Mechanism of emodin in treating hepatitis B virus-associated hepatocellular carcinoma: network pharmacology and cell experiments.

Introduction: Hepatocellular carcinoma (HCC) is a pressing global issue, with Hepatitis B virus (HBV) infection remaining the primary. Emodin, an anthraquinone compound extracted from the natural plant's. This study investigates the molecular targets and possible mechanisms of emodin in treating HBV-related HCC based on network pharmacology and molecular docking and validates the screened molecular targets through in vitro experiments. Methods: Potential targets related to emodin were obtained through PubChem, CTD, PharmMapper, SuperPred, and TargetNet databases. Potential disease targets for HBV and HCC were identified using the DisGeNET, GeneCards, OMIM, and TTD databases. A Venn diagram was used to determine overlapping genes between the drug and the diseases. Enrichment analysis of these genes was performed using GO and KEGG via bioinformatics websites. The overlapping genes were imported into STRING to construct a protein-protein interaction network. Cytoscape 3.9.1 software was used for visualizing and analyzing the core targets. Molecular docking analysis of the drug and core targets was performed using Schrodinger. The regulatory effects of emodin on these core targets were validate through in vitro experiments. Results: A total of 43 overlapping genes were identified. GO analysis recognized 926 entries, and KEGG analysis identified 135 entries. The main pathways involved in the KEGG analysis included cancer, human cytomegalovirus infection and prostate cancer. The binding energies of emodin with HSP90AA1, PTGS2, GSTP1, SOD2, MAPK3, and PCNA were all less than -5 kcal/mol. Compared to normal liver tissue, the mRNA levels of XRCC1, MAPK3, and PCNA were significantly elevated in liver cancer tissue. The expression levels of XRCC1, HIF1A, MAPK3, and PCNA genes were closely related to HCC progression. High expressions of HSP90AA1, TGFB1, HIF1A, MAPK3, and PCNA were all closely associated with poor prognosis in HCC. In vitro experiments demonstrated that emodin significantly downregulated the expression of HSP90AA1, MAPK3, XRCC1, PCNA, and SOD2, while significantly upregulating the expression of PTGS2 and GSTP1. Conclusion: This study, based on network pharmacology and molecular docking validation, suggests that emodin may exert therapeutic effects on HBV-related HCC by downregulating the expression of XRCC1, MAPK3, PCNA, HSP90AA1, and SOD2, and upregulating the expression of PTGS2 and GSTP1.

Novel Risk Factors for Cervical Facet Joint Degeneration in the Subaxial Cervical Spine: Correlation with Cervical Sagittal Alignment and Bone Mineral Density.

OBJECTIVE: The impact of cervical sagittal alignment on cervical facet joint degeneration (CFD) and the risk factors for CFD in patients with degenerative cervical myelopathy (DCM) were investigated in the current study. METHODS: A total of 250 surgical patients with DCM were recruited. The clinical data and radiographical characteristics, including CFD, cervical sagittal balance parameters, Hounsfield unit (HU) values, disc degeneration (DD), and modic change, were collected. The detailed correlation between these characteristics and CFD was analyzed. Characteristics, including CFD, were compared among the various cervical alignment types and different CFD groups. Finally, the risk factors for CFD were revealed via logistic regression. RESULTS: CFD was prevalent in DCM patients. Age, cervical sagittal vertical axis (cSVA), range of motion, T1 slope, thoracic inlet angle, DD, HU value, and modic change correlated with CFD segmentally and globally (P < 0.05). The lordosis and sigmoid types had a significantly higher CFD prevalence (P < 0.05). Furthermore, the average CFD threshold for the severe CFD group was 1.625 (area under the curve, 0.958). Additionally, 167 patients with average CFD <1.625 and 83 patients with CFD of ≥1.625 were classified into the mild CFD group and severe CFD group, respectively. Finally, multivariate analysis was performed, and age, cSVA, HU value, modic change, and DD were determined to be independent risk factors for CFD. CONCLUSIONS: The load distribution tends to shift to a more shear-like pattern in the sigmoid and kyphosis types and in those with a higher cSVA, thereby promoting CFD. Aging, cervical malalignment, low bone mineral density, DD, and modic change were revealed to result in high risks of CFD.

Glutamine suppresses senescence and promotes autophagy through glycolysis inhibition-mediated AMPKα lactylation in intervertebral disc degeneration.

Regulating metabolic disorders has become a promising focus in treating intervertebral disc degeneration (IDD). A few drugs regulating metabolism, such as atorvastatin, metformin, and melatonin, show positive effects in treating IDD. Glutamine participates in multiple metabolic processes, including glutaminolysis and glycolysis; however, its impact on IDD is unclear. The current study reveals that glutamine levels are decreased in severely degenerated human nucleus pulposus (NP) tissues and aging Sprague-Dawley (SD) rat nucleus pulposus tissues, while lactate accumulation and lactylation are increased. Supplementary glutamine suppresses glycolysis and reduces lactate production, which downregulates adenosine-5'-monophosphate-activated protein kinase α (AMPKα) lactylation and upregulates AMPKα phosphorylation. Moreover, glutamine treatment reduces NP cell senescence and enhances autophagy and matrix synthesis via inhibition of glycolysis and AMPK lactylation, and glycolysis inhibition suppresses lactylation. Our results indicate that glutamine could prevent IDD by glycolysis inhibition-decreased AMPKα lactylation, which promotes autophagy and suppresses NP cell senescence.

Rosuvastatin suppresses TNF-α-induced matrix catabolism, pyroptosis and senescence via the HMGB1/NF-κB signaling pathway in nucleus pulposus cells.

Intervertebral disc degeneration is mainly caused by irregular matrix metabolism in nucleus pulposus cells and involves inflammatory factors such as TNF-α. Rosuvastatin, which is widely used in the clinic to reduce cholesterol levels, exerts anti-inflammatory effects, but whether rosuvastatin participates in IDD remains unclear. The current study aims to investigate the regulatory effect of rosuvastatin on IDD and the potential mechanism. In vitro experiments demonstrate that rosuvastatin promotes matrix anabolism and suppresses catabolism in response to TNF-α stimulation. In addition, rosuvastatin inhibits cell pyroptosis and senescence induced by TNF-α. These results demonstrate the therapeutic effect of rosuvastatin on IDD. We further find that HMGB1, a gene closely related to cholesterol metabolism and the inflammatory response, is upregulated in response to TNF-α stimulation. HMGB1 inhibition or knockdown successfully alleviates TNF-α-induced ECM degradation, senescence and pyroptosis. Subsequently, we find that HMGB1 is regulated by rosuvastatin and that its overexpression abrogates the protective effect of rosuvastatin. We then verify that the NF-κB pathway is the underlying pathway regulated by rosuvastatin and HMGB1. In vivo experiments also reveal that rosuvastatin inhibits IDD progression by alleviating pyroptosis and senescence and downregulating HMGB1 and p65. This study might provide new insight into therapeutic strategies for IDD.

BRD9 Inhibition Attenuates Matrix Degradation and Pyroptosis in Nucleus Pulposus by Modulating the NOX1/ROS/NF-κB axis.

Intervertebral disc degeneration (IDD) is considered to be the leading cause of low back pain (LBP). The progression of IDD is closely related to the inflammatory microenvironment, which results in extracellular matrix degradation and cell death. One of the proteins, which have been shown to participate in the inflammatory response, is the bromodomain-containing protein 9 (BRD9). This study aimed to investigate the role and mechanism of BRD9 in regulating IDD. The tumor necrosis factor-α (TNF-α) was used to mimic the inflammatory microenvironment in vitro. Western blot, RT-PCR, immunohistochemistry, immunofluorescence, and flow cytometry were used to demonstrate the effect of BRD9 inhibition or knockdown on matrix metabolism and pyroptosis. We found that the expression of BRD9 was upregulated as IDD progressed. BRD9 inhibition or knockdown alleviated TNF-α-induced matrix degradation, reactive oxygen species (ROS) production, and pyroptosis in rat nucleus pulposus cells. Mechanistically, RNA-seq was used to investigate the mechanism of BRD9 in promoting IDD. Further investigation revealed that BRD9 regulated NOX1 expression. Inhibition of NOX1 could abrogate matrix degradation, ROS production, and pyroptosis caused by BRD9 overexpression. In vivo, the radiological and histological evaluation showed that the pharmacological inhibition of BRD9 alleviated IDD development in rat IDD model. Our results indicated that BRD9 could promote IDD via the NOX1/ROS/ NF-κB axis by inducing matrix degradation and pyroptosis. Targeting BRD9 may be a potential therapeutic strategy in treating IDD.

Chromobox homolog 4 overexpression inhibits TNF-α-induced matrix catabolism and senescence by suppressing activation of the NF-κB signaling pathway in nucleus pulposus cells.

Intervertebral disc degeneration (IDD) is featured as enhanced catabolism of extracellular matrix (ECM) in the nucleus pulposus (NP), in which tumor necrosis factor-alpha (TNF-α)-related cell senescence is involved. Chromobox homolog protein 4 (CBX4) exhibits anti-inflammatory effects and shows promising therapeutic potential. Thus, in the present study, we explore the role of CBX4 in IDD. Immunohistochemistry staining reveals that CBX4 expression is decreased in severe degenerative NP tissues compared to mild degenerative tissues, and real-time PCR and western blot analysis results show that CBX4 expression is downregulated under TNF-α stimulation in NP cells. siRNA and adenoviruses are used to knockdown or overexpress CBX4, respectively. The results demonstrate that CBX4 knockdown augments the catabolism of ECM in human NP cells, while CBX4 overexpression in rat NP cells restores the ECM degradation induced by TNF-α, as illustrated by immunofluorescence and western blot analysis. In addition, transcriptome sequencing results reveal the regulatory effect of CBX4 on the cell cycle, and further western blot analysis and senescence-associated ß-galactosidase staining assay indicate that CBX4 overexpression alleviates cell senescence in the presence of TNF-α. Moreover, the phosphorylation of p65, which indicates the activation of NF-κB signaling, is measured by western blot analysis and immunofluorescence assay, and the results reveal that CBX4 overexpression reduces the TNF-α-induced increase in the p-p65/p65 ratio. In addition, the effect of CBX4 overexpression in NP cells is suppressed by NF-κB agonist. In summary, our results indicate that CBX4 overexpression can suppress TNF-α-induced matrix catabolism and cell senescence in the NP by inhibiting NF-κB activation. This study may provide new approaches for preventing and treating IDD.

Atorvastatin inhibited TNF-α induced matrix degradation in rat nucleus pulposus cells by suppressing NLRP3 inflammasome activity and inducing autophagy through NF-κB signaling.

Intervertebral disc degeneration (IDD) is one of the main causes of lower back pain (LBP). It results from an imbalance between the degradation and synthesis of extracellular matrix (ECM) components in nucleus pulposus (NP) cells. Atorvastatin, an HMG-CoA reductase inhibitor, plays a vital role in many diseases, such as cardiovascular disease and osteoarthritis. However, the effect of atorvastatin on IDD is unclear. Herein, we demonstrated that atorvastatin affects matrix degradation induced by TNF-α and demonstrated the mechanism by which TNF-α modulates matrix metabolism in rat NP cells. Real-time PCR, western blotting and immunofluorescence staining were performed to detect the mRNA and protein expression of related genes. mRFP-GFP-LC3 adenovirus plasmid transfection and transmission electron microscopy (TEM) were used to detect cell autophagy. NLRP3 inhibitor and lentiviral vectors containing shRNA-NLRP3 were used to show the effect of NLRP3 on autophagic flux and the NF-κB signaling pathway. The results revealed that atorvastatin might suppress matrix degradation induced by TNF-α by suppressing NLRP3 inflammasome activity and inducing autophagic flux. Moreover, atorvastatin suppressed NF-κB signaling induced by TNF-α. NF-κB signaling inhibition suppressed NLRP3 inflammasome activity, and NLRP3 inhibition suppressed NF-κB signaling activation induced by TNF-α. NLRP3 inhibition or NLRP3 knockdown induced autophagic flux in the presence of TNF-α. Overall, the present study demonstrated that atorvastatin might suppress matrix degradation induced by TNF-α and further revealed the crosstalk among NLRP3 inflammasome activity, autophagy and NF-κB signaling.

TNF-α/NF-κB signaling epigenetically represses PSD4 transcription to promote alcohol-related hepatocellular carcinoma progression.

BACKGROUND: Chronic alcohol consumption is more frequently associated with advanced, aggressive hepatocellular carcinoma (HCC) tumors. Alcohol adversely impacts ER/Golgi membrane trafficking and Golgi protein N-glycosylation in hepatocytes; these effects have been attributed (in part) to dysregulated adenosine diphosphate-ribosylation factor (ARF) GTPase signaling. Here, we investigated the role of the ARF GTPase guanine exchange factor PSD4 in HCC progression. METHODS: R-based bioinformatics analysis was performed on publicly available array data. Modulating gene expression was accomplished via lentiviral vectors. Gene expression was analyzed using quantitative real-time PCR and immunoblotting. PSD4 promoter methylation was assessed using quantitative methylation-specific PCR. Phospho-p65(S276)/DNMT1 binding to the PSD4 promoter was analyzed via chromatin immunoprecipitation. We constructed ethanol/DEN-induced and DEN only-induced transgenic murine models of HCC. RESULTS: We identified PSD4 as a hypermethylated, suppressed gene in alcohol-related HCC tumors; however, PSD4 was not dysregulated in all-cause HCC tumors. Certain HCC cell lines also displayed varying degrees of PSD4 downregulation. PSD4 overexpression or knockdown decreased and increased cell migration and invasiveness, respectively. Mechanistically, PSD4 transcription was repressed by TNF-α-induced phospho-p65(S276)'s recruitment of DNA methyltransferase 1 (DNMT1), resulting in PSD4 promoter methylation. PSD4 inhibited pro-EMT CDC42 activity, resulting in downregulation of E-cadherin and upregulation of N-cadherin and vimentin. Hepatocyte-specific PSD4 overexpression reduced ethanol/DEN-induced HCC tumor progression and EMT marker expression in vivo. CONCLUSIONS: PSD4 is a hypermethylated, suppressed gene in alcohol-related HCC tumors that negatively modulated pro-EMT CDC42 activity. Furthermore, we present a novel phospho-NF-κB p65(S276)/DNMT1-mediated promoter methylation mechanism by which TNF-α/NF-κB signaling represses PSD4 transcription in HCC cells.

Bromodomain-containing protein 7 regulates matrix metabolism and apoptosis in human nucleus pulposus cells through the BRD7-PI3K-YAP1 signaling axis.

Intervertebral disc degeneration (IDD) results from dysregulated metabolism of the extracellular matrix of the nucleus pulposus (NP) and involves the participation of inflammatory factors such as TNF-α. Bromodomain-containing protein 7 (BRD7) shows considerable potential for anti-inflammatory applications. Herein, we investigated the role of BRD7 in IDD. The immunohistochemistry results demonstrated decreased BRD7 expression in severely degenerated human NP tissues compared to those showing mild degeneration. Lentiviruses and adenoviruses were used to knock down or overexpress BRD7 and YAP1, respectively. Our results revealed that BRD7 knockdown promoted matrix degradation and suppressed PI3K and YAP1 expression, while BRD7 overexpression alleviated matrix degradation and promoted YAP1 and PI3K expression. In addition, PI3K inhibition augmented matrix degradation, enhanced apoptosis, and reduced YAP1 expression, whereas YAP1 overexpression promoted matrix synthesis, suppressed apoptosis and promoted PI3K expression. Besides, BRD7 overexpression reversed the reductions in sulfated glycosaminoglycan levels induced by TNF-α, but this effect was blocked by PI3K or YAP1 inhibitors. Moreover, YAP1 and PI3K were shown to interact through coimmunoprecipitation analysis. In summary, our results demonstrate that BRD7 can regulate matrix metabolism and apoptosis in human NP cells through the BRD7-PI3K-YAP1 signaling axis. This study might provide new insights into the prevention and treatment of IDD.

Identification of key potential targets for TNF-α/TNFR1-related intervertebral disc degeneration by bioinformatics analysis.

BACKGROUND: Bioinformatics analysis was performed on gene expression profile microarray data to identify the key genes activated through the TNF-α/TNFR1 signaling pathway in intervertebral disc degeneration (IDD). The common differentially expressed genes (co-DEGs) were calculated in nucleus pulposus (NP) cells and annulus fibrosus (AF) cells under TNF-α treatment or TNFR1 knockdown, which reveals the potential mechanism of TNF-α involvement in IDD and may provide new therapeutic targets for IDD. METHODS: Differentially expressed genes (DEGs) in TNF-α-treated or TNFR1-knockdown NP cells and AF cells were identified. Further analysis of the gene ontology (GO), signaling pathways and interaction networks of the DEGs or co-DEGs were conducted using the Database for Annotation, Visualization and Integrated Discovery, STRING Database, and Cytoscape software. The relationship between genes and musculoskeletal diseases, including IDD, was assessed with the Comparative Toxicogenomics Database. The predicted microRNAs corresponding to the co-DEGs were also identified by microRNA Data Integration Portal. RESULTS: In NP cells, the DEGs (|log2FoldChange|>2, adj.P < 0.01) were identified including 48 DEGs by TNF-α treatment and 74 DEGs by TNFR1 knockdown; in AF cells, correspondingly, 105 DEGs were identified. The co-DEGs between NP cells and AF cells were calculated including CXCL8, ICAM1, BIRC3, RELB, NFKBIA, and TNFAIP3. They may be the hub genes that were significantly associated with both NP cells and AF cells through the TNF-α/TNFR1 signaling pathway. The co-DEGs and corresponding predicted miRNAs may be potential therapeutic targets for IDD. CONCLUSIONS: CXCL8, ICAM1, BIRC3, RELB, NFKBIA, and TNFAIP3 may have a synergistic effect on TNF-α-induced IDD development.Abbreviations: IDD: Intervertebral disc degeneration; NP: Nucleus pulposus; AF: Annulus fibrosus; co-DEG: Common differentially expressed gene; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; PPI: Protein-protein interaction.

Nicotinamide Phosphoribosyl Transferase Controls NLRP3 Inflammasome Activity Through MAPK and NF-κB Signaling in Nucleus Pulposus Cells, as Suppressed by Melatonin.

Intervertebral disc degeneration (IDD) is characterized by an imbalance between matrix synthesis and degradation in intervertebral discs. However, the causes of this imbalance remain elusive. Previous studies revealed that NLRP3 inflammasome plays a vital role in IDD and nicotinamide phosphoribosyl transferase (NAMPT) is involved in matrix degradation induced by IL-1ß. In the current study, real-time PCR, western blot and NAMPT knockdown, or overexpression experiments were used to detect the regulatory effects of NAMPT on NLRP3 inflammasome activity in nucleus pulposus (NP) cells. The results revealed that NAMPT downregulation or overexpression controlled the matrix degradation induced by TNF-α by modulating NLRP3 inflammasome activity. Moreover, the NAMPT inhibition study demonstrated MAPK and NF-κB signaling play a key role in above process. In addition, melatonin was reported to play a protective role in matrix metabolism of NP cells. Herein, real-time PCR, western blot analysis, and immunofluorescence staining experiments revealed that melatonin showed protective effects against TNF-α-induced matrix degradation by downregulating NAMPT and reducing NLRP3 inflammasome activity in NP cells. The current investigation verified that melatonin could alleviate matrix degradation induced by TNF-α by suppressing NAMPT and NLRP3 inflammasome activity. Moreover, NAMPT downregulation controlled the matrix degradation induced by TNF-α by suppressing NLRP3 inflammasome activity through MAPK and NF-κB signaling in NP cells.

Bromodomain-containing protein 4 inhibition alleviates matrix degradation by enhancing autophagy and suppressing NLRP3 inflammasome activity in NP cells.

An imbalance between matrix synthesis and degradation is the hallmark of intervertebral disc degeneration while inflammatory cytokines contribute to the imbalance. Bromodomain and extra-terminal domain (BET) family is associated with the pathogenesis of inflammation, and inhibition of BRD4, a vital member of BET family, plays an anti-inflammatory role in many diseases. However, it remains elusive whether BRD4 plays a similar role in nucleus pulposus (NP) cells and participates in the pathogenesis of intervertebral disc degeneration. The present study aims to observe whether BRD4 inhibition regulates matrix metabolism by controlling autophagy and NLRP3 inflammasome activity. Besides, the relationship was investigated among nuclear factor κB (NF-κB) signaling, autophagy and NLRP3 inflammasome in NP cells. Here, real-time polymerase chain reaction, western blot analysis and adenoviral GFP-LC3 vector transduction in vitro were used, and it was revealed that BRD4 inhibition alleviated the matrix degradation and increased autophagy in the presence or absence of tumor necrosis factor α. Moreover, p65 knockdown or treatment with JQ1 and Bay11-7082 demonstrated that BRD4 inhibition attenuated NLRP3 inflammasome activity through NF-κB signaling, while autophagy inhibition by bafilomycin A1 promoted matrix degradation and NLRP3 inflammasome activity in NP cells. In addition, analysis of BRD4 messenger RNA expression in human NP tissues further verified the destructive function of BRD4. Simply, BRD4 inhibition alleviates matrix degradation by enhancing autophagy and suppressing NLRP3 inflammasome activity through NF-κB signaling in NP cells.

Cancer Stem Cells of Diffuse Large B Cell Lymphoma Are Not Enriched in the CD45+CD19- cells but in the ALDHhigh Cells.

Although the existence of cancer stem cells (CSCs) has been suggested in diffuse large B cell lymphoma (DLBCL), there is still no definitive marker. CD45+CD19- has been regarded as a potential marker of CSCs in mantle cell lymphoma (MCL). So, we explored the role of CD45+CD19- in DLBCL. However, both CD45+CD19- cells and CD45+CD19+ cells did not generate tumors until more than 100,000 cells were inoculated in NOD/SCID mice, even CD45+CD19+ cells generated more and larger tumors, as well as the soft agar colony formation in vitro; The aldehyde dehydrogenase (ALDH) activity was also identified in this study. Only 1,500 ALDHhigh cells were enough to generate tumors in mice while the same number of ALDH- cells were not. Moreover, both groups formed tumors when more cells were inoculated, but ALDHhigh cells formed more and larger tumors. The similar result was obtained in vitro clonogenicity experiments. OCT4, SOX2, Nanog, and ABCG2 genes did not show any difference in CD45+CD19+, CD45+CD19-, ALDHhigh and ALDH- cells. Taken together, CSCs are not enriched in the CD45+CD19- cells but in the ALDHhigh cells in DLBCL cell lines.

Erdheim-Chester disease mimicking lumbar nerve schwannoma: case report and literature review.

Introduction: Erdheim-Chester disease (ECD) is a rare, non-Langerhans cell histiocytosis. The clinical spectrum of ECD is diverse, varying from asymptomatic focal lesion to life-threatening multisystem infiltration. Neurological manifestations of ECD are common, mostly due to the involvement of the central nerve system. However, spinal nerve or peripheral nerve involvement has rarely been mentioned. Case presentation: Herein, we present a case of a 32-year-old female patient complaining about radiating pain on the front and lateral side of her left thigh for 2 months. Spinal MRI with contrast enhancement showed a space-occupying lesion on the left L3/L4 intervertebral foramen, indicating an initial diagnosis of lumbar nerve schwannoma. The patient underwent surgery to remove the mass and decompress the lumbar nerve. Postoperative histological examination revealed the diffuse infiltration of foamy histiocytes that were CD68+, CD163+, and CD1a- on immunostaining, which confirmed the diagnosis of Erdheim-Chester disease. The radiating pain was gradually alleviated and PET-CT was performed but showed no further involvement of ECD. Discussion: To the best of our knowledge, this is the first case of ECD demonstrated as an infiltrative mass on the spinal nerve, with imaging manifestations and compression symptoms similar to those of peripheral nerve schwannoma.

Taurine Supplementation Ameliorates Arsenic-Induced Hepatotoxicity and Oxidative Stress in Mouse.

We previously reported that taurine treatment inhibited arsenic (As)-induced apoptosis in the liver of mice. This study was designed to explore the effect of taurine on liver function and its underlying mechanism in As-exposed mice. Mice were randomly divided into 3 groups, ten mice in each group. Group 1, control group, only orally received drinking water alone. Group 2, As intoxication group, was exposed to 4 mg/L As2O3 via drinking water for 60 days. Group 3, taurine protection group, was treated with 4 mg/L As2O3 and 150 mg/kg both. Taurine administration significantly revered the increases of alanine transaminase (ALT) and aspartate transaminase (AST) activities in serum. The decrease of glutathione (GSH) was inhibited with taurine treatment in the liver of As-exposed mice. At the same time, taurine significantly inhihibited As-induced enhancement of malondialdehyde (MDA) in the liver. Here we show that taurine protective effect on liver function in As-exposed mice maybe involve lipid peroxidation.

Protective Effect of Taurine on Paraquat-Induced Lung Epithelial Cell Injury.

The herbicide Paraquat induce oxidative stress-mediated lung injury. Taurine is a well-known antioxidant. This study was designed to explore the effect of taurine on paraquat-induced injury and its related mechanism in A549 cells. The cells were pretreated with various concentrations of taurine for 30 min prior to paraquat exposure. 24 h later, cell viability was examined by the MTT assay. The level of glutathione (GSH) and the activity of glutathione peroxidase (GPx) were analyzed. The results show that taurine treatment significantly attenuates the decrease in cell viability mediated by paraquat in A549 cells. Taurine also reversed paraquat-induced disturbances in GSH content and GPx activity. Taurine exerts protection against paraquat-mediated A549 cell toxicity likely through modulation of oxidative stress.

MicroRNA-145 overexpression attenuates apoptosis and increases matrix synthesis in nucleus pulposus cells.

AIMS: Lower back pain is often associated with intervertebral disc degeneration (IDD), which results from a decrease in nucleus pulposus (NP) cells and an imbalance between the degradation and synthesis of extracellular matrix (ECM) components. Multiple microRNAs play crucial roles in the modulation of NP cell apoptosis and matrix degradation. miR-145 is an important microRNA related to degenerative diseases such as osteoarthritis. Here, the effect of miR-145 in IDD was elucidated. The aim of this study was to explore the role and mechanism of miR-145 in the apoptosis of NP cells and in matrix metabolism in NP cells. MATERIALS AND METHODS: Real-time PCR, western blotting and flow cytometry analysis were used to observe the effect of miR-145 on NP cell apoptosis in the absence or presence of oxidative stress. Cell transfection, loss-of-function experiments using an ADAM17 inhibitor or lentiviral shADAM17, immunofluorescence, real-time PCR and western blotting were performed to demonstrate the role and mechanism of miR-145 in NP cell matrix metabolism. KEY FINDINGS: miR-145 attenuated NP cell apoptosis in the absence and presence of oxidative stress. Moreover, miR-145 overexpression increased and miR-145 suppression decreased matrix synthesis. ADAM17, which is expressed in degenerative discs, is the target of miR-145. ADAM17 gene suppression with lentiviral shRNA or an inhibitor enhanced matrix synthesis in NP cells. In addition, siADAM17 reversed the matrix degradation induced by miR-145 inhibition. SIGNIFICANCE: miR-145 suppresses apoptosis and promotes ECM synthesis in NP cells. miR-145 is thus a potential therapeutic microRNA for IDD.

Hepatitis B core antigen regulates dendritic cell proliferation and apoptosis through regulation of PKC/NF­κB signaling pathway.

Hepatitis B core antigen (HBcAg) possesses unusual immunologic features. However, the biological roles and mechanisms of HBcAg in dendritic cell proliferation and apoptosis remain to be elucidated. In the present study, DC2.4 cells were treated with different concentrations of HBcAg (10, 20 and 30 µg/ml). MTT assay and flow cytometry (Annexin V/propidium iodide analysis) were performed to investigate changes in cell proliferation and apoptosis. Western blot analysis was conducted to examine the changes in nuclear factor (NF)­κB and protein kinase C (PKC) signaling pathways. NF­κB inhibitor pyrrolidine dithiocarbamate (PDTC) and PKC inhibitor Chelerythrine were used to block these two signaling pathways. It was identified that HBcAg increased proliferation and decreased apoptosis in a dose­dependent manner. Western blotting results demonstrated that HBcAg upregulated p­PKC, p­IκB, p­P65, tumor necrosis factor­α and B­cell lymphoma 2 (Bcl­2) levels, and downregulated cleaved caspase 3, demonstrating that HBcAg activated the PKC and NF­κB signaling pathways. NF­κB inhibitor PDTC reduced the effects of HBcAg on DC2.4 proliferation (0.6 fold vs. 0.25 fold) and apoptosis (0.43 fold vs. 0.17 fold), and on Bcl­2 expression levels. PKC inhibitor Chelerythrine reduced the biological effects of HBcAg; it reduced proliferation (0.67 fold vs. 0.23 fold) and upregulated apoptosis (0.43 fold vs. 0.13 fold). Chelerythrine also blocked NF­κB activity and the HBcAg­induced Bcl­2 increase, suggesting the effect on Bcl­2 from HBcAg was dependent on the PKC/NF­κB signaling pathway. In conclusion, HBcAg promoted proliferation and inhibited apoptosis through the PKC/NF­κB/Bcl­2 signaling pathway in DC2.4 cells.

Transcription factor 7-like 2 controls matrix degradation through nuclear factor κB signaling and is repressed by microRNA-155 in nucleus pulposus cells.

AIM: TCF7L2, a key transcription factor in the canonical Wnt pathway, plays a vital role in the matrix degradation of chondrocytes. However, it is unknown whether TCF7L2 is important in the matrix metabolism of inner gel-like nucleus pulposus (NP) cells; thus, the aim of this study was to explore the effect and mechanism of TCF7L2 in this process. METHODS: Western blotting and immunofluorescence analyses were used to observe TCF7L2 expression in rat and human NP tissues. Real-time PCR and western blotting were performed to detect the expression of TCF7L2 stimulated by inflammatory cytokines. Dual luciferase reporter assay, real-time PCR, western blotting and knockdown experiments were performed to demonstrate the role of NF-κB signaling in matrix regulation by TCF7L2 and the regulation of TCF7L2 by miR-155 in intervertebral disc degeneration. KEY FINDINGS: TCF7L2 is present in rat and human NP tissues and is expressed in the nucleus of NP cells. TCF7L2 is refractory to stimulation of rat and human NP cells with the inflammatory cytokines TNF-α and IL-1ß, in contrast to the results in other cell types. Loss-of-function experiments using TCF7L2 siRNA or lentiviral shTCF7L2 showed that TCF7L2 knockdown suppresses matrix degradation through p65/NF-κB signaling in the absence and presence of TNF-α. In addition, TCF7L2 expression is repressed by miR-155 overexpression and promoted by miR-155 inhibition. SIGNIFICANCE: Overall, these results demonstrate that the suppression of TCF7L2, which is modulated by miR-155, inhibits matrix degradation through p65/NF-κB signaling. TCF7L2 suppression may have therapeutic potential in intervertebral disc degeneration.

[Effects of bone marrow mesenchymal stem cells on the proliferation of hepatocytes and cirrhotic fat-storing cells in vitro].

OBJECTIVE: To investigate the differentiation of bone marrow mesenchymal stem cells (BMSCs) and the effects of BMSCs on the proliferation of cirrhotic fat-storing cells (CFSC) and hepatocytes in vitro. METHODS: BMSCs and hepatocytes were isolated and harvested from the bone marrow and livers of rats. A co-culture system was set up by transwell inserts in which the two chambers were separated by a semipermeable membrane. BMSCs labeled with PKH26 were cultured with hepatocytes/CFSC in the co-culture system and also in a cell-cell direct contact culture system. Anti-albumin and anti-smooth muscle alpha-actin (alpha-SMA) antibodies were tested by using fluorescence immunocytochemistry. BMSCs and hepatocytes/CFSC cultured alone served as controls. The proliferation level of hepatocytes in the co-culture system was measured. CFSC were cultured with the conditional medium of BMSCs, and their quantities were measured microscopically. RESULTS: Expression of albumin was observed in the hepatocytes of the two culture systems after they were cultured for 72 h but the albumin levels were higher in the cell-cell direct contact culture system (P<0.01). As compared to the controls, the number of hepatocytes was larger in the co-culture system (P<0.01). No expression of alpha-SMA in CFSC was observed in either culture system. The proliferation of CFSC was inhibited by the conditional medium of BMSCs. The longer the time of the co-culturing the more significant was the CFSC growth suppression (P<0.01). CONCLUSIONS: BMSCs can be induced into hepatocytes by a local micro-environment formed by hepatocytes. BMSCs may promote proliferation of hepatocytes and inhibit proliferation of CFSC.