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.

Expression and clinical significance of hypoxia-induced long non-coding RNA TCONS_I2_00001955 in breast cancer.

BACKGROUND: Long non-coding RNAs (lncRNAs) have been found to play important roles in occurrence, development, and metastasis of various tumors. We aimed to screen long non-coding RNAs (lncRNAs) that promote invasion and metastasis of breast cancer cells under hypoxia, and investigate the relationship between lncRNA expression and clinicopathological features and prognosis in invasive breast cancer. METHODS: LncRNA microarray was used to screen the differentially expressed lncRNAs in MCF7, MDA-MB-231, and SKBR3 breast cancer cell lines cultured under normoxia and hypoxia, respectively. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to verify the microarray results. CCK8 and Transwell experiments were performed to identify the lncRNA that promote proliferation, migration, and invasion of breast cancer cells. Expression of the lncRNA and HIF-1α in invasive breast cancer was detected by RNAscope and immunohistochemistry, respectively. Correlation between the lncRNA expression and baseline characteristics was analyzed. Prognostic value of the lncRNA was evaluated using univariate and multivariate Cox regression. RESULTS: Expression of lncRNA TCONS_I2_00001955 in all the three breast cancer cells was increased under hypoxia. Overexpression of TCONS_I2_00001955 significantly enhanced proliferation, migration, and invasion of SKBR3 cells. Positive expression of TCONS_I2_00001955 was associated with recurrence, metastasis, and high expression of HIF-1α (P < 0.05), and it was an independent risk factor for poor disease-free survival of breast cancer. CONCLUSION: Hypoxia-induced lncRNA TCONS_I2_00001955 was associated with aggressive feature and poor prognosis of breast cancer.

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.

High BTBD7 expression positive is correlated with SLUG-predicted poor prognosis in hormone receptor-negative breast cancer.

BACKGROUND: Hormone receptor-negative breast cancer (HRNBC), which includes triple-negative breast cancer (TNBC) and human epidermal growth factor receptor 2 (HER-2) overexpressing breast cancer, is prone to metastasis and has a poor prognosis. BTB/POZ domain-containing protein 7 (Btbd7) is thought to regulate SLUG and the epithelial-mesenchymal transition (EMT) process. However, the role of Btbd7 in HRNBC is unclear. METHODS: Expression of BTBD7 and SLUG in HRNBC tumor tissue and normal adjacent tissue (NAT) as well as breast cancer cells were characterized by immunohistochemistry and immunofluorescence. MDA-MA-231 cells was transfected with BTBD7 siRNA and detected by qRT-PCR and western blot. Expression levels of Slug and EMT related proteins were detected western blot analysis. cell invasion assays were used to analyse cell invasion ability of MDA-MA-231. GO and KEGG analyses was used to analysis the gene function. RESULTS: The total positive rate of BTBD7 expression in HRNBC tumor tissue was 66.7%, which was higher than that in NAT (52.1%) and benign breast lesion tissues (20%). Co-expression of SLUG and BTBD7 proteins could be found in HRNBC tissue and MDA-MA-231 cells. BTBD7 silencing significantly up-regulated the epithelial marker E-cadherin, down-regulated the mesenchymal markers α-SMA and SLUG and suppressed the invasion abilities of MDA-MA-231 cells. GO and KEGG analyses based on 322 DEGs showed that BTBD7 may be associated with generic transcription in breast cancer. CONCLUSIONS: The study data indicated that BTBD7 was inversely associated with SLUG expression. Higher BTBD7 was associated with poor clinicopathologic features and prognosis in HRNBC patients. BTBD7 silencing inhibited EMT through regulation of SLUG expression. BTBD7 might act as a potential molecular target for gene therapy in HRNBC patients.

Naringin alleviates H2O2-induced apoptosis via the PI3K/Akt pathway in rat nucleus pulposus-derived mesenchymal stem cells.

Purpose: To investigate the protective effect of naringin (Nar) on H2O2-induced apoptosis of nucleus pulposus-derived mesenchymal stem cells (NPMSC) and the potential mechanism in this process. Methods: Rat NPMSC were cultured in MSC culture medium or culture medium with different concentrations of H2O2. Nar or the combination of Nar and LY294002 was added into the culture medium to investigate the effects of Nar. Cell viability was evaluated by cell counting kit-8 (CCK-8) assay. The apoptosis rate was determined using Annexin V/PI dual staining and terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assays. Additionally, the levels of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were analyzed by flow cytometry. ATP level in NPMSC was analyzed via ATP detection kit. Mitochondrial ultrastructure change was observed through transmission electron microscope (TEM). Levels of apoptosis-associated molecules (cleaved caspase-3, Bax and Bcl-2) were evaluated via RT-PCR and western blot, respectively. Results: The cells isolated from NP met the criteria for MSC. H2O2 significantly promoted NPMSC apoptosis in a dose and time-dependent manner. Nar showed no cytotoxicity effect on NPMSC up to a concentration of 100 µM for 24 h. Nar exhibited protective effects against H2O2-induced NPMSC apoptosis including apoptosis rate, expressions of proapoptosis and antiapoptosis related genes and protein. Nar could also alleviate H2O2-induced mitochondrial dysfunction of increased mitochondrial ROS production, reduced MMP, decreased intracellular ATP and mitochondrial ultrastructure change. However, these protected effects were inhibited after LY294002 treatment. Conclusions: Our results demonstrated that Nar efficiently attenuated H2O2-induced NPMSC apoptosis and mitochondrial dysfunction. The activation of ROS-mediated PI3K/Akt pathway may be the potential mechanism in this process.

The effect of intervertebral disc degenerative change on biological characteristics of nucleus pulposus mesenchymal stem cell: an in vitro study in rats.

Purpose: To evaluate the change on biological characteristics of mesenchymal stem cell (MSC) derived from normal and degenerative intervertebral disc (IVD). Methods: MSC was isolated from normal and degenerative IVD rat model. Immunophenotype detected by flow cytometric analysis, expression of stemness genes determined by reverse-transcription polymerase chain reaction (RT-PCR) and osteogenic, adipogenic and chondrogenic differentiation were compared between MSC derived from normal IVD (N-NPMSC) and degenerative IVD (D-NPMSC). The biological characteristics including cell proliferation, colony formation, apoptosis, caspase-3 activity and mRNA and protein expressions of hypoxia inducible factor-1α (HIF-1α), glucose transporter 1 (GLUT-1), vascular endothelial growth factor (VEGF), silent information regulator protein 1 (SIRT1) and silent information regulator protein 6 (SIRT6) were compared between N-NPMSC and D-NPMSC. Results: Both of N-NPMSC and D-NPMSC highly expressed CD105, CD90 and CD73, and lower expressed CD34 and CD45. There was no significant difference in cell morphology and multipotent differentiation ability between N-NPMSC and D-NPMSC. D-NPMSC showed significantly lower expressions of stemness genes, cell proliferation and colony formation ability. D-NPMSC also exhibited increased cell apoptosis rate and caspase-3 expression, and significantly lower expressions of HIF-1α, GLUT-1, VEGF, SIRT1 and SIRT6 in mRNA and protein levels compared with N-NPMSC. Conclusions: N-NPMSC showed significantly higher proliferation rate, better colony forming and stemness maintenance ability, whereas reduced cell apoptosis rate compared with D-NPMSC. HIF-1α-mediated signal pathway may be involved in the regulation of NPMSC proliferation. These findings indicated that degenerative change of IVD should be taken into account when selecting a source of NPMSC for clinical application.

A new method of subtotal thyroidectomy for Graves' disease leaving a unilateral remnant based on the upper pole.

BACKGROUND: The aim of this prospective randomized study was to evaluate the feasibility of subtotal thyroidectomy with leaving a unilateral remnant based on the upper pole. METHODS: Patients who underwent the subtotal thyroidectomy and isthmusectomy leaving either a unilateral remnant based on the upper pole (Group I, 79 patients) or the bilateral dorsal thyroid tissue remained (Group II, 89 patients) were compared in operation time, blood loss, recurrence, and postoperative complications. RESULTS: Among 168 patients analyzed, the operation time remained similar, but the blood loss, the reoperation time, and recurrence in Group I were much less than Group II. In addition, no postoperative hemorrhage occurred in Group I. Two patients (2.28%) in Group II underwent recurrent laryngeal nerve damages. Four patients (5.06%) in Group I and 3 patients (3.37%) in Group II experienced transient hypocalcemia. Recurrence only occurred in Group II. CONCLUSION: In terms of blood loss, reoperation time, postoperative complication, and the recurrence, subtotal thyroidectomy with recurrent laryngeal nerves identification and the unilateral superior pole remnant of the gland provides a better outcome than subtotal thyroidectomy with bilateral dorsal thyroid tissue remnant.

[Establishment of a NOD/SCID mouse model with human immune reconstitution bearing human triple-negative breast cancer].

OBJECTIVE: To establish a NOD/SCID mouse model with human immune reconstitution and observe its immune response to human triple-negative breast cancer xenograft. METHODS: Twenty-four NOD/SCID mice without immune leakage were subjected to cyclophosphamide (CTX) treatment 3 days prior to immune reconstitution with human peripheral blood mononuclear cell (PBMC) injection and subcutaneous transplantation of human triple-negative breast cancer MDA-MB-231 cells, CTX treatment and PBMC injection without tumor cell transplantation, MDA-MB-231 cell transplantation only, or no treatments. The tumor growth and immune responses of the mice were observed at regular intervals. RESULTS: Compared with the tumor-bearing mice, the tumor-bearing mice with immune reconstitution showed prolonged incubation period of tumor formation, slower tumor growth rate and increased survival rate. Human IgG and CD3(+) T cells were detected in the peripheral blood of the mice 1 week after human PBMC injection. The percentage of CD3(+) T cells in the spleen cells was 55.3% at 9 weeks in tumor-bearing mice with immune reconstitution and 52.7% in tumor-bearing mice without immune reconstitution. The spleen index of the tumor-bearing mice with immune reconstitution was much higher than that in mice with only immune reconstitution and the control mice (9.64 vs 3.82∓0.31 and 1.51∓0.14 mg/g). CONCLUSION: A stable NOD/SCID mouse model with immune reconstitution has been established successfully, which shows immune responses to triple-negative breast cancer xenografts and allows studies of immunological therapy study of triple-negative breast cancer.