Yaosen Wu

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.

The inhibition of EZH2 ameliorates osteoarthritis development through the Wnt/β-catenin pathway

The purpose of our study was to elucidate the role of the histone methyltransferase enhancer of zeste homologue 2 (EZH2) in the pathophysiology of osteoarthritis (OA) and to develop a strategy to modulate EZH2 activity for OA treatment. The expression of EZH2 in normal and OA human cartilage was compared by western blotting. The effect of EZH2 overexpression and inhibition on chondrocyte hypertrophy related gene expression was examined by real-time PCR, and histone methylation on the promoter of the Wnt inhibitor SFRP1 was analyzed using a chromatin immunoprecipitation (ChIP) PCR. Histological assessment of OA mice joint was carried out to assess the in vivo effects of EZH2 inhibitor EPZ005687. We found EZH2 level was significantly increased in the chondrocytes of OA patients compared to normal humans. Overexpression of EZH2 promoted Indian Hedgehog, MMP-13, ADAMTS-5 and COLX expression, while inhibition of EZH2 reversed this trend. Furthermore, the induction of EZH2 led to β-catenin signaling activation by increasing H3K27me3 on the promoter of SFRP1, while the inhibition of EZH2 silenced β-catenin signaling. Finally, intraarticular injection of EPZ005687 delayed OA development in mice. These results implicated EZH2 activity in OA development. Pharmacological inhibition of EZH2 may be an effective therapeutic approach for osteoarthritis.

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.

Microendoscopic anterior approach for irreducible atlantoaxial dislocation: surgical techniques and preliminary results.

Study Design Surgical techniques and preliminary results. Objective To describe and evaluate the safety and efficacy of a new minimal invasive technique for the irreducible atlantoaxial dislocation (IADD). Summary of Background Data Endoscope has been widely used in minimal invasive spinal surgery. However, there are no clinical reports regarding anterior approach for IADD in the literature. Methods Ten consecutive patients with IADD were treated by anterior release with microendoscopic aide and subsequently reduction, anterior transarticular screw fixation and morselized autologous bone grafts. There were 3 cases of odontiod dysplasia, 4, chronic odontiod fracture, 1, odontiod absence, 1 fasilar impression, and 1 malunion of odontoid fracture. According to Symon and Lavenders classification of disability, 6 cases were moderate disability, 3 severe nonbedbound, and 1 severe bedridden. The procedure was performed by the same surgeon (Yong-Long Chi). Results The new technique was performed successfully in all cases. All the patients underwent transarticular screw fixation and anterior morselized autograft fusion. The average operation time was 120u2009min (range, 90 to 150u2009min) and the mean estimated blood loss was 150u2009mL (range, 100 to 250u2009mL). Postoperative radiographs demonstrated that 9 cases restored anatomic position and 1 had partial reduction. According to the postoperative computed tomography all the screws were appropriately placed. Follow-up after surgery, longest is 16 months and minimal 8 months with a mean of 12 months, neurologic status was improved in all patients. There was no loss of fixation and solid fusion was achieved in all cases. Conclusions Surgical technique of microendoscopic anterior release, reduction, fixation, and fusion is safe and reliable minimally invasive for treating IADD.

Butein inhibits IL-1β-induced inflammatory response in human osteoarthritis chondrocytes and slows the progression of osteoarthritis in mice

Abstract Osteoarthritis (OA) is a progressive degenerative disease characterized by irreversible articular cartilage destruction. Butein, a polyphenolic compound isolated from the stem bark of cashews and Rhus verniciflua Stokes, has been reported to have anti‐inflammatory effects. This study aimed to assess the effect of butein on human OA chondrocytes and mice OA models induced by destabilization of the medial meniscus (DMM). In vitro, human OA chondrocytes were pretreated with butein at 10, 50 &mgr;M and subsequently stimulated with IL‐1&bgr; (10 ng/ml) for 24 h. Production of NO, PGE2, TNF‐&agr; and IL‐6 was evaluated by the Griess reaction and ELISAs. The mRNA expression of COX‐2, iNOS, TNF‐&agr;, IL‐6, MMP‐1, MMP‐3, MMP‐13, ADAMTS‐4, ADAMTS‐5, COL‐2 and SOX‐9 were measured by real‐time PCR. The protein expression of COX‐2, iNOS, MMP‐13, COL‐2, SOX‐9, p65 and I&kgr;B‐&agr; were detected by Western blot. P65 nuclear translocation was detected by immunofluorescence. In vivo, the severity of OA was determined by histological analysis. We found that butein significantly inhibited the IL‐1&bgr;‐induced production of NO and PGE2, expression of COX‐2, iNOS, TNF‐&agr;, IL‐6 and MMP‐13, degradation of COL‐2 and SOX‐9 at mRNA and protein levels as well as MMP‐1, MMP‐3, ADAMTS‐4 and ADAMTS‐5 gene expression. Furthermore, butein dramatically suppressed IL‐1&bgr;‐stimulated I&kgr;B‐&agr; degradation and NF‐kB p65 activation. In vivo, the cartilage in butein‐treated mice exhibited less Safranin O loss, cartilage erosion and lower OARSI scores. Butein also reduced subchondral bone plate thickness and alleviated synovitis. Taken together, these findings indicate that butein may be a potential agent in the treatment of OA. Graphical abstract Figure. No Caption available. HighlightsButein inhibited the IL‐1&bgr;‐induced inflammatory mediators in human osteoarthritis chondrocytes.Butein inhibited the IL‐1&bgr;‐induced degradation of type II collagen and SOX‐9 in human osteoarthritis chondrocytes.Butein inhibited the IL‐1&bgr;‐induced I&kgr;B‐&agr; degradation, NF‐kB p65 activation and NF‐&kgr;B p65 nuclear translocation in human osteoarthritis chondrocytes.Butein attenuated the arthritis in mice osteoarthritis models.

Fisetin inhibits IL-1β-induced inflammatory response in human osteoarthritis chondrocytes through activating SIRT1 and attenuates the progression of osteoarthritis in mice

&NA; Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage degradation and inflammation. Fisetin, a polyphenol extracted from fruits and vegetables, has been reported to have anti‐inflammatory effects. Our study aimed to investigate the effect of fisetin on OA both in vitro and in vivo. In vitro, chondrocytes were pretreated with fisetin alone or fisetin combined with sirtinol (an inhibitor of SIRT1) for 2 h before IL‐1&bgr; stimulation. Production of NO, PGE2, TNF‐&agr; and IL‐6 were evaluated by the Griess reaction and ELISAs. The mRNA (COX‐2, iNOS, MMP‐3, MMP‐13, ADAMTS‐5, Sox‐9, aggrecan and collagen‐II) and protein expression (COX‐2, iNOS, MMP‐3, MMP‐13, ADAMTS‐5 and SIRT1) were measured by qRT‐PCR and Western blot respectively. Immunofluorescence was used to assess the expression of collagen‐II and SIRT1. SIRT1 activity was quantified with SIRT1 fluorometric assay kit. The in vivo effect of fisetin was evaluated by gavage in mice OA models induced by destabilization of the medial meniscus (DMM). We found that fisetin inhibited IL‐1&bgr;‐induced expression of NO, PGE2, TNF‐&agr;, IL‐6, COX‐2, iNOS, MMP‐3, MMP‐13, ADAMTS‐5. Besides, fisetin remarkably decreased IL‐1&bgr;‐induced degradation of Sox‐9, aggrecan and collagen‐II. Furthermore, fisetin significantly inhibited IL‐1&bgr;‐induced SIRT1 decrease and inactivation. However, the inhibitory effect of fisetin was obvious abolished by sirtinol, suggesting that fisetin exerts anti‐inflammatory effects through activating SIRT1. In vivo, fisetin‐treated mice exhibited less cartilage destruction and lower OARSI scores. Moreover, fisetin reduced subchondral bone plate thickness and alleviated synovitis. Taken together, these findings indicate that fisetin may be a potential agent in the treatment of OA. Graphical abstract Figure. No caption available. HighlightsFisetin inhibited the IL‐1&bgr;‐induced inflammatory mediators in human osteoarthritis chondrocytes.Fisetin inhibited the IL‐1&bgr;‐induced degradation of Sox‐9, aggrecan and collagen‐II in human osteoarthritis chondrocytes.Fisetin inhibited the IL‐1&bgr;‐induced SIRT1 decrease and inactivation in human osteoarthritis chondrocytes.Sirtinol reversed the the inhibitory effect of fisetin on IL‐1&bgr;‐induced inflammatory response in human osteoarthritis chondrocytes.Fisetin alleviated the progression of osteoarthritis in mice models.

Stabilization of HIF-1α by FG-4592 promotes functional recovery and neural protection in experimental spinal cord injury

Previous studies have shown that inhibition of prolyl hydroxylase(PHD) stabilizes Hypoxia-inducible factor 1, alpha subunit(HIF-1α), increases tolerance to hypoxia, and improves the prognosis of many diseases. However, the role of PHD inhibitor (PHDI) in the recovery of spinal cord injury remains controversial. In this study, we investigated the protective role of a novel PHDI FG-4592 both in vivo and in vitro. FG-4592 treatment stabilized HIF1α expression both in PC12 cells and in spinal cord. FG-4592 treatment significantly inhibited tert-Butyl hydroperoxide(TBHP)-induced apoptosis and increases the survival of neuronal PC-12 cells. FG-4592 administration also improved recovery and increased the survival of neurons in spinal cord lesions in the mice model. Combination therapy including the specific HIF-1α blocker YC-1 down-regulated the HIF-1α expression and partially abolished the protective effect of FG-4592. Taken together, our results revealed that the role of FG-4592 in SCI recovery is related to the stabilization of HIF-1α and inhibition of apoptosis. Overall, our study suggests that PHDIs may be feasible candidates for therapeutic intervention after SCI and central nervous system disorders in humans.

Hidden blood loss and the influential factors after percutaneous kyphoplasty surgery

PurposePercutaneous kyphoplasty (PKP) is a minimally invasive procedure for the treatment of osteoporotic vertebral compression fractures (OVCFs). It is generally considered that there is little blood loss during the surgery. However, a significant perioperative hidden blood loss (HBL) is neglected. This study was to examine the amount of HBL and determine the influential factors during PKP.MethodsFrom January 2015 to January 2016, 115 patients with OVCFs who were scheduled to have a PKP were enrolled in this study. The factors analyzed included gender, age, body mass index (BMI), percentage of vertebral height loss, percentage of vertebral height restoration, number of fracture levels, bone mineral density (BMD), duration of symptom, cement leakage, and other internal diseases (hypertension, diabetes mellitus). According to Gross’s formula, each patient’s height, weight, and pre-operative and post-operative hematocrit were recorded and used for calculating the blood loss. Influential factors were further analyzed by multivariate linear regression analysis and t test.ResultsThe mean HBL was 282xa0±xa0162xa0mL (mL) and the post-operative Hb loss was 8.7xa0±xa05.4xa0g per liter (g/L). According to multivariate linear regression analysis, patients with severe vertebral height loss (Pxa0=xa00.016), better vertebral height restoration (Pxa0=xa00.038), and multi-segmental vertebral fractures (Pxa0=xa00.000) had a higher amount of HBL. Fresh fractures (Pxa0=xa00.008) and cement leakage (Pxa0=xa00.004) were also important factors to increase HBL, whereas gender (Pxa0=xa00.642), age (Pxa0=xa00.203), BMI (Pxa0=xa00.075), hypertension (Pxa0=xa00.099), diabetes mellitus (Pxa0=xa00.905), and BMD (Pxa0=xa00.521) were not correlate with HBL. When we compared the incidence of anemia between pre-operative and post-operative, we found that the incidence of anemia was significantly associated with HBL (Pxa0=xa00.000).ConclusionsHBL cannot be ignored in perioperative period, especially for poor physical condition and multiple fractures patients. Having a correct understanding of HBL can help improve clinical assessment capabilities, ensuring patients’ safety.

Celastrol reduces IL-1β induced matrix catabolism, oxidative stress and inflammation in human nucleus pulposus cells and attenuates rat intervertebral disc degeneration in vivo

Celastrol has been reported to exert therapeutic potential on pro-inflammatory diseases including asthma, Crohns disease, arthritis and neurodegenerative disorders via inhibiting NF-κB pathway. While the effect of celastrol on intervertebral disc degeneration (IDD), which is also a pro-inflammatory disease, remains unknown. In this study, we evaluated the effect of celastrol on IDD in IL-1β treated human nucleus pulposus cells in vitro as well as in puncture induced rat IDD model in vivo. Our results showed that celastrol reduced the expression of catabolic genes (MMP-3, 9, 13, ADAMTS-4, 5), oxidative stress factors (COX-2, iNOS) and pro-inflammatory factors (IL-6, TNF-a) induced by IL-1β in nucleus pulposus cells, also phosphorylation of IκBα and p65 were attenuated by celastrol, indicating NF-κB pathway was inhibited by celastrol in nucleus pulposus cells. In vivo study showed that celastrol treated rats had stronger T2-weighted signal than vehicle-treated rats at 2 weeks and 6 weeks time point, suggesting celastrol could attenuate intervertebral disc degeneration in vivo. Together, our study demonstrates that celastrol could reduce IL-1β induced matrix catabolism, oxidative stress and inflammation in human nucleus pulposus cells and attenuates rat intervertebral disc degeneration in vivo, which shows its potential to be a therapeutic drug for IDD.