Effects of Expression of Matrix Metalloproteinases and Discoidin Domain Receptors in Ligamentum Flavum Fibrosis in Patients with Degenerative Lumbar Canal Stenosis.

STUDY DESIGN: This is a retrospective cohort study. PURPOSE: This study aimed to clarify the role of crosstalk between discoidin domain receptors (DDRs) and matrix metalloproteinases (MMPs) in the ligamentum flavum (LF) fibrosis obtained from patients with degenerative lumbar canal stenosis (DLCS). OVERVIEW OF LITERATURE: The DDRs, DDR1 and DDR2, are cell surface receptors and have an essential role in collagen fiber accumulation in several fibrotic diseases. MMPs are one of the critical factors in extracellular matrix remodeling and elastic fiber degradation in LF tissues. However, the crosstalk between DDRs and MMPs and the role of this molecular signal in LF fibrosis remain unclear. METHODS: A total of 35 patients were divided into two groups in this study. Spinal surgery was performed in 23 of these patients with the diagnosis of DLCS. Twelve patients with lumbar disk herniation (LDH) were included in the control group. On axial T2-weighted magnetic resonance imaging, LF thickness was measured bilaterally at the level of the facet joint. Histology, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot analyses were performed on LF tissue samples. LF tissues were stained with hematoxylin and eosin. In addition, the grade of fibrosis was histologically assessed using Masson trichrome triple staining. DDR1 and DDR2 Western blot analyses were performed. DDR1, DDR2, MMP2, MMP3, MMP9, and MMP13 expression levels were measured using qRT-PCR analysis. RESULTS: The grade of fibrosis and LF thickness were significantly higher in the DLCS patients than in the LDH patients. DDR1 and DDR2 gene expression and protein levels in LF tissues are significantly greater in DLCS samples than in control samples, according to both qRT-PCR and Western blot analyses. In addition, we detected a significant expression of the MMP3, MMP9, and MMP13, which are known to have important roles in extracellular matrix remodeling in DLCS. Furthermore, we discovered a link between DDR protein levels and LF thickness, fibrosis, and MMP3/MMP9. CONCLUSIONS: Our results indicate that DDR1, DDR2, and MMP3 and MMP9 signals can be correlated with each other in LF tissues and be promoted LF fibrosis leading to spinal canal narrowing in patients with DLCS.

Protective effect of pyrrolidine dithiocarbamate against methotrexate-induced testicular damage.

The aim of the study was to investigate the protective effect of pyrrolidine dithiocarbamate (PDTC) against methotrexate (MTX)-induced testicular damage in rats. Forty Wistar albino male rats were divided into equally four groups: Control group (saline solution, IP), PDTC group (100 mg/kg PDTC,IP, 10 days), MTX group (20 mg/kg MTX, IP, single dose, on the 6th day) and MTX + PDTC group (100 mg/kg PDTC, IP, 10 days and 20 mg/kg MTX, IP, single dose, on the 6th day). After 10 days, testicular tissues were excised for morphometric, histological and immunohistochemical evaluations. Serum testosterone, follicle stimulating hormone (FSH), luteinizing hormone (LH) and prokineticin 2 (PK2) levels were determined. Body and testicular weights were measured. Testicular damage was assessed by histological evaluation. Nuclear factor kappa B (NFkB), nuclear factor erythroid 2 related factor 2 (Nrf2) and PK2 immunoreactivities were evaluated by HSCORE. Body and testicular weights, serum FSH, LH, testosterone levels, seminiferous tubule diameter and germinal epithelial thickness were significantly decreased in the MTX group. However, serum PK2 level, histologically damaged seminiferous tubules and interstitial field width were significantly increased. Additionally, there was an increase in NFkB and PK2 immunoreactivity, whereas there was a significant decrease in Nrf2 immunoreactivity. PDTC significantly improved hormonal, morphometric, histological and immunohistochemical findings. Taken together, we conclude that PDTC may reduce MTX-induced testicular damage via NFkB, Nrf2 and PK2 signaling pathways.

The Role of JAK-STAT Signaling Activation in Hypertrophied Ligamentum Flavum.

BACKGROUND: Although previous studies have reported the expression of JAK1, STAT3, and phosphorylated STAT3 in hypertrophied ligamentum flavum (LF), the role of the Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling pathway in hypertrophied LF has not been fully elucidated. The aim of this study was to identify the important JAK/STAT gene expression patterns of the 3 main receptors involved in this pathway: interferon (IFN)-γ receptor (IFN-γR), IFN-α receptor (IFNAR), and interleukin (IL)-6 receptor (IL-6R). METHODS: The human LF specimens were obtained from 28 patients who underwent lumbar spine surgery for either degenerative lumbar canal stenosis (DLCS) (n = 28) or lumbar disc herniation (LDH) (n = 20). In this design, patients with LDH served as the control group. The degree of fibrosis was demonstrated by Masson's trichrome staining. The location and expression profiling of the JAK/STAT pathway were analyzed by quantitative real-time polymerase chain reaction and Western blotting. The thickness of the LF was measured with axial T1-weighted magnetic resonance imaging. RESULTS: The most severe fibrotic changes were on the dorsal side of the LF. IL-6 and IFN-I expression levels were significantly increased on the dorsal side of the LF. While expression levels of IL-6R and IFNAR on the dural and dorsal side were significantly higher in the DLCS samples, IFN-γR and endothelial epidermal growth factor receptor in LF samples showed a significant increase only on the dorsal side. JAK/STAT genes were significantly expressed, especially on the dorsal side. CONCLUSIONS: Our data suggest that IFNAR- and IL-6R-dependent JAK/STAT signaling pathways may be significant targets in drug development strategies for the treatment of LF hypertrophy.

Inhibition of the Invasion of Human Glioblastoma U87 Cell Line by Ruxolitinib: A Molecular Player of miR-17 and miR-20a Regulating JAK/STAT Pathway.

AIM: To determine the interaction between ruxolitinib, JAK/STAT signalling, and two angio-microRNAs (miRs) to expose potential target molecules in the inhibition of glioblastoma invasion. MATERIAL AND METHODS: The invasion properties of glioblastoma were analyzed using a cancer cell spheroid invasion assay. Following treatment of 195 nM ruxolitinib, the relative expression levels of miR-17 and miR-20a and genes of IL-6/JAK/STAT3 receptor signaling belonging to the JAK/STAT pathway were measured by qRT-PCR in treated and untreated three-dimensional tumor spheres of U87 cells. RESULTS: Our results indicated that a therapeutic dose of ruxolitinib (195 nM) significantly increased miR-17 and miR-20a expression. Ruxolitinib treatment resulted in the production of IL-6 and active formation of IL-6 receptor complex for the subsequent activation of the IL-6R/JAK2/STAT3 axis. However, ruxolitinib treatment significantly decreased the expression of JAK2 and PI3K. Pearson correlation analyses revealed a strong negative correlation of miR-17 with JAK2, STAT3, and PI3K expressions, and also miR-20a has a negative correlation with expression levels of JAK2 and PI3K. The only positive correlation was found to be between miR-20a and IL-6, gp130 expressions. CONCLUSION: The specific JAK2 inhibitor ruxolitinib plays an important role in glioblastoma angiogenesis biology via inhibiting IL-6 receptor-dependent JAK/STAT signaling. Additionally, both miR-17a-3p and miR-20a overexpression induced by ruxolitinib treatment may be playing a major role in downregulated JAK2, STAT3, and PI3K proteins. Our results suggest that miR-17-3p and miR-20a-5p may serve as new therapeutic targets for the treatment of glioblastoma.