Nai-Feng Tian

Apoptosis, senescence, and autophagy in rat nucleus pulposus cells: Implications for diabetic intervertebral disc degeneration†‡

This research was aimed to study the mechanisms by which diabetes aggravates intervertebral disc degeneration (IDD) and to discuss the relationship between autophagy and IDD in nucleus pulposus (NP) cells. Sixteen weeks after injecting streptozotocin (STZ), the intervertebral discs (IVDs) were studied by histology, Alcian blue, 1,9‐dimethylmethylene blue (DMMB), immunohistochemistry, and RT‐PCR to explore the IDD. The apoptosis and senescence of NP cells was investigated by terminal deoxyribonucleotidyl transferase (TDT)‐mediated dUTP‐digoxigenin nick end labeling (TUNEL) assay, immunohistochemistry, and Western blot for caspase3, caspase8, caspase9, and p16lnk4A (increased in cellular senescence). The level of autophagy in NP cells was detected by Western blot, immunohistochemistry, and transmission electron microscopy (TEM). The proteoglycan and collagen II in the extracellular matrix and the aggrecan and collagen II mRNA expression in NP cells of diabetic rats were decreased compared with the control group. Diabetes increased apoptosis of NP cells and led to activations of initiators of intrinsic (caspases‐9) and extrinsic (caspase‐8) pathways as well as their common executioner (caspase‐3). Cellular senescence was increased about twofold in NP of diabetic rats. In addition, the Western blot, immunohistochemistry, and TEM demonstrated higher level of autophagy in NP cells of diabetic rats than control rats to a statistically significant extent. These findings support that diabetes induced by STZ can cause IDD by accelerating the apoptosis and senescence of NP cells excluding the overweight influence. And the results suggest that the autophagy may be a response mechanism to the change of NP cells in diabetic rats.

The Relationship Between Vitamin A and Risk of Fracture: Meta-Analysis of Prospective Studies

Osteoporotic fracture is a significant cause of morbidity and mortality and is a challenging global health problem. Previous reports of the relation between vitamin A intake or blood retinol and risk of fracture were inconsistent. We searched Medline and Embase to assess the effects of vitamin A (or retinol or beta‐carotene but not vitamin A metabolites) on risk of hip and total fracture. Only prospective studies were included. We pooled data with a random effects meta‐analysis with adjusted relative risk (adj.RR) and 95% confidence interval (CI). We used Q statistic and I2 statistic to assess heterogeneity and Eggers test to assess publication bias. Eight vitamin A (or retinol or beta‐carotene) intake studies (283,930 participants) and four blood retinol level prospective studies (8725 participants) were included. High intake of vitamin A and retinol were shown to increase risk of hip fracture (adj.RR [95% CI] = 1.29 [1.07, 1.57] and 1.40 [1.03, 1.91], respectively), whereas beta‐carotene intake was not found to increase the risk of hip fracture (adj.RR [95% CI] = 0.82 [0.59, 1.14]). Both high or low level of blood retinol was shown to increase the risk of hip fracture (adj.RR [95% CI] = 1.87 [1.31, 2.65] and 1.56 [1.09, 2.22], respectively). The risk of total fracture does not differ significantly by level of vitamin A (or retinol) intake or by blood retinol level. Dose‐response meta‐analysis shows a U‐shaped relationship between serum retinol level and hip fracture risk. Our meta‐analysis suggests that blood retinol level is a double‐edged sword for risk of hip fracture. To avoid the risk of hip fracture caused by too low or too high a level of retinol concentration, we suggest that intake of beta‐carotene (a provitamin A), which should be converted to retinol in blood, may be better than intake of retinol from meat, which is directly absorbed into blood after intake.

Metformin protects against apoptosis and senescence in nucleus pulposus cells and ameliorates disc degeneration in vivo

Intervertebral disc degeneration (IDD) is a complicated process that involves both cellular apoptosis and senescence. Metformin has been reported to stimulate autophagy, whereas autophagy is shown to protect against apoptosis and senescence. Therefore, we hypothesize that metformin may have therapeutic effect on IDD through autophagy stimulation. The effect of metformin on IDD was investigated both in vitro and in vivo. Our study showed that metformin attenuated cellular apoptosis and senescence induced by tert-butyl hydroperoxide in nucleus pulposus cells. Autophagy, as well as its upstream regulator AMPK, was activated by metformin in nucleus pulposus cells in a dose- and time-dependent manner. Inhibition of autophagy by 3-MA partially abolished the protective effect of metformin against nucleus pulposus cells’ apoptosis and senescence, indicating that autophagy was involved in the protective effect of metformin on IDD. In addition, metformin was shown to promote the expression of anabolic genes such as Col2a1 and Acan expression while inhibiting the expression of catabolic genes such as Mmp3 and Adamts5 in nucleus pulposus cells. In vivo study illustrated that metformin treatment could ameliorate IDD in a puncture-induced rat model. Thus, our study showed that metformin could protect nucleus pulposus cells against apoptosis and senescence via autophagy stimulation and ameliorate disc degeneration in vivo, revealing its potential to be a therapeutic agent for IDD.

Stimulation of autophagy promotes functional recovery in diabetic rats with spinal cord injury

In this study we examined the relationship between autophagy and apoptosis in diabetic rats after spinal cord injury (SCI), also we determined the role of autophagy in diabetes-aggravated neurological injury in vivo and in vitro. Our results showed that diabetes decreased the survival of neurons, promoted astrocytes proliferation, increased inflammatory cells infiltration and inhibited functional recovery after SCI. Diabetes was shown to confer increased activation of apoptotic pathways, along with an increase in autophagy; similar effects were also observed in vitro in neuronal PC12 cells. Treatment with rapamycin, an autophagy activator, partially abolished the adverse effect of diabetes, suggesting that diabetes may enhance neurological damage and suppress locomotor recovery after SCI, in addition to its effects on apoptosis and autophagy. In contrast, further stimulation of autophagy improved neurological function via inhibition of apoptosis. These results explained how diabetes exacerbates SCI in cellular level and suggested autophagy stimulation to be a new therapeutic strategy for diabetic SCI.

Metformin Improves Functional Recovery After Spinal Cord Injury via Autophagy Flux Stimulation.

Spinal cord injury (SCI) is a severe neurological disease with few efficacious drugs. Autophagy is a cellular process to confront with stress after SCI and considered to be a therapeutic target of SCI. In this study, we investigated the therapeutic effect of metformin on functional recovery after SCI and its underlying mechanism of autophagy regulation. Using a rat model of traumatic SCI, we found improved function recovery which was paralleled by a reduction of apoptosis after metformin treatment. We further examined autophagy via detecting autophagosomes by transmission electron microscopy and immunofluorescence, as well as autophagy markers by western blot in each groups. The results showed that the number of autophagosomes and expression of autophagy markers such as LC3 and beclin1 were increased in SCI group, while autophagy substrate protein p62 as well as ubiquitinated proteins were found to accumulate in SCI group, indicating an impaired autophagy flux in SCI. But, metformin treatment attenuated the accumulation of p62 and ubiquitinated proteins, suggesting a stimulative effect of autophagy flux by metformin. Blockage of autophagy flux by chloroquine partially abolished the apoptosis inhibition and functional recovery effect of metformin on SCI, which suggested that the protective effect of metformin on SCI was through autophagy flux stimulation. Activation of AMPK as well as inhibition of its downstream mTOR signaling were detected under metformin treatment in vivo and in vitro; inhibition of AMPK signaling by compound C suppressed autophagy flux induced by metformin in vitro, indicating that AMPK signaling was involved in the effect of metformin on autophagy flux regulation. Together, these results illustrated that metformin improved functional recovery effect through autophagy flux stimulation and implied metformin to be a potential drug for SCI therapy.

Fusion versus Nonfusion for Surgically Treated Thoracolumbar Burst Fractures: A Meta-Analysis

Background Posterior pedicle screw fixation has become a popular method for treating thoracolumbar burst fractures. However, it remains unclear whether additional fusion could improve clinical and radiological outcomes. This meta-analysis was performed to evaluate the effectiveness of fusion as a supplement to pedicle screw fixation for thoracolumbar burst fractures. Methodology/Principal Findings MEDLINE, OVID, Springer, and Google Scholar were searched for relevant randomized and quasi-randomized controlled trials that compared the clinical and radiological efficacy of fusion versus nonfusion for thoracolumbar burst fractures managed by posterior pedicle screw fixation. Risk of bias in included studies was assessed using the Cochrane Risk of Bias tool. We generated pooled risk ratios or weighted mean differences across studies. Based on predefined inclusion criteria, 4 eligible trials with a total of 220 patients were included in this meta-analysis. The mean age of the patients was 35.1 years. 96.8% of the fractures were located at T12 to L1 level. Baseline characteristics were similar between the fusion and nonfusion groups. No significant difference was identified between the two groups regarding radiological outcome, functional outcome, neurologic improvement, and implant failure rate. The pooled data showed that the nonfusion group was associated with significantly reduced operative time (p<0.0001) and blood loss (p  = 0.0003). Conclusions/Significances The results of this meta-analysis suggested that fusion was not necessary when thoracolumbar burst fracture was treated by posterior pedicle screw fixation. More randomized controlled trials with high quality are still needed in the future.

Pooled Analysis of Non-Union, Re-Operation, Infection, and Approach Related Complications after Anterior Odontoid Screw Fixation

Background Anterior odontoid screw fixation (AOSF) has been one of the most popular treatments for odontoid fractures. However, the true efficacy of AOSF remains unclear. In this study, we aimed to provide the pooled rates of non-union, reoperation, infection, and approach related complications after AOSF for odontoid fractures. Methods We searched studies that discussed complications after AOSF for type II or type III odontoid fractures. A proportion meta-analysis was done and potential sources of heterogeneity were explored by meta-regression analysis. Results Of 972 references initially identified, 63 were eligible for inclusion. 54 studies provided data regarding non-union. The pooled non-union rate was 10% (95% CI: 7%–3%). 48 citations provided re-operation information with a pooled proportion of 5% (95% CI: 3%–7%). Infection was described in 20 studies with an overall rate of 0.2% (95% CI: 0%–1.2%). The main approach related complication is postoperative dysphagia with a pooled rate of 10% (95% CI: 4%–17%). Proportions for the other approach related complications such as postoperative hoarseness (1.2%, 95% CI: 0%–3.7%), esophageal/retropharyngeal injury (0%, 95% CI: 0%–1.1%), wound hematomas (0.2%, 95% CI: 0%–1.8%), and spinal cord injury (0%, 95% CI: 0%–0.2%) were very low. Significant heterogeneities were detected when we combined the rates of non-union, re-operation, and dysphagia. Multivariate meta-regression analysis showed that old age was significantly predictive of non-union. Subgroup comparisons showed significant higher non-union rates in age ≥70 than that in age ≤40 and in age 40 to <50. Meta-regression analysis did not reveal any examined variables influencing the re-operation rate. Meta-regression analysis showed age had a significant effect on the dysphagia rate. Conclusions/Significances This study summarized the rates of non-union, reoperation, infection, and approach related complications after AOSF for odontoid factures. Elderly patients were more likely to experience non-union and dysphagia.

Differentiation of Menstrual Blood–derived Stem Cells Toward Nucleus Pulposus-like Cells in a Coculture System With Nucleus Pulposus Cells

Study Design. Human stromal stem cells derived from menstrual blood (MenSCs) and nucleus pulposus (NP) cells were cocultured under normal or low oxygen (O2) condition. Objective. To assess the differentiation capability of MenSCs toward nucleus pulposus cells under normal or low oxygen condition. Summary of Background Data. Given the proliferative capacity and pluripotentiality of mesenchymal stem cells, mesenchymal stem cells transplantation is thought to be a promising approach to managing intervertebral disc degeneration. Methods. Using coculture plates with 0.4-&mgr;m pore size polyethylene terephthalate track-etched inserts, MenSCs and NP cells (1:1 ratio) were cocultured with cell-to-cell contact for 2 weeks in normal (20% O2) or low oxygen tension (2% O2), respectively. Extracellular matrix accumulation was quantified by dimethylmethylene blue assay, histological staining, and quantitative reverse-transcription polymerase chain reaction. Novel characteristic human NP markers cytokeratin-19 (KRT19), carbonic anhydrase XII (CA12), and forkhead box F1 (FoxF1) were also detected by quantitative reverse-transcription polymerase chain reaction. Results. The result of quantitative reverse-transcription polymerase chain reaction showed that aggrecan and COL2A1 genes expression was significantly increased in differentiated MenSCs (P < 0.05). There was significantly more COL2A1 gene expression in normoxic group than that in low O2 group (P < 0.05). But no significant difference was observed in aggrecan gene expression between normoxic group and low O2 group. These aforementioned results were also confirmed by histological analysis. We also found that the characteristic NP markers (KRT19, CA12, FoxF1) were significantly upregulated in differentiated MenSCs. Moreover, low O2 tension (2%) further enhanced these genes expression (P < 0.05). Conclusion. In our study, MenSCs were successfully differentiated into NP-like cells and may become a new source of seed cells for the treatment of intervertebral disc degeneration in the future. Level of Evidence: N/A

Menopause is associated with lumbar disc degeneration: a review of 4230 intervertebral discs

Abstract Objective The main objective of this study was to investigate, in a population of normal postmenopausal women, the association between menopause and severity of lumbar disc degeneration from the first lumbar to the first sacral vertebra on magnetic resonance imaging. Methods Between January 2010 and May 2013, 846 normal women and 4230 intervertebral discs were retrospectively analyzed. Age, height, weight and years since menopause (YSM) were recorded. Disc degeneration was evaluated using the modified Pfirrmann grading system. Results Compared to premenopausal and perimenopausal women, postmenopausal women had more severe disc degeneration after removal of age, height and weight effects (p < 0.0001). Postmenopausal women were divided into six subgroups for every 5 YSM. When YSM was below 15 years, there was a significant difference between every two groups, i.e. groups 1–5 YSM, 6–10 YSM and 11–15 YSM (p < 0.01). A positive trend was observed between YSM and severity of disc degeneration, respectively, i.e. L1/L2 (r = 0.235), L2/L3 (r = 0.161), L3/L4 (r = 0.173), L4/L5 (r = 0.146), L5/S1 (r = 0.137) and all lumbar discs (r = 0.259) (p < 0.05 or 0.01). However, when YSM was above 15, there was no difference, i.e. groups 16–20 YSM, 21–25 YSM and 26–30 YSM (p > 0.05), and the significance correlation also disappeared (p > 0.05). Conclusion Menopause is associated with disc degeneration in the lumbar spine. The association almost entirely occurred in the first 15 years since menopause, suggesting estrogen decrease may be a risk factor for lumbar disc degeneration.

Lactate down‐regulates matrix systhesis and promotes apoptosis and autophagy in rat nucleus pulposus cells

The intervertebral disk (IVD) is avascular and anaerobic glycolysis has been recognized as the main source of energy. Due to anaerobic glycolysis, there are high levels of lactate production in disk. Previous study shows lactate concentration is elevated in the degenerated IVD. However, it is not clear how lactate causes degeneration of disks. In this study, we found that 2 mM lactate promote proliferation of NP cells, while 6 mM lactate slightly inhibit their proliferation. By detection under transmission electron microscopy, and western bolt for autophagy related protein beclin‐1, LC3 and p62, we demonstrated that 6 mM lactate leads to autophagy induction of NP cells. TUNEL results showed that the apoptosis incidence was increased. High lactate concentration induced the degradation in protein expression and mRNA level of GAG content, type II collagen and slight increase of type I collagen. Based on these observations, we conclude that high lactate concentration is a pathogenic factor for IVD degeneration, and lactate metabolism may be a new therapeutic target for IVD degeneration.