Xinghuo Wu

TNF-a mediated inflammatory macrophage polarization contributes to the pathogenesis of steroid-induced osteonecrosis in mice

The phenotypic polarization of macrophages are involved in steroid-induced osteonecrosis (ON). This study tried to investigate the detrimental and beneficial roles of M1/M2 macrophages associated with TNF-a in ON. Mice ON model was induced by the injection of methylprednisolone. After that, flow cytometry technique, immunohistochemistry, immunofluorescence, ELISA, and RT-PCR methods were used to investigate the expression pattern of macrophages and the expression of inflammatory cytokines. During the progression of ON, massive chronic inflammatory cells infiltrated into the necrotic zone, represented by the infiltration of macrophages. In the early stage of ON, there was high TNF-a activity; and a large population of M1 macrophages infiltrated into the necrotic zone. On the contrary, the expression of TNF-a gradually decreased; simultaneously, a larger M2 cell population presented in the necrotic zone in the late stage of ON. The increased M2 macrophages could be beneficial for resolving inflammation and promoting tissue repair, confirmed by the histologic findings of appositional new bone formation around the necrotic bone. Thus, it showed that TNF-a-mediated alteration of M1/M2 macrophage polarization contributed to the pathogenesis of steroid-induced osteonecrosis. M1-polarized macrophages appeared to be disruptive in the early stage of ON, while M2-polarized macrophages played an important role in the late stage during the pathogenesis of ON.

A Combination of Granulocyte Colony-Stimulating Factor and Stem Cell Factor Ameliorates Steroid-Associated Osteonecrosis in Rabbits

Objective Bone marrow-derived stem cells (BMSC) have been highlighted for the treatment of osteonecrosis (ON) before collapse of the femoral head. In our study, the potential of granulocyte colony-stimulating factor/stem cell factor (G-CSF/SCF)-mobilized BMSC to repair steroid-associated ON was assessed in rabbits. Methods ON was induced by low-dose lipopolysaccharide and subsequent pulsed high-dose methylprednisolone. Rabbits in the treated group were subjected to subcutaneous injections of GCSF at a dose of 100 μg/kg and SCF 25 μg/kg per day for 5 days; rabbits in the control group were given saline. Blood samples were collected and serum osteocalcin was detected by ELISA. Radiological analysis was performed by magnetic resonance imaging (MRI). Then bilateral femora and humeri were harvested and processed to paraffin sections and hard-tissue sections for immunohistochemical, histologic, and histomorphometric analysis. Results The mean number of leukocytes and relative numbers of mononuclear cells increased significantly after mobilization. All rabbits displayed a marked increase in osteocalcin protein expression in response to G-CSF/SCF. MRI scans showed a reactive interface between the necrotic and reparative zones after G-CSF/SCF administration. Quantitative analysis showed that new vessel formation was 3.3-fold greater and vessel density was 2.6-fold greater in the treatment group than the control group. The histologic and histomorphometric analysis revealed that the new bone volume was significantly higher in the G-SCF/SCF group than in the control group at 4 weeks. Conclusion G-CSF/SCF-induced mobilization of BMSC in the necrotic foci may represent a promising strategy for promoting functional bone repair of early-stage ON.

Structural Augmentation with Biomaterial-Loaded Allograft Threaded Cage for the Treatment of Femoral Head Osteonecrosis

Seventy-six patients with femoral head necrosis were allocated to a program of either core decompression (control group) or core decompression and implantation of a biomaterial-loaded allograft threaded cage (treatment group). All patients were followed up prospectively clinically and radiographically. In the control group, no significant improvement in Harris hip score was found, and 13 of the 22 hips had deteriorated to stage III. In the treatment group, the mean Harris hip score was improved from 62.8 to 81.6; the clinical success rate at 36 months postoperatively was 91%. Collapse was seen in 1 hip, and another 3 hips exhibited progressive collapse. The procedure is attractive as a minimally invasive and salvage procedure, which shows encouraging success rates and early clinical results in patients with Steinberg stage I-II osteonecrosis.

Advanced glycation end products regulate anabolic and catabolic activities via NLRP3-inflammasome activation in human nucleus pulposus cells

Intervertebral disc degeneration is widely recognized as a cause of lower back pain, neurological dysfunction and other musculoskeletal disorders. The major inflammatory cytokine IL‐1β is associated with intervertebral disc degeneration; however, the molecular mechanisms that drive IL‐1β production in the intervertebral disc, especially in nucleus pulposus (NP) cells, are unknown. In some tissues, advanced glycation end products (AGEs), which accumulate in NP tissues and promote its degeneration, increase oxidative stress and IL‐1β secretion, resulting in disorders, such as obesity, diabetes mellitus and ageing. It remains unclear whether AGEs exhibit similar effects in NP cells. In this study, we observed significant activation of the NLRP3 inflammasome in NP tissues obtained from patients with degenerative disc disease compared to that with idiopathic scoliosis according to results detected by Western blot and immunofluorescence. Using NP cells established from healthy tissues, our in vitro study revealed that AGEs induced an inflammatory response in NP cells and a degenerative phenotype in a NLRP3‐inflammasome‐dependent manner related to the receptor for AGEs (RAGE)/NF‐κB pathway and mitochondrial damage induced by mitochondrial reactive oxygen species (mtROS) generation, mitochondrial permeability transition pore (mPTP) activation and calcium mobilization. Among these signals, both RAGE and mitochondrial damage primed NLRP3 and pro‐IL‐1β activation as upstream signals of NF‐κB activity, whereas mitochondrial damage was critical for the assembly of inflammasome components. These results revealed that accumulation of AGEs in NP tissue may initiate inflammation‐related degeneration of the intervertebral disc via activation of the NLRP3 inflammasome.

The Involvement of Protease Nexin-1 (PN1) in the Pathogenesis of Intervertebral Disc (IVD) Degeneration

Protease nexin-1 (PN-1) is a serine protease inhibitor belonging to the serpin superfamily. This study was undertaken to investigate the regulatory role of PN-1 in the pathogenesis of intervertebral disk (IVD) degeneration. Expression of PN-1 was detected in human IVD tissue of varying grades. Expression of both PN-1 mRNA and protein was significantly decreased in degenerated IVD, and the expression levels of PN-1 were correlated with the grade of disc degeneration. Moreover, a decrease in PN-1 expression in primary NP cells was confirmed. On induction by IL-1β, the expression of PN-1 in NP cells was decreased at day 7, 14, and 21, as shown by western blot analysis and immunofluorescence staining. PN-1 administration decreased IL-1β-induced MMPs and ADAMTS production and the loss of Agg and Col II in NP cell cultures through the ERK1/2/NF-kB signaling pathway. The changes in PN-1 expression are involved in the pathogenesis of IVD degeneration. Our findings indicate that PN-1 administration could antagonize IL-1β-induced MMPs and ADAMTS, potentially preventing degeneration of IVD tissue. This study also revealed new insights into the regulation of PN-1 expression via the ERK1/2/NF-kB signaling pathway and the role of PN-1 in the pathogenesis of IVD degeneration.

Association between interleukin 8 -251 A/T and +781 C/T polymorphisms and osteoarthritis risk.

PURPOSE Interleukin 8 (IL-8), as a member of the CXC chemokine family, has a regulatory role in joint inflammation and cartilage degradation, and contribute to the pathophysiology of osteoarthritis. The aim of the current study was to examine the influence of the IL-8 gene polymorphisms at positions -251 (rs4073) and +781 (rs2227306) on the risk of osteoarthritis. METHODS This hospital-based case-control study comprised 150 patients with osteoarthritis and 150 age- and gender-matched controls. IL-8 251 A/T and +781 C/T polymorphisms were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS Patients with osteoarthritis had a significantly higher frequency of IL-8 -251 TT genotype [odds ratio (OR)=2.16, 95% confidence interval (CI)=1.09, 4.26; P=0.03], IL-8 -251 T allele (OR=1.41, 95% CI=1.02, 1.94; P=0.04), IL-8 +781 TT genotype (OR=2.79, 95% CI=1.10, 7.08; P=0.03) and IL-8 +781 T allele (OR=1.48, 95% CI=1.02, 2.14; P=0.04) than controls. But the findings are less emphatic by the Bonferroni correction. When stratifying by body mass index, type, articular involvement, and Kellgren-Lawrence grade, no significant differences were found in any groups. CONCLUSIONS For the first time, the current data suggested that the TT genotype and T allele of the IL-8 gene polymorphisms at positions -251 and +781 might confer a high risk of osteoarthritis. In the future, additional well-designed large studies were required for the validation of our results.

G-CSF/SCF exert beneficial effects via anti-apoptosis in rabbits with steroid-associated osteonecrosis

OBJECTIVES Osteonecrosis is also known as avascular necrosis, and two types of cell death are included in the pathogenesis of osteonecrosis: necrosis and apoptosis. Apoptosis in the osteonecrosis of femoral head is thought to be the key determinant of glucocorticoid-induced cortical bone loss. The present study was implemented to evaluate the anti-apoptotic effect of Granulocyte colony-stimulating factor and stem cell factor (G-CSF/SCF) in rabbits with steroid-induced osteonecrosis. METHODS In the experiment, osteonecrosis was induced by low-dose lipopolysaccharide and subsequent pulsed high-dose methylprednisolone. Rabbits in preventive medicine group were treated with 100 μg/kg/d G-CSF and 25 μg/kg/d SCF. Then hematological and histomorphometric methods were used to investigate the treatment effects of osteonecrosis. Apoptosis was assessed via quantitative TUNEL staining and activated caspase-3 immunostaining and immunoblotting. RESULTS The results showed that G-CSF/SCF treatment could increase the secretion of serum osteocalcin, but inhibit the expression of serum tartrate-resistant acid phosphatase (TRAP5b). The incidence of osteonecrosis was significantly decreased in Preventive group when compared with Steroid group (42.1% vs. 88.2%). Histomorphometric analysis showed that G-CSF/SCF pre-disposal treatment was able to increase trabecular mineral appositional rate (MAR) and bone formation rate (BFR). Quantitative TUNEL and activated caspase-3 levels showed lower apoptosis in the Preventive group. CONCLUSIONS In conclusion, G-CSF/SCF treatment could inhibit caspase-3-dependent apoptosis in osteocytes to exert beneficial effects in preventing steroid-induced ON in rabbit models.

Simvastatin Inhibits IL-1β-Induced Apoptosis and Extracellular Matrix Degradation by Suppressing the NF-kB and MAPK Pathways in Nucleus Pulposus Cells

Statins are widely used hypocholesterolemic drugs that block the mevalonate pathway. Some studies have shown that statins may have the potential to inhibit intervertebral disk (IVD) degeneration (IDD). Interleukin (IL)-1β, a catabolic cytokine, is a key regulator of IDD. This study aimed to investigate the mechanism underlying the effect of simvastatin on IDD. The viability of nucleus pulposus (NP) cells was determined by the methyl-thiazolyl-tetrazolium (MTT) assay. The apoptosis of NP cells was measured by flow cytometric analysis, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and western blotting of relevant apoptotic proteins. The protein levels of catabolic factors and anabolic factors were determined by western blotting. The cells were stimulated with IL-1β in the absence or presence of simvastatin to investigate the effects on matrix metalloproteinase (MMP)-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, ADAMTS-5, type II collagen, and aggrecan expression. Our findings indicate that simvastatin considerably inhibited IL-1β-induced apoptosis in NP cells. We also found that simvastatin attenuated IL-1β-induced expression and MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5 activities and also reduced the decrease in type II collagen and aggrecan expression. In addition, simvastatin considerably suppressed the nuclear translocation and activation of nuclear factor-kappa B (NF-KB) by inhibiting p65 phosphorylation and translocation and blocking inhibitor kB-α degradation. It also inhibited MAPK pathway activation by blocking c-Jun N-terminal kinase (JNK), p38, and ERK phosphorylation. The results of our study revealed that simvastatin is a potential agent for IDD prevention and treatment.

MicroRNA-132 upregulation promotes matrix degradation in intervertebral disc degeneration

&NA; MicroRNAs (miRNAs) have been shown to be involved in the pathogenesis of intervertebral disc degeneration (IDD). This experiment was designed to study the expression and role of the miRNA, miR‐132, in IDD. MiR‐132 expression in human nucleus pulposus (NP) tissue was assessed by quantitative real‐time PCR. The methylation status of the miR‐132 was assessed with methylation‐specific PCR and bisulfite sequencing PCR. The regulation of growth differentiation factor5 (GDF5) expression by miR‐132 was evaluated by luciferase reporter assay. Moreover, we investigated the function of miR‐132 on IDD in vivo using a classic needle‐punctured rat tail model. These results showed that miR‐132 expression was upregulated during IDD and this upregulation was associated with hypomethylation of its promoter. MiR‐132 overexpression led to increased expression of ECM catabolic factors, including MMP13 and ADAMTS4, in NP cells while levels of anabolic proteins, such as type II collagen and aggrecan, were diminished. GDF5 was identified as a direct target of negative regulation by miR‐132. MAPK/ERK signaling was also found to be associated with miR‐132‐induced ECM degradation. In addition, we showed that miR‐132 inhibition effectively attenuated NP ECM degradation in IDD in vivo. Our findings demonstrated that miR‐132 promotes ECM degradation by human NP cells by direct targeting of GDF5. Hence, miR‐132 represents a potential therapeutic target in the treatment of IDD. HighlightsGDF5 is a direct target of miR‐132.Aberrant miR‐132 expression in IDD is related to the hypomethylation of its promoter region.miR‐132 promotes NP ECM degradation in vitro and in vivo.miR‐132 exerts its function in human NP cells by targeting GDF5.

IAPP modulates cellular autophagy, apoptosis, and extracellular matrix metabolism in human intervertebral disc cells.

The pathogenic process of intervertebral disc degeneration (IDD) is characterized by imbalance in the extracellular matrix (ECM) metabolism. Nucleus pulposus (NP) cells have important roles in maintaining the proper structure and tissue homeostasis of disc ECM. These cells need adequate supply of glucose and oxygen. Islet amyloid polypeptide (IAPP) exerts its biological effects by regulating glucose metabolism. The purpose of this study was to investigate the expression of IAPP in degenerated IVD tissue, and IAPP modulation of ECM metabolism in human NP cells, especially the crosstalk mechanism between apoptosis and autophagy in these cells. We found that the expression of IAPP and Calcr-RAMP decreased considerably during IDD progression, along with the decrease in the expression of AG, BG, and Col2A1. Induction of IAPP in NP cells by transfection with pLV-IAPP enhanced the synthesis of aggrecan and Col2A1 and attenuated the expression of pro-inflammatory factors, tumor necrosis factor (TNF)-α, and interleukin (IL)-1. Upregulation of IAPP also affected the expression of the catabolic markers—matrix metalloproteinases (MMPs) 3, 9 and 13 and ADAMTS 4 and 5. Downregulation of IAPP by siRNA inhibited the expression of anabolic genes but increased the expression of catabolic genes and inflammatory factors. The expressions of autophagic and apoptotic markers in NP cells transfected with pLV-IAPP were upregulated, including BECLIN1, ATG5, ATG7, LC3 II/I and Bcl-2, while significantly increase in the expression of Bax and Caspase-3 in NP cells transfected with pLV-siIAPP. Mechanistically, PI3K/AKT-mTOR and p38/JNK MAPK signal pathways were involved. We propose that IAPP might play a pivotal role in the development of IDD, by regulating ECM metabolism and controlling the crosstalk between apoptosis and autophagy in NP, thus potentially offering a novel therapeutic approach to the treatment of IDD.