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.

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.

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.

[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.

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.

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.

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.

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.

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.

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.

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.

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.

Upregulation of tumor necrosis factor α and ADAMTS-5, but not ADAMTS-4, in human intervertebral cartilage endplate with modic changes.

STUDY DESIGN: This study investigated the expression of 2 types of ADAMTS in human intervertebral cartilage endplate (CEP) and related mechanisms concerning tumor necrosis factor α (TNF-α)-induced nuclear factor κB signaling pathway. OBJECTIVE: To determine which type of ADAMTS is more strongly expressed and the role of TNF-α in CEP. SUMMARY OF BACKGROUND DATA: ADAMTS-4 and ADAMTS-5 were proven to be essential in the degeneration of articular cartilage and intervertebral disc. CEP is an important structure adjacent to the disc. However, the activities of ADAMTS in CEP are unclear. METHODS: CEPs were obtained from subjects after spinal surgery and categorized as members of either the Modic change group or the control group. Sections of these tissues were stained with hematoxylin-eosin, safranin O, and immunohistochemistry procedures for ADAMTS-4, ADAMTS-5, and TNF-α. Transcriptional levels of aggrecan, type I collagen, type II collagen, type X collagen, ADAMTS-4, ADAMTS-5, and TNF-α were investigated by quantitative real-time polymerase chain reaction. In addition, the effect of TNF-α on ADAMTS-5 and its potential mechanisms are investigated in cultured bovine endplate chondrocytes in vitro. RESULTS: Our data demonstrated that the degenerative changes associated with the expression of extracellular matrix proteins were correlated with increased levels of ADAMTS-5, but not ADAMTS-4, in the CEP of patients with Modic changes. The expression levels of TNF-α in the Modic change group were significantly increased, which was correlated with the enhanced expression of ADAMTS-5. Additional in vitro investigation confirmed that TNF-α could upregulate the expression of ADAMTS-5 by activating nuclear factor κB pathway in cultured bovine endplate chondrocytes. CONCLUSION: We conclude that the upregulation of TNF-α and ADAMTS-5, but not ADAMTS-4, may play an important role in degenerative CEP-induced low back pain. LEVEL OF EVIDENCE: N/A.

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.

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.

Clinical observation of whole brain radiotherapy concomitant with targeted therapy for brain metastasis in non-small cell lung cancer patients with chemotherapy failure.

OBJECTIVE: To investigate the clinical effects of whole brain radiotherapy concomitant with targeted therapy for brain metastasis in non-small cell lung cancer (NSCLC) patients with chemotherapy failure. MATERIALS AND METHODS: Of the 157 NSCLC patients with chemotherapy failure followed by brain metastasis admitted in our hospital from January 2009 to August 2012, the combination group (65 cases) were treated with EGFR-TKI combined with whole brain radiotherapy while the radiotherapy group (92 cases) were given whole brain radiotherapy only. Short-term effects were evaluated based on the increased MRI in brain 1 month after whole brain radiotherapy. Intracranial hypertension responses, hematological toxicity reactions and clinical effects of both groups were observed. RESULTS: There were more adverse reactions in the combination group than in radiotherapy group, but no significant differences were observed between the two groups in response rate (RR) and disease control rate (DCR) (P>0.05). Medium progression free survival (PFS), medium overall survival (OS) and 1-year survival rate in combination group were 6.0 months, 10.6 months and 42.3%, while in the radiotherapy group they were 3.4 months, 7.7 months and 28.0%, respectively, which indicated that there were significant differences in PFS and OS between the two groups (P<0.05). Additionally, RPA grading of each factor in the combination group was a risk factor closely related with survival, with medium PFS in EGFR and KRAS mutation patients being 8.2 months and 11.2 months, and OS being 3.6 months and 6.3 months, respectively. CONCLUSIONS: Whole brain radiotherapy concomitant with target therapy is favorable for adverse reaction tolerance and clinical effects, being superior in treating brain metastasis in NSCLC patients with chemotherapy failure and thus deserves to be widely applied in the clinic.

Effect and possible mechanism of muscle-splitting approach on multifidus muscle injury and atrophy after posterior lumbar spine surgery.

BACKGROUND: Multifidus muscle injury and atrophy are common after posterior lumbar spine surgery and are associated with low back pain and functional disability. In theory, muscle-splitting and retraction with a self-retaining retractor are considered to be the major surgical factors. The effects and mechanisms of retraction have been well studied, but the exact effect and possible mechanism of injury and atrophy after muscle-splitting still lack experimental evidence. METHODS: New Zealand White rabbits were divided into two groups. In group S, through a skin and lumbodorsal fascial incision, the bilateral multifidus muscles were dissected from the osseous structures in the standard fashion, while in group C, only a skin and lumbodorsal fascial incision was made. In each group, the multifidus muscle was evaluated by magnetic resonance imaging (MRI) and by histological analysis at three and forty-eight hours and at one, three, six, twelve, and twenty-four weeks after surgery. RESULTS: In group C, there was no injury or atrophy of the multifidus muscle after surgery. In group S, the mean T2-weighted signal intensity ratios of gross multifidus to psoas on fat-suppressed T2-weighted cross-sectional MRI scans peaked on week 3 and returned to baseline on week 24. Necrosis and inflammation of the multifidus muscle were evident and became more severe at one week. Fibrotic change was mainly seen at three and six weeks after surgery, and fatty degeneration mainly occurred at twelve and twenty-four weeks. Decreased acetylcholine activity and granular degeneration of the neuromuscular junction were observed at all follow-up times, and the numbers of degenerating neuromuscular junctions increased significantly with time after surgery. CONCLUSIONS: The splitting approach is an important cause of multifidus muscle injury and atrophy in posterior lumbar spine surgery. Denervation and disuse may be important factors in multifidus muscle atrophy in the splitting approach. CLINICAL RELEVANCE: This study provides a basis for the prevention of multifidus muscle injury and atrophy after posterior lumbar surgery.

Low bone mineral density in chinese adults with nonalcoholic Fatty liver disease.

Aim. To investigate bone metabolic characteristics in Chinese adults with nonalcoholic fatty liver disease (NAFLD). Methods. A total of 224 patients (99 males and 125 postmenopausal females) were recruited and divided into 4 groups: males without NAFLD, males with NAFLD, females without NAFLD, and females with NAFLD. Bone mineral density (BMD) was evaluated according to body mass index (BMI), waist circumference (WC), and serum biomarkers. ß cell function was evaluated by HOMA2%B, HOMA2%S, and HOMA2IR. Results. Males in the NAFLD group had lower BMD of the right hip and the femoral neck (0.852 ± 0.117 versus 0.930 ± 0.123, P = 0.002; 0.736 ± 0.119 versus 0.812 ± 0.132, P = 0.004), and females had lower BMD of the right hip (0.725 ± 0.141 versus 0.805 ± 0.145, P = 0.002) even after adjusted for weight, BMI, waist, HDL, and ALT. There was no significant difference in bone metabolic markers between patients with and without NAFLD. NAFLD was an important factor that affected the bone; moreover, the effect attenuated when HOMA2IR entered into the model (R (2) = 0.160, ß = -0.172, and P = 0.008). Conclusions. NAFLD exerts a detrimental effect on BMD in both males and females. Insulin resistance may play an important role in this pathophysiological process.