SR9009 attenuates inflammation-related NPMSC pyroptosis and IVDD through NR1D1/NLRP3/IL-1ß pathway.

Intervertebral disc is a highly rhythmical tissue. As a key factor linking biorhythm and inflammatory response, the shielding effect of NR1D1 in the process of intervertebral disc degeneration remains unclear. Here, we first confirmed that NR1D1 in the nucleus pulposus tissue presents periodic rhythmic changes and decreases in expression with intervertebral disc degeneration. Second, when NR1D1 was activated by SR9009 in vitro, NLRP3 inflammasome assembly and IL-1ß production were inhibited, while ECM synthesis was increased. Finally, the vivo experiments further confirmed that the activation of NR1D1 can delay the process of disc degeneration to a certain extent. Mechanistically, we demonstrate that NR1D1 can bind to IL-1ß and NLRP3 promoters, and that the NR1D1/NLRP3/IL-1ß pathway is involved in this process. Our results demonstrate that the activation of NR1D1 can effectively reduce IL-1ß secretion, alleviate LPS-induced NPMSC pyroptosis, and protect ECM degeneration.

Melatonin alleviates oxidative stress-induced injury to nucleus pulposus-derived mesenchymal stem cells through activating PI3K/Akt pathway.

Background: The changes in the microenvironment of degenerative intervertebral discs cause oxidative stress injury and excessive apoptosis of intervertebral disc endogenous stem cells. The purpose of this study was to explore the possible mechanism of the protective effect of melatonin on oxidative stress injury in NPMSCs induced by H2O2. Methods: The Cell Counting Kit-8 assay was used to evaluate the cytotoxicity of hydrogen peroxide and the protective effects of melatonin. ROS content was detected by 2'7'-dichlorofluorescin diacetate (DCFH-DA). Mitochondrial membrane potential (MMP) was detected by the JC-1assay. Transferase mediated d-UTP Nick end labeling (TUNEL) and Annexin V/PI double staining were used to determine the apoptosis rate. Additionally, apoptosis-associated proteins and PI3K/Akt signaling pathway-related proteins were evaluated by immunofluorescence, immunoblotting and PCR. ECMs were evaluated by RT‒PCR and immunofluorescence. In vivo, X-ray, Magnetic resonance imaging (MRI) and Histological analyses were used to evaluate the protective effect of melatonin. Results: Melatonin had an obvious protective effect on NPMSCs treated with 0-10 µM melatonin for 24 h. In addition, melatonin also had obvious protective effects on mitochondrial dysfunction, decreased membrane potential and cell senescence induced by H2O2. More importantly, melatonin could significantly reduce the apoptosis of nucleus pulposus mesenchymal stem cells induced by H2O2 by regulating the expression of apoptosis-related proteins and decreasing the rate of apoptosis. After treatment with melatonin, the PI3K/Akt pathway was significantly activated in nucleus pulposus mesenchymal stem cells, while the protective effect was significantly weakened after PI3K-IN-1 treatment. In vivo, the results of X-ray, MRI and histological analyses showed that therapy with melatonin could partially reduce the degree of intervertebral disc degeneration. Conclusion: Our research demonstrated that melatonin can effectively alleviate the excessive apoptosis and mitochondrial dysfunction of nucleus pulposus mesenchymal stem cells induced by oxidative stress via the PI3K/Akt pathway, which provides a novel idea for the therapy of intervertebral disc degeneration. The translational potential of this article: This study indicates that melatonin can effectively alleviate the excessive apoptosis and mitochondrial dysfunction of NPMSCs through activating the PI3K/Akt pathway. Melatonin might serve as a promising candidate for the prevention and treatment of Intervertebral disc degeneration disease (IVDD) in the future.

Penfluridol targets acid sphingomyelinase to inhibit TNF signaling and is therapeutic against inflammatory autoimmune diseases.

BACKGROUND: Penfluridol, isolated from an FDA-approved small-molecule drug library as an inhibitor of tumor necrosis factor α (TNFα)-stimulated NF-κB activation, is clinically used to treat chronic schizophrenia and related disorders. This study is aimed to investigate the therapeutic effect of penfluridol on TNFα-stimulated inflammatory autoimmune diseases, particularly inflammatory arthritis. METHODS: Various in vitro studies to confirm the inhibitory effect of penfluridol on TNFα-induced NF-κB activity in bone marrow-derived macrophages or Raw 264.7 macrophage cell line. In vivo studies assessed the therapeutic effects of penfluridol in various disease models, including TNFα transgenic mice, collagen-induced arthritis, DSS-induced colitis, and TNBS-induced colitis. Identification and characterization of the binding of penfluridol to acid sphingomyelinase using bioinformatics and drug affinity responsive target stability assay. Acid sphingomyelinase activity assays to reveal penfluridol-mediated inhibition of acid sphingomyelinase activity. siRNA knockdown experiments to illustrate the dependence of penfluridol's anti-TNF activity on acid sphingomyelinase. RESULTS: Penfluridol effectively inhibited TNFα-induced NF-κB activation in vitro and alleviated the severity of arthritis and colitis in vivo. Mechanistic studies revealed that penfluridol bound to acid sphingomyelinase and inhibited its activation. In addition, knockdown of acid sphingomyelinase largely abolished the inhibitory effects of penfluridol on TNFα-induced inflammatory cytokine production. Furthermore, penfluridol suppressed the differentiation of spleen naive CD4+T cells to TH1 and TH17 and inhibited M1 macrophage polarization. CONCLUSION: This study provides the rationale for the possible innovative use of penfluridol as a newly identified small-molecule drug for TNFα-driven diseases, such as inflammatory arthritis and colitis.

Association Between Dentin Matrix Protein 1 (rs10019009) Polymorphism and Ankylosing Spondylitis in a Chinese Han Population from Shandong Province.

BACKGROUND: Ankylosing spondylitis (AS) is the most common rheumatic condition that is slowly progressive and predominantly affects adolescents. Pathological bone formation associated with AS is an important cause of disability. The aim of the study was to investigate the possible involvement of the genes related to endochondral ossification and ectopia ossification in genetic susceptibility to AS in a Chinese Han population. METHODS: Sixty-eight single nucleotide polymorphisms (SNPs) from 13 genes were genotyped in discovery cohorts including 300 AS patients and 180 healthy controls. The rs10019009 in dentin matrix protein 1 (DMP1) gene shown as association with AS after multiple testing corrections in discovery cohorts was replicated in a validation independent cohort of 620 AS patients and 683 healthy controls. The rs10019009 was assessed with bioinformatics including phylogenetic context, F-SNP and FastSNP functional predictions, secondary structure prediction, and molecular modeling. We performed a functional analysis of rs10019009 via reverse transcription-polymerase chain reaction, alkaline phosphatase (ALP) activity in human osteosarcoma U 2 OS cells. RESULTS: Interestingly, the SNP rs10019009 was associated with AS in both the discovery cohort (P = 0.0012) and validation cohort (P = 0.0349), as well as overall (P = 0.0004) in genetic case-control association analysis. After a multivariate logistic regression analysis, the effect of this genetic variant was observed to be independent of linkage disequilibrium. Via bioinformatics analysis, it was found that the amino acid change of the rs10019009 led to changes of SNP function, secondary structure, tertiary conformation, and splice mode. Finally, functional analysis of rs10019009 in U 2 OS cells demonstrated that the risk T allele of the rs10019009 increased enzymatic activity of ALP, compared to that of the nonrisk allele (P = 0.0080). CONCLUSIONS: These results suggested that the DMP1 gene seems to be involved in genetic predisposition to AS, which may contribute to the ectopic mineralization or ossification in AS. In addition, DMP1 gene may be a promising intervention target for AS in the future.

Serum complement C3f and fibrinopeptide A are potential novel diagnostic biomarkers for non-alcoholic fatty liver disease: a study in Qingdao Twins.

AIMS: To compare the different serum peptidome patterns between twins with and without non-alcoholic fatty liver disease (NAFLD) in order to help understand the pathogenesis of NAFLD and to identify potential diagnostic and therapeutic targets. METHODS: The peptidomics patterns of 63 cases with NAFLD were compared with their twin healthy controls in Qingdao, China. Peptides between 800 Da and 3,500 Da were captured and concentrated using C18 reversed-phase columns, followed by MALDI-TOF mass spectrometry. The sequences of peptides associated with NAFLD were further identified by MALDI-TOF-TOF. Further validation studies were conducted. One hundred additional serum samples were detected by commercially available ELISA kits to calculate the concentrations of complement C3f and fibrinopeptide A, respectively. The differences of these two peptides in the NAFLD and control groups were compared using SPSS 17.0, respectively. RESULTS: Compared with healthy controls, eleven peaks (861.1, 877.07, 904.5, 1206.57, 1350.64, 1518.7, 1690.9, 1777.94, 2931.29, 3190.4, 3261.4) were up-regulated and 7 peaks (942.44, 1020.47, 1060.06, 1211.7, 1263.63, 1449.76, 2768.3) were down-regulated in the NAFLD group. Two peptides derived from complement C3f and fibrinopeptide A, respectively, had the highest ROC values indistinguishing NAFLD cases from their normal controls. In the validation group, the concentrations of complement C3f and fibrinopeptide A (1466.929 ± 78.306 pg/ml, 4.189 ± 0.326 ng/ml, respevtively) in NAFLD group was higher than in control group (complement C3f 1159.357 ± 99.624 pg/ml, FPA 3.039 ± 0.483 ng/ml; P<0.05). CONCLUSIONS: In this study, we established apeptidomics pattern that could help distinguish NAFLD patients from their twin controls. The differently-regulated peptides identified in our study may be potential diagnostic markers or therapeutic targets for NAFLD.

Proteomic analysis identifies MMP-9, DJ-1 and A1BG as overexpressed proteins in pancreatic juice from pancreatic ductal adenocarcinoma patients.

BACKGROUND: There is an urgent need to discover more sensitive and specific biomarkers to improve early diagnosis and screen high-risk patients for pancreatic ductal adenocarcinoma (PDAC). Pancreatic juice is an ideal specimen for PDAC biomarkers discovery, because it is an exceptionally rich source of proteins released from pancreatic cancer cells. METHODS: To identify novel potential biomarkers for PDAC from pancreatic juice, we carried out difference gel electrophoresis (DIGE) and tandem mass spectrometry (MS/MS) to compare the pancreatic juice profiling from 9 PDAC patients and 9 cancer-free controls. Of the identified differently expressed proteins, three up-regulated proteins in pancreatic cancer juice, matrix metalloproteinase-9 (MMP-9), oncogene DJ1 (DJ-1) and alpha-1B-glycoprotein precursor (A1BG), were selected for validation by Western blot and immunohistochemistry. Serum MMP-9 levels were also detected by enzyme linked immunosorbent assay (ELISA). RESULTS: Fourteen proteins were up-regulated and ten proteins were down-regulated in cancerous pancreatic juice compared with cancer-free controls. Increased MMP-9, DJ-1 and A1BG expression in cancerous pancreatic juice were confirmed by Western blot. Immunohistochemical study showed MMP-9, DJ-1 and A1BG positively expressed in 82.4%, 72.5% and 86.3% of pancreatic cancer tissues, significantly higher than that in normal pancreas tissues. Up-regulation of DJ-1 was associated with better differentiation (p < 0.05). Serum MMP-9 levels were significantly higher in PDAC (255.14 ng/ml) than those in chronic pancreatitis (210.22 ng/ml, p = 0.009) and healthy control (203.77 ng/ml, p = 0.027). CONCLUSION: The present proteome analysis revealed MMP-9, DJ-1 and A1BG proteins as elevated in pancreatic juice from PDAC, which suggest their further utility in PDAC diagnosis and screening. This is the first time A1BG was identified as a potential biomarker in pancreatic cancer associated samples. The measurement of serum MMP-9 might be clinically useful for PDAC diagnosis.