)-3, MMP-13 in synovium was measured by Western blot. RESULT: The India ink score and Mankin score of AOPPs/ACLT + MMx group were both higher than the other two groups at the two time points. Western blot have revealed that intra-articular injection of AOPPs upregulated the protein expression of MMP-3 and MMP-13 in synovium. CONCLUSION: AOPPs participated in the occurrence and development of OA by upregulating the protein expression of MMP-3 and MMP-13 in synovium.

Effect of low-magnitude whole-body vibration combined with alendronate in ovariectomized rats: a random controlled osteoporosis prevention study.

BACKGROUND: Alendronate (ALE) is a conventional drug used to treat osteoporosis. Low-magnitude whole-body vibration (WBV) exercise has been developed as a potential treatment for osteoporosis. The aim of this study was to investigate whether low-magnitude WBV could enhance the protective effect of ALE on bone properties in ovariectomized rats. METHODS: A total of 128 Sprague-Dawley rats were randomly divided into five groups (SHAM, OVX+VEH, OVX+WBV, OVX + ALE, OVX+WBV+ALE). The level of WBV applied was 0.3 g at 45-55 Hz for 20 min/day, 5 day/week and for 3 months. ALE was administered in dose of 1 mg/Kg once a week. Every four weeks eight rats from each group were sacrificed and their blood and both tibiae were harvested. The expression of osteocalcin and CTX in serum was measured by enzyme-linked immunosorbent assay (ELISA) and the tibiae were subjected to metaphyseal three-point bending and µCT analysis. RESULTS: Osteocalcin rose after ovariectomy and was not appreciably changed by either alendronate or WBV alone or in combination. Alendronate treatment significantly prevented an increase in CTX. WBV alone treatment did not alter this effect. Compared with the OVX+WBV group, nearly all tested indices such as the BV/TV, TV apparent, Tb.N, Tb.Th, and Conn.D were higher in the OVX+ALE group at week 12.Compared with the OVX+WBV group, certain tested indices such as BV/TV, TV apparent, Tb.N, and Con.D, were higher in the OVX+WBV+ALE group at week 12. At week 12, tibiae treated with WBV+ALE exhibited a significantly higher Fmax compared to the OVX+VEH group, and a significant difference was also found in energy absorption between the OVX+WBV+ALE and OVX+VEH groups. CONCLUSIONS: Compared with the WBV, ALE was more effective at preventing bone loss and improved the trabecular architecture. However, WBV enhanced the effect of alendronate in ovariectomized rats by inducing further improvements in trabecular architecture.