ANGPTL3 is part of the machinery causing dyslipidemia majorily via LPL inhibition in mastitis mice.

Previous investigations have shown that inflammation induces changes in lipid and lipoprotein metabolism, and increased expression of angiopoietin-like protein 3 (ANGPTL3) contributes to the development of dyslipidemia. Here we investigated whether there is a correlation between increased ANGPTL3 expression and dyslipidemia in mastitis mice. Thirty mice were divided into two groups: control group and Staphylococcus aureus (S. aureus)-induced mastitis mice group. Changes in the levels of blood lipids [total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C)]; activity of myeloperoxidase (MPO); concentrations of plasma inflammation biomarkers [interferon-γ (IFNγ), tumor necrosis factor α (TNFα), and interleukin-1α (IL-1α)]; concentration of plasma ANGPTL3 protein; lipoprotein lipase (LPL) activities in postheparin plasma; expressions of hepatic N-acetylgalactosaminyltransferase 2 (GALNT2), hepatic ANGPTL3 and adipose LPL were determined. The major results indicated specific pathological mammary tissue changes, elevated MPO activity, reduced GALNT2 mRNA expression, elevated ANGPTL3 mRNA and protein expression and reduced LPL mRNA and protein expression. In plasma samples the S.aureus infused mice displayed elevated ANGPTL3 protein concentration, TG, TC and LDL-C levels, and reduced postheparin LPL activities and HDL-C level. The data suggests that ANGPTL3 is part of the machinery causing dyslipidemia majorily via LPL inhibition in mastitis mice.

Effect of pure muscle retraction on multifidus injury and atrophy after posterior lumbar spine surgery with 24 weeks observation in a rabbit model.

PURPOSE: To evaluate the effect of pure muscle retraction on multifidus injury and atrophy. MATERIALS AND METHODS: Sixty-three adult New Zealand white rabbits were divided evenly into three groups: 1-h retraction (group R1), 2-h retraction (R2), and sham surgery (C). The multifidus muscle was evaluated using magnetic resonance imaging (MRI) and histology after 3 and 48 h, and 1, 3, 6, 12, and 24 weeks after surgery. RESULTS: Multifidus muscle injury and atrophy were not observed in group C, but were obvious in groups R1 and R2. Edema, necrosis, and inflammation mainly occurred in the first week postoperatively, and were more severe in R2 than in R1 (P < 0.01). Muscle fiber regeneration began at week 1, fibrotic changes mainly occurred at weeks 3 and 6, and fat degeneration became obvious at weeks 12 and 24 postoperatively. The fibrosis and fat degeneration scores of R2 were higher than those of R1 (P < 0.01). Decreased acetylcholine activity and granular degeneration of the neuromuscular junction were observed in both retraction groups, but was more severe in R2 than in R1 (P < 0.01). CONCLUSION: Muscle retraction was an important factor not only for multifidus injury, but also for long-term multifidus atrophy after posterior lumbar surgery; a longer retraction time caused more severe multifidus injury and atrophy. Muscle fibers can be regenerated postoperatively, and denervation might be the reason for muscle atrophy.

PHD/HIF-1 upregulates CA12 to protect against degenerative disc disease: a human sample, in vitro and ex vivo study.

Intervertebral disc degeneration is a major cause of low back pain. The nucleus pulposus (NP) is an important intervertebral disc component. Recent studies have shown that carbonic anhydrase 12 (CA12) is a novel NP marker. However, the mechanism by which CA12 is regulated and its physiological function are unclear. In our study, CA12, hypoxia-inducible factor 1α (HIF-1α) and HIF-2α expression levels were examined in 81 human degenerated NP samples using real-time RT-PCR, immunohistochemistry and western blot. Rat NP cells were cultured in a hypoxic environment, and hypoxia-induced CA12 expression was examined. Rat NP cells were treated with HIF-1α siRNA or the prolyl hydroxylase (PHD) inhibitor dimethyloxalylglycine (DMOG) to evaluate the role of PHD/HIF-1 in regulating CA12 expression. Rat NP cells were treated with CA12 siRNA to determine the function of CA12. A rat ex vivo model was established to confirm that PHD, HIF-1, and CA12 have important roles in disc degeneration. We found that CA12 was significantly downregulated in degenerated human NP samples at the mRNA and protein levels. CA12 expression sharply increased by ~30-fold in response to hypoxia. The expression of HIF-1α, but not HIF-2α, also decreased in degenerated human NP samples and was positively correlated with CA12 expression. HIF-1α knockdown under hypoxia reduced the CA12 mRNA and protein expression levels. DMOG treatment increased HIF-1α and CA12 expression. CA12 knockdown significantly inhibited anabolic protein expression, whereas catabolic enzymes remained unchanged. The ex vivo experiments supported our in vitro studies of the role of PHD/HIF-1/CA12. In conclusion, CA12 is downregulated in degenerated NPs, and its expression may be regulated by the PHD/HIF-1 axis. Decreased CA12 expression may lead to decreased extracellular matrix synthesis, which contributes to degenerative disc disease progression.

The NLRP3/Caspase-1/Interleukin-1ß Axis Is Active in Human Lumbar Cartilaginous Endplate Degeneration.

BACKGROUND: Modic changes are the MRI signal changes of degenerative lumbar vertebral endplate and which lead to or accelerate intervertebral disc degeneration. NLRP3, caspase-1, and interleukin-1ß (IL-1ß) play a pivotal role in the pathogenesis of many inflammatory diseases, such as osteoarthritis. However, the roles of IL-1ß and its activators caspase-1 and NLRP3 are unclear in the degenerative endplate. QUESTIONS/PURPOSES: We asked: (1) What are the degenerative changes of the histologic features and chondrogenic markers' gene expressions between the cartilaginous endplates of patients with Modic changes and trauma (control)? (2) How does the NLRP3/caspase-1/IL-1ß axis in the cartilaginous endplates of patients with Modic changes compare with control (trauma) specimens? METHODS: Surgical specimens of cartilaginous endplates were divided into Modic changes (n = 56) and the trauma control (n = 16) groups. Hematoxylin and eosin and safranin O staining of cartilaginous endplate tissues were done to evaluate the extracellular matrix. Reverse transcription-polymerase chain reaction was performed on these tissues to investigate mRNA expression of type II collagen (Col II), SOX-9, matrix metalloproteinase-3, and a disintegrin like and metalloproteinase thrombospondin type I motifs-5. NLRP3, caspase-1, and IL-1ß were evaluated by reverse transcription-polymerase chain reaction and immunohistochemistry. RESULTS: Hematoxylin and eosin and safranin O staining showed the extracellular matrix degraded in the cartilaginous endplates of patients with Modic changes but not in the control cartilaginous endplates. Chondrogenic Col II (p = 0.024) and SOX9 (p = 0.053) were downregulated in the Modic changes group compared with the control group. In contrast to the control group, the transcriptional levels of NLRP3 (p < 0.001), caspase-1 (p = 0.054), and IL-1ß (p = 0.001) were all upregulated in the Modic changes group. CONCLUSIONS: The expression of NLRP3, caspase-1, and IL-1ß was upregulated in the patients with low back pain and Modic changes on MRI compared with patients with vertebral burst fracture without degenerative changes on MRI. The data suggest the NLRP3/caspase-1/IL-1ß axis may be implicated in lumbar cartilaginous endplate degeneration. CLINICAL RELEVANCE: The NLRP3/caspase-1/IL-1ß axis is active in cartilaginous endplates of patients with Modic changes and inflammatory cascades can exacerbate the cartilaginous endplate degeneration which may act as a trigger for intervertebral disc degeneration and low back pain. If these findings can be confirmed by others, we hope that new and effective therapy could be developed directed against this target.

Serum vitamin D status and in vitro fertilization outcomes: a systematic review and meta-analysis.

OBJECTIVE: To assess the correlation between serum 25-hydroxy vitamin D [25(OH)D] status and outcomes of in vitro fertilization (IVF) in infertile women through review systematically. METHODS: We used Embase, Pubmed, and Cochrane database to identify all studies that assessed the correlation between serum vitamin D levels and IVF outcomes in infertile women up until 30 June 2015, with the restricted language of English. We included studies that compared IVF outcomes between infertile women vitamin D <20 ng/ml and vitamin D ≥20 ng/ml. The results were summarized using Stata 12.0 software. For studies reported dichotomous outcomes (clinical pregnancy rate and live birth rate), we pooled the relative risks ratios (RRs) and 95 % confidence intervals (CIs) in a random effects model. RESULTS: Our search resulted in the retrieval and screening of 134 studies. Of those, five studies were included in our meta-analysis. The risk for lower clinical pregnancy rate was not significantly increased in the deficient group (RR 0.88, 95 % CI 0.69-1.11). Lower vitamin D status was associated with lower live birth rate (RR 0.76, 95 % CI 0.61-0.93). CONCLUSION: There is no significant correlation between deficient serum vitamin D level and lower clinical pregnancy rate in infertile woman undergoing in vitro fertilization. On the other hand, deficient vitamin D level was related to lower live birth rate.

Angiopoietin-like protein 2 may mediate the inflammation in murine mastitis through the activation of interleukin-6 and tumour necrosis factor-α.

Mastitis is the inflammation of the mammary gland. Recent research has shown that Angiopoietin-like protein 2 (ANGPTL2) is a key inflammatory mediator. In the present study, we tested whether there is a correlation between increased ANGPTL2 expression and inflammation in response to Staphylococcus aureus in murine mastitis and the mechanisms involved. Thirty mice were divided into two groups: blank control group, challenged group. The entire infused mammary glands were removed to observe the changes of histopathology, myeloperoxidase (MPO) activity, production of tumour necrosis factor-α (TNF-α) and interleukin (IL)-6, and genes expression of ANGPTL2, TNF-α and IL-6. In challenged group, the structure of mammary glands was damaged and the large areas of cell fragments were observed. The MPO activity, IL-6 and TNF-α concentrations, ANGPTL2, IL-6, and TNF-α mRNA levels were significantly elevated in challenged group compared with blank control group. The present findings indicate ANGPTL2 may mediate the inflammation in murine mastitis through the activation of IL-6 and TNF-α.

Gene expression analysis of porcine peripheral blood mononuclear cell in response to immune stimulation of Poly I:C.

Polyinosinic-polycytidylic acid (Poly I:C) is an analogue of natural double strand RNA (dsRNA), which can simulate the viral dsRNA and stimulate the immune response. In the present study, peripheral blood mononuclear cells (PBMC) were isolated from piglets of Dapulian and Landrace with different disease resistance, and stimulated with 20 µg/mL Poly I:C for 24 hours in vitro culture. The expression of several cytokines (IL6, IL8, TNFα, IL10, IRF3, IFNα and IFNγ) and three pattern recognition receptors (TLR3, TLR4 and RIG1) was determined by qRT-PCR. The results showed that, most of the cytokines or receptors had obvious expression change compared with the control (without Poly I:C stimulation), especially the three cytokine genes IL6, IL8 and IL10, whose average expression change times were 20.71, 10.87 and 5.18, respectively. Expression comparison between breeds and among individuals of the same breed indicated that there was obvious difference not only between Dapulain and Landrace (Dapulain higher than Landrace) but also among the three individuals of the same breed. Our study simulated the infection of dsRNA to host cells using Poly I:C, and provided experimental foundation for further study on selecting the immune genes in response to Poly I:C stimulation and identifying the unique disease-resistance genes of Dapulian.

X-ray repair cross-complementing group 1 codon 399 polymorphism and lung cancer risk: an updated meta-analysis.

Many epidemiologic studies have investigated the association between x-ray repair cross-complementing group 1 gene (XRCC1) codon 399 polymorphism and lung cancer risk, but the results were inconsistent. We performed a meta-analysis of 46 studies on XRCC1 codon 399 polymorphism and lung cancer risk published before June 2013. In general population, the M allele and MM genotype were associated with increased risk of lung cancer compared with C allele and CC genotype, and the ORs were 1.06 (95% CI 1.01-1.12) and 1.19 (95% CI 1.05-1.34), respectively. When it was stratified according to Asian population, the association between XRCC1 codon 399 polymorphism and lung cancer risk was further strengthened. The ORs of comparison between M vs. C, MM vs. CC, and MM vs. CM + CC were 1.14 (95% CI 1.03-1.26), 1.41 (95% CI 1.11-1.78), and 1.38 (95% CI 1.12-1.71), respectively. The association between codon 399 polymorphism and lung cancer risk in nonsmoking Chinese women was stronger than any other subgroups. However, no associations were found in the Caucasian and African population. This meta-analysis has demonstrated that codon 399 polymorphism of XRCC1 gene might contribute to individual's susceptibility to lung cancer in Asian population, and especially in nonsmoking Chinese women. Future studies focused on interactions between combined genes and environmental risk factors are warranted.

Branched-Chain Amino Acids Deficiency Promotes Diabetic Neuropathic Pain Through Upregulating LAT1 and Inhibiting Kv1.2 Channel.

Diabetic neuropathic pain (DNP), one of the most common complications of diabetes, is characterized by bilateral symmetrical distal limb pain and substantial morbidity. To compare the differences  is aimed at serum metabolite levels between 81 DNP and 73 T2DM patients without neuropathy and found that the levels of branched-chain amino acids (BCAA) are significantly lower in DNP patients than in T2DM patients. In high-fat diet/low-dose streptozotocin (HFD/STZ)-induced T2DM and leptin receptor-deficient diabetic (db/db) mouse models, it is verified that BCAA deficiency aggravated, whereas BCAA supplementation alleviated DNP symptoms. Mechanistically, using a combination of RNA sequencing of mouse dorsal root ganglion (DRG) tissues and label-free quantitative proteomic analysis of cultured cells, it is found that BCAA deficiency activated the expression of L-type amino acid transporter 1 (LAT1) through ATF4, which is reversed by BCAA supplementation. Abnormally upregulated LAT1 reduced Kv1.2 localization to the cell membrane, and inhibited Kv1.2 channels, thereby increasing neuronal excitability and causing neuropathy. Furthermore, intraperitoneal injection of the LAT1 inhibitor, BCH, alleviated DNP symptoms in mice, confirming that BCAA-deficiency-induced LAT1 activation contributes to the onset of DNP. These findings provide fresh insights into the metabolic differences between DNP and T2DM, and the development of approaches for the management of DNP.

[Arsenic trioxide enhances TRAIL inducing human lung cancer cell line A549 cells apoptosis by down-regulate the expression of NF-kappaB].

OBJECTIVE: To study the influence of arsenic trioxide combined with human tumor necrosis factor related apoptosis inducing ligand (TRAIL) on the apoptosis and the expression of NF-kappaB of human non-small-cell lung cancer cell line. METHODS: The proliferation of human non-small-cell lung cancer cell line A549 cultured in vitro were treated by As2O3, TRAIL alone and combined. The cell proliferation was detected by the assay of MTT, flow cytometry with PI stain was used to detect the apoptosis rate, NF-kappaB mRNA level of A549 cells were detected by RT-PCR. The expression of NF-kappaB protein were detected by Western blot. The activity of NF-kappaB was measured by ELISA. RESULTS: Compared with As2O3 alone, As2O3 combined with TRAIL could increase the inhibition and apoptosis ratio significantly (P<0.05). The expression of NF-kappaB in combined group was obviously less than that in As2O3 alone and control; the activity of NF-kappaB was inhibited by combined groups. The NF-kappaB mRNA and protein expression and the activity of NF-kappaB were separately negative related with the apoptosis ratio (P<0.05). CONCLUSION: As2O3 can enhance TRAIL inducing of human lung cancer cell lines A549 apoptosis by inhibition of NF-kappaB signaling pathway.

Low-expression of E-cadherin in leukaemia cells causes loss of homophilic adhesion and promotes cell growth.

E-cadherin (epithelial cadherin) belongs to the calcium-dependent adhesion molecule superfamily and is implicated in the interactions of haematopoietic progenitors and bone marrow stromal cells. Adhesion capacity to bone marrow stroma was impaired for leukaemia cells, suggesting that a breakdown of adhesive mechanisms governed by an adhesion molecule may exist in leukaemic microenvironment. We previously found that E-cadherin was low expressed in primary acute leukaemia cells compared with normal bone marrow mononuclear cells. In this study, we investigate the functional importance of low E-cadherin expression in leukaemia cell behaviours and investigate its effects in the abnormal interaction of leukaemic cells with stromal cells. After expression of E-cadherin was restored by a demethylating agent in leukaemia cells, E-cadherin-specific adhesion was enhanced. Additionally, siRNA (small interfering RNA)-mediated silencing of E-cadherin in Raji cells resulted in a reduction of cell homophilic adhesion and enhancement of cell proliferation and colony formation. These results suggest that low expression of E-cadherin contributes to the vigorous growth and transforming ability of leukaemic cells.

[Genomic DNA extraction from hair sacs of pigs using modified phenol-chloroform method].

In referring to various methods for genomic DNA extraction from different tissues, we modified the classical phenol-chloroform procedure and reaction system for use in genomic DNA extraction from pig hair sacs. With the modified the phenol-chloroform method we successfully obtained high quality genomic DNA from pig hair sacs. Genomic DNA can be extracted from sacs of one to six pig hairs with satisfied quantity and quality for the need of PCR-based molecular ex-periment.

[Effect of Heterozygosity Loss in HLA Region before Transplantation on HLA Typing in Patients with Leukemia].

OBJECTIVE: To investigate the effect of loss of heterozygosity(LOH) in HLA region at initial diagnosis and remission of leukemia patient before transplantation on HLA typing. METHODS: The HLA typing was performed in DNA extracted from peripheral blood obtained at diagnosis (Sample 1 and Sample 2) and remission (Sample 3) in one pretransplant male patient with mixedphenotype acute leukemia (MPAL). HLA typing for HLA-A, B, C, DQB1, DRB1 was performed by Sequence-based typing (SBT), Sequence-specific oligonucleotide probe hybridization (SSO) and Sequence-specific primers (SSP). To define more precisely a cutoff limit for the detection of a heterozygous DNA present in a fraction of the cells by the SBT technology, DNA mixing experiments were performed. RESULTS: SBT results showed that Sample 1 and Sample 2 were both homozygous HLA results at five loci (lost one haplotype) although the sequencing background of Sample 1 was a little high. Except HLA-C locus was homozygous, Sample 3 was heterozygous HLA results at four loci. Based on DNA mixing experiments, a cutoff limit for the detection of heterozygous DNA was 20% by SBT technology, and a detection threshold for HLA-A, B, C, DQB1, DRB1 heterozygosity in blood samples was <75% blasts. CONCLUSION: Because LOH may be partial, any homozygous HLA result obtained during a blast crisis, especially ≥75% blasts, would have to be confirmed by a second typing on a buccal swab or on peripheral blood from the patient in complete remission.

Honokiol alleviates the degeneration of intervertebral disc via suppressing the activation of TXNIP-NLRP3 inflammasome signal pathway.

Intervertebral disc degeneration (IVDD) is a multifactorial disease and responsible for many spine related disorders, causes disability in the workforce and heavy social costs all over the world. Honokiol, a low molecular weight natural product, could penetrate into and distribute in IVDs to achieve therapeutic effect in a rat tail model. Therefore, the present study was undertaken to examine the antiinflammatory, antioxidation and IVD-protective effect of honokiol using nucleus pulposus cells and investigate its mechanisms to provide a new basis for future clinical treatment of IVDD. In the current study, we demonstrated that honokiol inhibits the H2O2-induced apoptosis (caspase-9, caspase-3, and bax), levels of oxidative stress mediators (ROS, MDA), expression of inflammatory mediators (Interleukin-6, COX-2, and iNOS), major matrix degrading proteases (MMP-3, MMP-13, ADAMTS5, and ADAMTS4) associated with nucleus pulposus degradation. Furthermore, we found nucleus pulposus protective ability of honokiol by up-regulating extra cellular matrix anabolic factors like type II collagen (Col II) and SOX9 in nucleus pulposus. We also found that honokiol suppressed the phosphorylation of NF-kB and JNK, and activation of TXNIP-NLRP3 inflammasome in H2O2-stimulated nucleus pulposus cells, thereby inhibiting the activation of downstream inflammatory mediators such as Interleukin-1ß. Furthermore, honokiol showed a cartilage protective effect in the progression of IVDD in a rat model induced by puncture. Thus, our results demonstrate that honokiol inhibited the H2O2 induced apoptosis, oxidative stress, and inflammatory responses through the depression of TXNIP/NLRP3/caspase-1/ Interleukin - 1ß signaling axis and the activation of NF-kB and JNK. Honokiol possess nucleus pulposus protective properties and may be of value in suppressing the pathogenesis of IVDD.

Ascorbic Acid Attenuates Multifidus Muscles Injury and Atrophy After Posterior Lumbar Spine Surgery by Suppressing Inflammation and Oxidative Stress in a Rat Model.

STUDY DESIGN: A rat model of multifidus muscles injury and atrophy after posterior lumbar spine surgery. OBJECTIVE: We determined the effect of ascorbic acid (AA) on the postoperative multifidus muscles in rat model. SUMMARY OF BACKGROUND DATA: Previous studies show oxidative stress and inflammation are two main molecular mechanisms in multifidus muscle injury and atrophy after posterior lumbar surgery. AA may have a protective effect in postoperative multifidus muscles. METHODS: Rats were divided into sham surgery, control surgery, and surgery plus AA groups. Multifidus muscles of the control and AA groups were excised from the osseous structures. The muscles were retracted continuously for 2 hours. In the sham and AA groups, AA was administered via oral gavage daily in the first week. In each group, the oxidative stress was evaluated by measuring malondialdehyde (MDA) and Total superoxide dismutase (T-SOD). The inflammation, fat degeneration, or fibrosis of multifidus muscle were evaluated by quantitative real-time polymerase chain reaction (q-PCR), histology, or immunohistochemical analysis. RESULTS: T-SOD activity was significantly lower in the control group than that in the AA group in the first week. MDA levels were significantly higher in the AA group. Interleukin-6 and tumor necrosis factor-α in multifidus muscles also showed significant differences when treated with AA. The inflammation score on histology was significantly lower in the AA group postoperatively in the first week. In the long run, marker genes for fibrosis and fat degeneration, and fibrosis and fat degeneration scores, were significantly lower in the AA than the control group on days 14 and 28 postoperatively. CONCLUSION: In conclusion, AA attenuated the oxidative stress and inflammation response in the postoperative multifidus muscles, and remarkable differences were observed from the histological assessment and related marker genes expression. Our results provided important insight into the anti-inflammatory and anti-oxidative effects of AA in the postoperative multifidus muscles. LEVEL OF EVIDENCE: N/A.

Lycorine protects cartilage through suppressing the expression of matrix metalloprotenases in rat chondrocytes and in a mouse osteoarthritis model.

Extracellular matrix (ECM) degrading enzymes, including matrix metalloproteinases (MMPs), are critical for cartilage destruction in the progression of osteoarthritis (OA). Thus, identifying novel drugs, which suppress the synthesis of MMPs may facilitate the treatment of OA. The cytotoxicity of lycorine was determined using a CCK8 assay. The effects of lycorine on IL­1ß­induced upregulation of MMPs and activation of mitogen­activated protein kinase pathways were detected by western blot analysis and reverse transcription­quantitative polymerase chain reaction. Hematoxylin and eosin staining and Safranin O staining were used to evaluate the effect of lycorine in a mouse anterior cruciate ligament transection model. In the present study, it was demonstrated for the first time, to the best of our knowledge, that lycorine (LY) suppressed interleukin­1ß (IL­1ß)­induced synthesis of MMP­3 and MMP­13 in vitro. Molecular analysis revealed that LY abrogated the phosphorylation of c­Jun N­terminal kinase (JNK) and the activation of the nuclear factor (NF)­κB signaling pathway caused by IL­1ß stimulation. In addition, in vivo experiments in a mouse anterior cruciate ligament transection model confirmed the protective role of LY on cartilage. Taken together, the data obtained in the present study demonstrated that LY suppressed the IL­1ß­induced expression of MMP­3 and MMP­13 through inhibition of the JNK and NF­κB pathways, suggesting that LY may be used as a potential drug for the treatment of OA.

[Study on the relationship between polymorphisms of peroxisome proliferators-activated receptor-gamma coactivator-1alpha gene and type 2 diabetes in Shanghai Hans in China].

OBJECTIVE: To observe the association between single nucleotide polymorphism (SNP) of peroxisome proliferators-activated receptor-gamma coactivator-1alpha (PGC-1alpha ) gene and type 2 diabetes mellitus(T2DM). METHODS: Four common SNPs of PGC-1alpha gene were genotyped with polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) and then analyzed with transmission-disequilibrium test (TDT) and sib transmission-disequilibrium test (STDT) in 69 T2DM pedigrees (310 individuals). Furthermore, the authors performed a case-control study to genotype Gly482Ser in 156 patients with T2DM and 111 normal glucose tolerance people without family history. RESULTS: (1)There were no positive results in four variances in TDT-STDT analysis(P> 0.05). (2)The Gly482Ser exhibited a significant difference between the two groups. GA genotype carriers were at increased risk for T2DM (OR=1.85), and there was statistically significant difference in the allele frequency between the case and control groups(P=0.046). (3) The subjects with GG genotype at position Gly482Ser had a higher HDL-C and lower LDL-C and TG levels when compared against those with GA+AA genotype in the control group without family history(P=0.043,lzP=0.046, P=0.037 respectively). CONCLUSION: This study suggested that the PGC-1alpha gene might be implicated in the pathogenesis of T2DM. But the studied SNPs in PGC-1alpha gene may not be major susceptibility ones of T2DM mellitus in Han people of Shanghai.

Evaluation of 12 Novel Molecular Markers for Degenerated Nucleus Pulposus in a Chinese Population.

STUDY DESIGN: A transcriptional expression assessment of human samples. OBJECTIVE: To evaluate 12 new candidate nucleus pulposus (NP) markers in degenerative disc disease in a Chinese population. SUMMARY OF BACKGROUND DATA: Disc degeneration is a major contributor of low back pain. However, no specific and reliable markers of degeneration of NP are available. METHODS: Specimens of NP were collected from 81 patients and grouped into the degenerated disc group (undergoing discectomy and fusion with significant signs of disc degeneration) and the trauma control group (undergoing anterior vertebral body and disc excision and fusion without signs of disc degeneration). Lumbar spine magnetic resonance imaging, hematoxylin-eosin staining, and safranin O staining of sections of NP tissues were conducted to evaluate the severity of the disc degeneration in all samples. Quantitative reverse transcription polymerase chain reaction was performed to investigate the levels of mRNA expression of these genes, as well as those of aggrecan, type II collagen, and SRY-box 9 (SOX-9). Degenerated samples were also divided into groups according to Pfirrmann grading system to elucidate the association of severity of degeneration and gene transcriptional levels. We also tested the relationship between mRNA levels of these genes and clinical characteristics such as hypertension and diabetes mellitus. RESULTS: We demonstrated that 11 of the 12 candidates showed significant differential expression in degenerated discs. Changes in the expression of these 11 genes were determined to be risk factors in degenerative disc diseases. The expression of neurochondrin (NCDN), keratin 8 (KRT8), and matrix Gla protein (MGP) even showed significant changes among subgroups of patients with degenerative disc disease stratified according to the Pfirrmann grading system. The expression of keratin 18 (KRT18), cadherin 2 (CDH2), synaptosomal-associated protein 25 (SNAP25), KRT8, and NCDN was significantly decreased in patients with hypertension. In contrast, the expression of MGP and cartilage oligomeric matrix protein was significantly upregulated in patients with diabetes mellitus. CONCLUSION: Overall, we demonstrated the clinical utility of 11 novel NP markers for degenerative disc disease. Among them, the expression of NCDN, KRT8, and MGP may indicate the severity of disc degeneration. LEVEL OF EVIDENCE: N/A.

Lycorine suppresses RANKL-induced osteoclastogenesis in vitro and prevents ovariectomy-induced osteoporosis and titanium particle-induced osteolysis in vivo.

Osteoclasts play an important role in diseases involving bone loss. In this study, we assessed the effect of a plant-derived natural alkaloid (lycorine, or LY) on osteoclastogenesis in vitro and in vivo. Our in vitro study showed that receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis could be inhibited by LY; this effect was due to inhibition of mitogen-activated protein kinase (MAPK) signalling via MAP kinase kinases (MKKs). The MAPK agonist anisomycin could partially rescue the inhibitory effect of LY. Furthermore, LY also played a protective role in both a murine ovariectomy (OVX)-induced osteoporosis model and a titanium particle-induced osteolysis model. These results confirmed that LY was effective in preventing osteoclast-related diseases in vivo. In conclusion, our results show that LY is effective in suppressing osteoclastogenesis and therefore could be used to treat OVX-induced osteoporosis and wear particle-induced osteolysis.

Selection of suitable reference genes for normalization of quantitative real-time polymerase chain reaction in human cartilage endplate of the lumbar spine.

The quantitative real-time polymerase chain reaction (qRT-PCR) is one of the most widely used methods to study gene expression profiles, and it requires appropriate normalization for accurate and reliable results. Although several genes are commonly used as reference genes (such as GAPDH, ACTB, and 18S rRNA), they are also regulated and can be expressed at varying levels. In this study, we evaluated twelve well-known reference genes to identify the most suitable housekeeping gene for normalization of qRT-PCR in human lumbar vertebral endplate with Modic changes, by using the geNorm, NormFinder, and BestKeeper algorithms. Our results showed that the rarely-used SDHA was the most stable single reference gene, and a combination of three, SDHA, B2M, and LDHA, was the most suitable gene set for normalization in all samples. In addition, the commonly-used genes, GAPDH, ACTB and 18S rRNA, were all inappropriate as internal standards. The rankings of reference genes for the three types of Modic change differed, although SDHA and RPL13A uniformly ranked in the first and last position, respectively. Further simulated expression analysis validated that the arbitrary use of a reference gene could lead to the misinterpretation of data. Our study confirmed the necessity of exploring the expression stability of potential reference genes in each specific tissue and experimental situation before quantitative evaluation of gene expression by qRT-PCR.