Anjing Liang

Melatonin inhibits adipogenesis and enhances osteogenesis of human mesenchymal stem cells by suppressing PPARγ expression and enhancing Runx2 expression

Abstract:  Adipogenesis and osteogenesis, a reciprocal relationship in bone marrow, are complex processes including proliferation of precursor cells, commitment to the specific lineage, and terminal differentiation. Accumulating evidence from in vitro and in vivo studies suggests that melatonin affects terminal differentiation of osteoblasts and adipocytes, but little is known about the effect of melatonin on the process of adipogenesis and osteogenesis, especially adipogenesis. This study was performed to determine the effect of melatonin on adipogenesis and osteogenesis in human mesenchymal stem cells (hMSCs). Cell proliferation assays demonstrated that melatonin had no apparent effect on the proliferation of hMSCs. When melatonin was added to the adipogenic/osteogenic medium, it directly inhibited adipogenesis and simultaneously promoted osteogenesis of hMSCs in a dose‐dependent manner. Furthermore, quantitative RT‐PCR demonstrated that melatonin significantly suppressed peroxisome proliferator‐activated receptor gamma (PPARγ) expression (day 3, 25% decrease; day 6, 47% decrease), but promoted Runx2 expression (day 3, 87% increase; day 6, 56% increase) in the early stages of adipogenesis and osteogenesis of hMSCs. Moreover, melatonin down‐regulated several markers of terminal adipocyte differentiation, including leptin (30%), lipoprotein lipase (LPL, 41%), adiponectin (51%), and adipocyte protein 2 (αP2, 45%). Meanwhile, melatonin up‐regulated several markers of osteoblast differentiation, including alkaline phosphatase (110%), osteopontin (218%), and osteocalcin (310%). These results suggest that melatonin directly inhibits hMSCs adipogenic differentiation and significantly enhances hMSCs osteogenic differentiation by suppressing PPARγ expression and enhancing Runx2 expression; this provides further evidence for melatonin as an anti‐osteoporosis drug.

Age-Related Increases of Macroautophagy and Chaperone-Mediated Autophagy in Rat Nucleus Pulposus

Objective: Excessive apoptosis plays an important role in the progression of intervertebral disc degeneration. However, the effect of autophagy, another type of programmed cell death, on the pathogenesis of disc degeneration is still unclear. Macroautophagy and chaperone-mediated autophagy (CMA) change and intervertebral disc degeneration aggravates with age. This study aims at examining the expression changes of light chain 3 (LC3), lysosome-associated membrane protein 2A (LAMP-2A), and Hsc70, the indicator substrates of macroautophagy and CMA, in rat nucleus pulposus (NP) to prove that macroautophagy and CMA are both related with age. Methods: Female Sprague–Dawley rats of 3, 12, and 24 months (n = 8 per age) were used in this study. Autophagic vacuoles in NP cells were detected by transmission electron microscopy. In NP, the expressions of LC3-II and LAMP-2A protein and mRNA were examined by immunohistochemistry and reverse transcription polymerase chain reaction, respectively. LC3-II, LC3-I, and LAMP-2A protein were also measured by western blot. The mRNA and protein level of myocyte enhancer factor-2D regulated by LAMP-2A and Hsc70 were detected by reverse transcriptase polymerase chain reaction and western blot, respectively. Results: Transmission electron microscopy showed more autophagic vacuoles in 12- and 24-month groups than in 3-month group. Expression of LC3-II and LC3-II/LC3-I in 24-month group was significantly higher than in 3-month group (p < 0.05). Meanwhile, LAMP-2A expression was significantly higher in 24-month group than in 3-month group (p < 0.05). However, lower expression of Hsc70 and myocyte enhancer factor-2D was found in the 24-month rats than in 3-month group (p < 0.05, p < 0.05, respectively). Conclusion: Macroautophagy and CMA were present and increased with age in rat NP.

Sustained release of melatonin from poly (lactic-co-glycolic acid) (PLGA) microspheres to induce osteogenesis of human mesenchymal stem cells in vitro

Abstract:  Melatonin promotes bone formation and prevents bone degradation via receptor‐dependent or receptor‐independent actions. The aim of this study is to encapsulate melatonin into poly (lactic‐co‐glycolic acid) (PLGA) microspheres (PLGA‐MEL‐MS) and create a melatonin sustained release system, then to evaluate its effect on the osteogenesis of human mesenchymal stem cells (hMSCs) in vitro. PLGA‐MEL‐MS were prepared by single emulsion solvent evaporation technique. Scanning electron microscopy demonstrated the incorporation of melatonin did not disturb the conventional generation of PLGA microspheres in size and morphology. In vitro drug release assay showed that PLGA‐MEL‐MS exhibited a biphasic drug release pattern: a low initial burst release effect with approximately 40% drug release at the first 3 days and a relatively retarded and continuous release with about 85% drug release over the 25 days. Cell proliferation assay demonstrated that PLGA‐MEL‐MS had no apparent effect on proliferation of human MSCs. In an osteogenesis assay, PLGA‐MEL‐MS obviously enhanced alkaline phosphatase (ALP) mRNA expression and increased ALP activity compared to that in the control group. Meanwhile, several markers of osteoblast differentiation were also significantly upregulated, including runx2, osteopontin, and osteocalcin. Furthermore, quantificational alizarin red‐based assay demonstrated that PLGA‐MEL‐MS significantly enhanced calcium deposit of hMSCs compared to the controls. Therefore, this simple melatonin sustained release system can control released melatonin to generate a microenvironment with a relatively stable concentration of melatonin for a period of time to support osteogenic differentiation of hMSCs in vitro. This suggests that this system may be used as bone growth stimulator in bone healing in vivo.

Association between Common Variants near LBX1 and Adolescent Idiopathic Scoliosis Replicated in the Chinese Han Population

Background Adolescent idiopathic scoliosis (AIS) is one of the most common spinal deformities found in adolescent populations. Recently, a genome-wide association study (GWAS) in a Japanese population indicated that three single nucleotide polymorphisms (SNPs), rs11190870, rs625039 and rs11598564, all located near the LBX1 gene, may be associated with AIS susceptibility [1]. This study suggests a novel AIS predisposition candidate gene and supports the hypothesis that somatosensory functional disorders could contribute to the pathogenesis of AIS. These findings warrant replication in other populations. Methodology/Principal Findings First, we conducted a case-control study consisting of 953 Chinese Han individuals from southern China (513 patients and 440 healthy controls), and the three SNPs were all found to be associated with AIS predisposition. The ORs were observed as 1.49 (95% CI 1.23–1.80, P = 5.09E-5), 1.70 (95% CI 1.42–2.04, P = 1.17E-8) and 1.52 (95% CI 1.27–1.83, P = 5.54E-6) for rs625039, rs11190870 and rs11598564, respectively. Second, a case-only study including a subgroup of AIS patients (N = 234) was performed to determine the effects of these variants on the severity of the condition. However, we did not find any association between these variants and the severity of curvature. Conclusion This study shows that the genetic variants near the LBX1 gene are associated with AIS susceptibility in Chinese Han population. It successfully replicates the results of the GWAS, which was performed in a Japanese population.

Genomic polymorphisms of G-Protein Estrogen Receptor 1 are associated with severity of adolescent idiopathic scoliosis

PurposeAdolescent idiopathic scoliosis (AIS) is reported to be associated with the two traditional estrogen receptor genes, ESR1 and ESR2. Yet, the novel estrogen receptor G protein-coupled estrogen receptor 1 (GPER) has not been studied. To investigate the association of GPER gene polymorphisms with the onset and deterioration of AIS, we performed a case-control study.MethodsClinical information was recorded, blood samples were taken and genomic DNA was extracted. After resequencing the gene in 45 cases and 45 controls who were randomly selected, 16 tag single nucleotide polymorphisms (SNPs) were selected. Then the association study was extended by an additional 344 patients and 293 controls with direct sequencing and a TaqMan-based genotyping assay. The chi-square test and logistic regression were used to analyse the genotypic and allelic association. One-way analysis of variance was used to compare the mean maximum Cobb angles and ages with different genotypes in the case-only data set.ResultsNo association was observed between the polymorphisms of the GPER gene and susceptibility to AIS. However, heterozygotes in three SNPs of the gene (rs3808351, rs10269151 and rs426655s3) were related significantly with the curve severity in AIS patients (P = 0.004, 0.048 and 0.028, respectively).ConclusionsOur results demonstrate that GPER gene polymorphisms are associated with the severity of curvature in AIS; deficits of GPER may contribute to the deterioration of spine deformity.

Increased Macroautophagy in the Pathological Process of Intervertebral Disc Degeneration in Rats

Objective: Macroautophagy increases with age in rat intervertebral discs; however, the effect of macroautophagy on the process of intervertebral disc degeneration (IVDD) is still unclear. The aim of this study was to examine the presence of autophagosome, as well as the levels of Beclin-1 and LC3 proteins, in vivo. Additionally, in vitro evidence of macroautophagy and GRP78 and GADD153 protein levels were investigated to explore the mechanism of macroautophagy in the process of IVDD. Methods: Male Sprague–Dawley rats, aged 2 months, were randomly divided into six groups (three control and three model groups, n = 8 per group). At the 6-, 12-, and 18-week time points, autophagosomes in nucleus pulposus cells were detected with transmission electron microscope (TEM). Expression of Beclin-1 and LC3 protein levels within intervertebral disc was detected using Western blotting analysis. Then, the rat annulus fibrosus cells were isolated and cultured with Earle’s balanced salt solution. At 1, 2, and 3 hr of culture, autophagosomes were detected using monodansylcadaverine assay, and LC3, Beclin-1, GRP78, and GADD153 protein levels were detected using Western blotting analysis. Results: Transmission electron microscopy revealed autophagosomes within nucleus pulposus cells in both the control and model groups. At 6-, 12-, and 18-week posttreatments, the levels of Beclin-1 and the LC3-II/LC3-I protein ratio in the model groups were higher than those in the control groups (p < 0.05). Compared with the control rats, amino acid starvation increased the number of monodansylcadaverine-positive cells and the LC3-II/LC3-I protein ratio in the model rats. Moreover, the in vitro levels of Beclin-1, GRP78, and GADD153 proteins were increased with the prolongation of amino acid starvation (p < 0.05). Conclusions: Macroautophagy was present and was associated with increased pathological process of IVDD in rats. Macroautophagy of intervertebral disc cells is possibly secondary to endoplasmic reticulum stress.

Autophagy attenuates the catabolic effect during inflammatory conditions in nucleus pulposus cells, as sustained by NF-κB and JNK inhibition

Proteoglycan degradation contributing to the pathogenesis of intervertebral disc (IVD) degeneration is induced by inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). Cell autophagy exists in degenerative diseases, including osteoarthritis and inter-vertebral disc degeneration. However, the autophagy induced by TNF-α and IL-1β and the corresponding molecular mechanism appear to be cell-type dependent. The effect and mechanism of autophagy regulated by TNF-α and IL-1β in IVDs remains unclear. Additionally, the impact of autophagy on the catabolic effect in inflammatory conditions also remains elusive. In the present study, autophagy activator and inhibitor were used to demonstrate the impact of autophagy on the catabolic effect induced by TNF-α. A critical role of autophagy was identified in rat nucleus pulposus (NP) cells: Inhibition of autophagy suppresses, while activation of autophagy enhances, the catabolic effect of cytokines. Subsequently, the autophagy-related gene expression in rat NP cells following TNF-α and IL-1β treatment was observed using immunofluorescence, quantitative polymerase chain reaction and western blot analysis; however, no association was present. In addition, nuclear factor κB (NF-κB), c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases and p38 mitogen-activated protein kinase inhibitors and TNF-α were used to determine the molecular mechanism of autophagy during the inflammatory conditions, and only the NF-κB and JNK inhibitor were found to enhance the autophagy of rat NP cells. Finally, IKKβ knockdown was used to further confirm the effect of the NF-κB signal on human NP cells autophagy, and the data showed that IKKβ knockdown upregulated the autophagy of NP cells during inflammatory conditions.

Endoplasmic reticulum stress-unfolding protein response-apoptosis cascade causes chondrodysplasia in a col2a1 p.Gly1170Ser mutated mouse model.

The collagen type II alpha 1 (COL2A1) mutation causes severe skeletal malformations, but the pathogenic mechanisms of how this occurs are unclear. To understand how this may happen, a col2a1 p.Gly1170Ser mutated mouse model was constructed and in homozygotes, the chondrodysplasia phenotype was observed. Misfolded procollagen was largely synthesized and retained in dilated endoplasmic reticulum and the endoplasmic reticulum stress (ERS)-unfolded protein response (UPR)-apoptosis cascade was activated. Apoptosis occurred prior to hypertrophy, prevented the formation of a hypertrophic zone, disrupted normal chondrogenic signaling pathways, and eventually caused chondrodysplasia. Heterozygotes had normal phenotypes and endoplasmic reticulum stress intensity was limited with no abnormal apoptosis detected. Our results suggest that earlier chondrocyte death was related to the ERS-UPR-apoptosis cascade and that this was the chief cause of chondrodysplaia. The col2a1 p.Gly1170Ser mutated mouse model offered a novel connection between misfolded collagen and skeletal malformation. Further investigation of this mouse mutant model can help us understand mechanisms of type II collagenopathies.

Normal leptin expression, lower adipogenic ability, decreased leptin receptor and hyposensitivity to Leptin in Adolescent Idiopathic Scoliosis.

Leptin has been suggested to play a role in the etiology of Adolescent Idiopathic Scoliosis (AIS), however, the leptin levels in AIS girls are still a discrepancy, and no in vitro study of leptin in AIS is reported. We took a series of case-control studies, trying to understand whether Leptin gene polymorphisms are involved in the etiology of the AIS or the change in leptin level is a secondary event, to assess the level of leptin receptor, and to evaluate the differences of response to leptin between AIS cases and controls. We screened all exons of Leptin gene in 45 cases and 45 controls and selected six tag SNPs to cover all the observed variations. Association analysis in 446 AIS patients and 550 healthy controls showed no association between the polymorphisms of Leptin gene and susceptibility/severity to AIS. Moreover, adipogenesis assay of bone mesenchymal stem cells (MSCs) suggested that the adipogenic ability of MSCs from AIS girls was lower than controls. After adjusting the differentiation rate, expressions of leptin and leptin receptor were similar between two groups. Meanwhile, osteogenesis assay of MSC showed the leptin level was similar after adjusting the differentiation rate, but the leptin receptor level was decreased in induced AIS osteoblasts. Immunocytochemistry and western blot analysis showed less leptin receptors expressed in AIS group. Furthermore, factorial designed studies with adipogenesis and osteogenesis revealed that the MSCs from patients have no response to leptin treatment. Our results suggested that Leptin gene variations are not associated with AIS and low serum leptin probably is a secondary outcome which may be related to the low capability of adipogenesis in AIS. The decreased leptin receptor levels may lead to the hyposensitivity to leptin. These findings implied that abnormal peripheral leptin signaling plays an important role in the pathological mechanism of AIS.

The relationship between concave angle of vertebral endplate and lumbar intervertebral disc degeneration.

Study Design. A retrospective study of magnetic resonance imaging of the lower lumbar spine. Objective. To describe the characteristics of the concave angle of vertebral endplate (CAVE) and study the association between CAVE and lumbar intervertebral disc degeneration (IVDD). Summary of Background Data. The vertebral endplate is responsible for transferring stress between disc and vertebral body, and its concavity is important in dispersing compression stress. However, the characteristics of CAVE and the relationship between CAVE and IVDD have not been investigated. Methods. Magnetic resonance images of the lower lumbar spine in 511 patients with low back pain were examined by 2 experienced spine surgeons. The grades of IVDD and lumbar disc herniation (LDH) were evaluated, several parameters including CAVE, height, and the sagittal diameter of vertebral body were measured, and the association between IVDD or LDH and CAVE was analyzed. Results. At L3–L4, L4–L5, and L5–S1, CAVE was smaller in the upper endplate (i.e., the inferior endplate of the superior vertebra) than in the lower endplate (i.e., the superior endplate of the inferior vertebra). There was no male/female difference in the size of CAVE in any of the segments. According to partial correlation analysis, CAVE was moderately related to IVDD, but no association between CAVE and LDH was found. Conclusion. When lumbar IVDD occurs, the CAVE increases and the endplate tends to flatten. The degree of flattening is related to the severity of the degeneration.