Correlation exploration among CT imaging, pathology and genotype of pulmonary ground-glass opacity.

To analyse the clinical features, imaging manifestation, pathological typing and genetic testing results of patients undergoing surgery for ground-glass opacity (GGO) nodules, and explore the reasonable diagnosis and treatment program for GGO patients as to provide the basis for the establishment of GGO treatment process. This study is an exploratory study. 465 cases with GGO confirmed by HRCT, undergoing surgery and approved by pathologic diagnosis in Shanghai pulmonary hospital were enrolled in this study. All the patients with GGO were cases with single lesion. The relationship between the clinical, imaging, pathological and molecular biological data of single GGO were statistically studied. (1) Among 465 cases, the median age was 58 years and females were 315 (67.7%); there were 397 (85.4%) non-smoking, and 354 cases (76.1%) had no clinical symptoms. There were 33 cases of benign and 432 cases of malignant GGO. Significant differences were observed on the size, vacuole sign, pleural indentation and blood vessel sign of GGO between two groups (p < 0.05). Of 230 mGGO, there were no AAH, 13 cases of AIS, 25 cases of MIA and 173 cases of invasive adenocarcinoma. The probability of solid nodules in invasive adenocarcinoma was higher than that in micro invasive carcinoma, and the difference was statistically significant (p < 0.05). 360 cases were followed up with the average follow-up time of 6.05 months, and GGO of 34 cases (9.4%) increased. (2) In 428 adenocarcinoma samples approved by pathologic diagnosis, there were 262 (61.2%) lesions of EGFR mutation, 14 (3.3%) lesions of KRAS mutation, 1 (0.2%) lesion of Braf mutation, 9 (2.1%) lesions of EML4-ALK gene fusion and 2 (0.5%) lesions of ROS1 fusion. The detection rate of gene mutation in mGGO was higher than that of pGGO. During the follow-up period, genetic testing results of 32 GGO showed that EGFR mutation rate was 53.1%, ALK positive rate of 6.3%, KRAS mutation rate of 3.1% and no ros1 and BRAF gene mutation. No statistically significant difference was observed in comparison with unchanged GGO. (3) EGFR mutation rate of invasive adenocarcinoma was the highest (168/228, 73.7%), mainly in the 19Del and L858R point mutations. No KRAS mutation was found in atypical adenoma hyperplasia. No significant difference was observed on the mutation rate of KRAS between different types of GGO (p = 0.811). EML4-ALK fusion gene was mainly detected in invasive adenocarcinoma (7/9). GGO tends to occur in young, non-smoking women. The size of GGO is related to the degree of malignancy. Pleural depression sign, vacuole sign and vascular cluster sign are all characteristic images of malignant GGO. pGGO and mGGO reflect the pathological development of GGO. During the follow-up, it is found that GGO increases and solid components appear, which is the indication of surgical resection. The detection rate of EGFR mutations in mGGO and invasive adenocarcinoma is high. pGGO has heterogeneity in imaging, pathology and molecular biology. Heterogeneity research helps to formulate correct individualized diagnosis and treatment plans.

ERK1 loss accelerates the progression of osteoarthritis in aged mice via NRF2/BACH1 signaling.

Osteoarthritis (OA) is now a common degenerative joint related disease. However, the clinical efficacy of drugs associated with cartilage regeneration remains limited. In our study, we firstly explored the role of ERK1 in the progression of OA. We clarified that ERK1-deficient mice were susceptible to age-related OA. The higher OARSI scores and more severe cartilage degeneration was observed in the ERK1-deficient mice. ERK1 deficiency decreased the nuclear transportation of Nrf2 in the chondrocytes and accelerated chondrocyte aging in vitro. Moreover, chondrocytes with ERK1 deficiency elevated the nuclear expression of BACH1, resulting in lowered expression of antioxidant enzymes in ERK1-deficient chondrocytes. The Nrf2 activator dimethyl fumarate (DMF) was used. Our experiments demonstrated the protective function of DMF against OA in ERK1 knockout mice. Above all, we confirmed the effects of ERK1 on the progression of OA and clarified the mechanisms underlying these effects. DMF might has significant use in the development of novel drugs for the therapy of OA in the future.

Circular RNA circPDE4D Protects against Osteoarthritis by Binding to miR-103a-3p and Regulating FGF18.

Osteoarthritis (OA) is a common, age-related, and painful disease characterized by cartilage destruction, osteophyte formation, and synovial hyperplasia. This study revealed that circPDE4D, a circular RNA derived from human linear PDE4D, plays a critical role in maintaining the extracellular cellular matrix (ECM) during OA progression. circPDE4D was significantly downregulated in OA cartilage tissues and during stimulation with inflammatory cytokines. The knockdown of circPDE4D predominantly contributed to Aggrecan loss and the upregulation of matrix catabolic enzymes, including MMP3, MMP13, ADAMTS4, and ADAMTS5, but not proliferation or apoptosis. In a murine model of destabilization of the medial meniscus (DMM), the intraarticular injection of circPDE4D alleviated DMM-induced cartilage impairments. Mechanistically, we found that circPDE4D exerted its effect by acting as a sponge for miR-103a-3p and thereby regulated FGF18 expression, which is a direct target of miR-103a-3p. In conclusion, our findings highlight a novel protective role of circPDE4D in OA pathogenesis and indicate that the targeting of the circPDE4D-miR-103a-3p-FGF18 axis might provide a potential and promising approach for OA therapy.

Circ_0000808 promotes the development of non-small cell lung cancer by regulating glutamine metabolism via the miR-1827/SLC1A5 axis.

BACKGROUND: Circular RNA (circRNA) has been proved to be an important molecular target for cancer treatment. However, the function and molecular mechanism of circ_0000808 in non-small cell lung cancer (NSCLC) are still unclear. METHODS: Quantitative real-time PCR was used to detect the expression of circ_0000808, miR-1827, and solute carrier family 1 member 5 (SLC1A5). Cell proliferation, apoptosis, migration, and invasion were measured by cell counting kit 8 assay, colony formation assay, EdU staining, flow cytometry, wound healing assay, and transwell assay. The protein expression was measured by Western blot analysis. Dual-luciferase reporter assay and RIP assay were used to investigate the interactions between miR-1827 and circ_0000808 or SLC1A5. Cell glutamine metabolism was assessed by determining glutamine uptake, glutamate production, and α-ketoglutarate production. Xenograft mouse model was used to assess the in vivo effects of circ_0000808. RESULTS: Circ_0000808 expression was upregulated in NSCLC tissues and cancer cells, and its silencing inhibited NSCLC cell proliferation, migration, and invasion and led to apoptosis. Further results confirmed that circ_0000808 interacted with miR-1827 to positively regulate SLC1A5. The rescue experiments showed that miR-1827 inhibitor reversed the suppressive effect of circ_0000808 knockdown on the malignant behaviors of NSCLC cells. Also, SLC1A5 overexpression abolished the inhibition effect of miR-1827 on NSCLC cell progression. In addition, circ_0000808/miR-1827/SLC1A5 axis positively regulated the glutamine metabolism process in NSCLC cells. Moreover, circ_0000808 knockdown reduced the NSCLC tumor growth in vivo. CONCLUSION: In summary, our data showed that circ_0000808 enhanced the progression of NSCLC by promoting glutamine metabolism through the miR-1827/SLC1A5 axis.

Identification of a promoter polymorphism affecting GPAT3 gene expression that is likely related to intramuscular fat content in pigs.

The intramuscular fat content (IMF) is an economically important trait in pigs and the Laiwu pig is famous for its excessively extremely high level of IMF. Our previous transcriptome study revealed that the dynamic expression of glycerol-phosphate acyltransferase 3 (GPAT3) is consistent with changes in the IMF of Laiwu pigs. In this study, we further analyzed the expression and polymorphism of GPAT3 in its promoter region. The results indicated that the expression of GPAT3 increased dramatically from 120 to 240 days and is consistent with changes in IMF deposition, and at both mRNA and protein levels, GPAT3 expression was markedly higher in the LD muscle of Laiwu pigs than that of Duroc × Landrace × Yorkshire pigs. Deletion from -1695 to -1187 of porcine GPAT3 greatly increased its transcription. Of the two SNPs identified, the transition from C to T at -1526 site increased the transcription of porcine GPAT3 and allele T mainly distributed in Laiwu pig population. These results collectively suggest that the SNP at -1526 site of GPAT3 may contribute to IMF deposition by affecting its expression in pigs.

Xanthorrhizol inhibits non-small cell carcinoma (A549) cell growth and promotes apoptosis through modulation of PI3K/AKT and NF-κB signaling pathway.

Xanthorrhizol (XNT) is a sesquiterpenoid agent isolated from Curcuma xanthorrhiza; It is known to exhibit various pharmacological activities including anti-cancer. We investigated the anti-cell proliferative and proapoptotic effects of XNT on Non-small cell carcinoma (A549) cells were analyzed by the generation of reactive oxygen species (ROS), alteration of mitochondrial membrane potential (ΔΨm), oxidative DNA damage, and apoptosis morphological changes were explored by Hoechst and AO/EtBr staining. Our study demonstrated that XNT treatment significantly reduced the viability of A549 cells in a concentration-dependent manner. We observed that XNT-induced oxidative stress-mediated apoptotic cell death by increasing intracellular ROS generation, depleting antioxidant levels, enhancing lipid peroxidation, increased apoptotic morphological changes, and % of DNA damage on human lung cancer cells. Furthermore, we observed that the XNT induce apoptosis through inhibits phosphorylation of PI3K, AKTand inhibit NF-κBp65 transcriptional signaling activity. In addition, XNT treatment alters the ΔΨm, thereby induces apoptosis was closely coordinated with the induction of pro-apoptotic markers Bax, Bad, caspase- 3, 9 and cytochrome c, and suppression of anti-apoptotic (Bcl-2, Bcl-XL) protein expression. According to our results, XNT-inducing apoptosis in A549 cells by causing oxidative damage and modulating apoptotic signaling events. Finally, XNT-induced apoptotic cell death was confirmed by the TUNEL assay. Therefore, XNT might be used as a chemotherapeutic agent for the treatment of lung cancer.

ML264 inhibits osteosarcoma growth and metastasis via inhibition of JAK2/STAT3 and WNT/ß-catenin signalling pathways.

Osteosarcoma, the most common bone malignancy, has a high morbidity rate and poor prognosis. Krüppel-like factor 5 (KLF5) is a key transcriptional regulator of cellular proliferation whose overexpression is observed in osteosarcoma cell lines (U2OS, 143B, MG63 and SAOS2). ML264, a small-molecule inhibitor of KLF5, exerts antiproliferative effects in colorectal cancer; however, its function in osteosarcoma remains unknown. Here, we explored the possible antitumour effects of ML264 on 143B and U2OS cell lines and murine tumour xenograft model. ML264 suppressed proliferation and clonogenic ability of osteosarcoma cells in a dose-dependent manner. Moreover, ML264 induced G0/G1 cell cycle arrest, with no influence on apoptosis, and inhibited the migratory and invasive abilities of osteosarcoma cells, as demonstrated by wound-healing and Transwell assays. Exposure to ML264 reduced the mRNA and protein levels of molecules associated with epithelial-mesenchymal transition phenotype, including N-cadherin, vimentin, Snail, matrix metalloproteinase (MMP) 9 and MMP13. Inhibition of signal transducer and activator of transcription (STAT) 3 phosphorylation and Wnt signalling was also observed. In the murine model of osteosarcoma, tumour growth was efficiently suppressed following a 10-day treatment with ML264. Collectively, our findings demonstrate the potential value of ML264 as a novel anticancer drug for osteosarcoma.

Octyl itaconate inhibits osteoclastogenesis by suppressing Hrd1 and activating Nrf2 signaling.

The endogenous metabolite itaconate has emerged as a regulator of macrophage function that limits inflammation. However, its effect on cell differentiation and osteoclast-related diseases is unclear. Here, for the first time, we explored the effect of itaconate and its cell-permeable itaconate derivative, 4-octyl itaconate (OI) on osteoclast differentiation in vitro and in vivo. Firstly, we demonstrated that itaconate concentration was lower in estrogen-deficient mice. OI released itaconate and induced the expression of nuclear factor-erythroid 2-related factor 2 (Nrf2) in bone marrow-derived macrophages during osteoclastogenesis. Furthermore, OI significantly suppressed the early, middle, and late stages of osteoclastogenesis induced by receptor activator of NF-κB ligand in vitro, as confirmed by tartrate-resistant acid phosphatase staining. Moreover, it significantly inhibited fibrous actin ring formation and bone resorption in vitro. Mechanistically, we observed that OI enhanced Nrf2 expression by suppressing its association with ubiquitin via inhibition of the E3 ubiquitin ligase (Hrd1). OI also inhibited LPS-induced the reactive oxygen species and inflammatory responses via Hrd1. An estrogen deficiency (via ovariectomy)-induced osteoporosis model was also established. Here, on micro-computed tomography and histologic analysis showed that OI effectively suppressed ovariectomy-induced bone loss. In summary, OI, an itaconate derivative, can inhibit osteoclastogenesis in vitro and in vivo, indicating that OI could be a potential drug to treat osteoclast-related diseases; our results also link itaconate to the development of osteoporosis.-Sun, X., Zhang, B., Pan, X., Huang, H., Xie, Z., Ma, Y., Hu, B., Wang, J., Chen, Z., Shi, P. Octyl itaconate inhibits osteoclastogenesis by suppressing Hrd1 and activating Nrf2 signaling.

Effects of microRNA-513b on cell proliferation, apoptosis, invasion, and migration by targeting HMGB3 through regulation of mTOR signaling pathway in non-small-cell lung cancer.

This study aimed to explore the underlying mechanism of miR-513b and HMGB3 in regulating non-small-cell lung cancer (NSCLC). NSCLC tumor, adjacent tissues, and cell lines were extracted, and the expression of miR-513b and HMGB3 were determined by quantitative real-time polymerase chain reaction (RT-qPCR) and western blot analysis. Then, miR-513b was overexpressed in NSCLC cell, and the proliferation, migration, invasion, and apoptosis of cells were determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), wound healing, transwell, and flow cytometry, respectively. Regulatory relationship between miR-513b and HMGB3 was determined using luciferase activity reporter assay. Lastly, HMGB3 and/or miR-513b were overexpressed in NSCLC cells, and the proliferation, migration, invasion, and apoptosis of cells were determined. Compared with the controls, the expression of miR-513b was significantly downregulated in the NSCLC tissues and cells lines by RT-qPCR ( p < 0.05). However, the expression of HMGB3 was significantly downregulated at both messenger RNA and protein levels ( p < 0.05). Overexpression of miR-513b could significantly inhibit the proliferation, invasion, migration, and promote apoptosis of NSCLC cells ( p < 0.05). HMGB3 was a target of miR-513b, and overexpression of HMGB3 could obviously reverse the effect of miR-513 on the proliferation, invasion, migration, and apoptosis of NSCLC cells ( p < 0.05). The present results could suggest miR-513b was downregulated in NSCLC and could regulate the proliferation, invasion, migration, and apoptosis of NSCLC cells via HMGB3.

Circular RNA circTADA2A promotes osteosarcoma progression and metastasis by sponging miR-203a-3p and regulating CREB3 expression.

BACKGROUND: As a subclass of noncoding RNAs, circular RNAs (circRNAs) have been demonstrated to play a critical role in regulating gene expression in eukaryotes. Recent studies have revealed the pivotal functions of circRNAs in cancer progression. However, little is known about the role of circTADA2A, also named hsa_circ_0043278, in osteosarcoma (OS). METHODS: CircTADA2A was selected from a previously reported circRNA microarray comparing OS cell lines and normal bone cells. QRT-PCR was used to detect the expression of circTADA2A in OS tissue and cell lines. Luciferase reporter, RNA immunoprecipitation (RIP), RNA pull-down and fluorescence in situ hybridization (FISH) assays were performed to confirm the binding of circTADA2A with miR-203a-3p. OS cells were stably transfected with lentiviruses, and Transwell migration, Matrigel invasion, colony formation, proliferation, apoptosis, Western blotting, and in vivo tumorigenesis and metastasis assays were employed to evaluate the roles of circTADA2A, miR-203a-3p and CREB3. RESULTS: Our findings demonstrated that circTADA2A was highly expressed in both OS tissue and cell lines, and circTADA2A inhibition attenuated the migration, invasion and proliferation of OS cells in vitro as well as tumorigenesis and metastasis in vivo. A mechanistic study revealed that circTADA2A could readily sponge miR-203a-3p to upregulate the expression of CREB3, which was identified as a driver gene in OS. Furthermore, miR-203a-3p inhibition or CREB3 overexpression could reverse the circTADA2A silencing-induced impairment of malignant tumor behavior. CONCLUSIONS: CircTADA2A functions as a tumor promoter in OS to increase malignant tumor behavior through the miR-203a-3p/CREB3 axis, which could be a novel target for OS therapy.

A comparison of methods for effective differentiation of the frozen-thawed 3T3-L1 cells.

The differentiation efficiency of 3T3-L1 preadipocytes is an essential factor affecting studies on cellular mechanisms associated with obesity, diabetes, and related disorders. Differentiation of 3T3-L1 cells is commonly induced by an adipogenic cocktail containing insulin, dexamethasone (DEX), and 3-isobutyl-1-methylxanthine (IBMX). However, 3T3-L1 cells after freezing and thawing for many times always have a low differentiation efficiency. To solve this problem, we compared the differentiation efficiency of six commonly used adipogenic cocktails and protocols published in 2017. On this basis, we further compared 18 adipogenic cocktails with 2 µM rosiglitazone added and/or with a prolonged treatment with IBMX. The results revealed that the adipogenic cocktail containing 0.5 mM IBMX, 1 µM DEX, and 10 µg/mL insulin was the most effective for the frozen-thawed 3T3-L1 cells differentiation. Rosiglitazone, and IBMX under a prolonged treatment, could improve the differentiation efficiency of the frozen-thawed 3T3-L1 cells. However, the effect was closely related to concentrations of agents in the adipogenic cocktails.

Comparative gene expression profiling of muscle reveals potential candidate genes affecting drip loss in pork.

BACKGROUND: Drip loss is a key aspect of meat quality. Transcriptome profiles of muscle with divergent drip loss would offer important insight into the genetic factors responsible for the trait. In this study, drip loss and other meat quality traits of 28 purebred Duroc pigs were measured, muscles of these individuals were RNA sequenced, and eight individuals with extremely low and high drip loss were selected for analyzing their transcriptome differences and identifying potential candidate genes affecting drip loss. RESULTS: As a result, 363 differentially expressed (DE) genes were detected in the comparative gene expression analysis, of which 239 were up-regulated and 124 were down-regulated in the low drip loss group. The DE genes were further filtered by correlation analysis between their expression and drip loss values in the 28 Duroc pigs measured and comparison of them with QTLs affecting drip loss. Consequently, of the 363 DE genes, 100 were identified as critical DE genes for drip loss. Functional analysis of these critical DE genes revealed some GO terms (extracellular matrix, cell adhesion mediated by integrin, heterotypic cell-cell adhesion), pathway (ECM-receptor interaction), and new potential candidate genes (TNC, ITGA5, ITGA11, THBS3 and CD44) which played an important role in regulating the variation of drip loss, and deserved to carry further studies to unravel their specific mechanism on drip loss. CONCLUSIONS: Our study revealed some GO terms, pathways and potential candidate genes affecting drip loss. It provides crucial information to understand the molecular mechanism of drip loss, and would be of help for improving meat quality of pigs.

RIPK2-Mediated Autophagy and Negatively Regulated ROS-NLRP3 Inflammasome Signaling in GMCs Stimulated with High Glucose.

BACKGROUND: Hyperglycemia plays a vital role in diabetic nephropathy (DN); autophagy and its potential upregulator receptor-interacting protein kinase 2 (RIPK2) are associated with ROS, which play a potential role in regulating NLRP3, and may be involved in inflammation in DN. AIM: In this study, we aimed to explore the mechanisms mediated by RIPK2 in autophagy and the relationship with ROS-NLRP3 of DN, by investigating the levels of RIPK2 and autophagy in glomerular mesangial cells (GMCs) stimulated with high glucose. MATERIAL AND METHODS: GMCs were divided into the following groups: normal group (NC), high glucose group (HG), and RIPK2 siRNA group. RIPK2, LC3, caspase1, and IL-1ß levels were measured by western blotting and RT-PCR. Autophagosomes were measured by GFP-RFP-LC3; ROS were detected by DCFH-DA. RESULTS: High glucose upregulated RIPK2 and LC3 in GMCs during short periods (0-12 h) (p < 0.01), while RIPK2 and LC3 were significantly downregulated in the long term (12-72 h) (p < 0.01); these changes were positively correlated with glucose concentration (p < 0.01). In addition, levels of ROS, caspase1, and IL-1ß increased in a time- and dose-dependent manner in the high glucose group, even with an increased expression of LC3 (p < 0.01). However, LC3 expression decreased in the siRIPK2 group, while levels of ROS, caspase1, and IL-1ß increased (p < 0.01). CONCLUSIONS: Autophagy was activated by high glucose at short time periods but was inhibited in the long term, demonstrating a dual role for high glucose in autophagy of GMCs. RIPK2 regulates ROS-NLRP3 inflammasome signaling through autophagy and may be involved in the pathogenesis of DN.

Genome-wide association study for intramuscular fat content in Chinese Lulai black pigs.

OBJECTIVE: Intramuscular fat (IMF) content plays an important role in meat quality. Identification of single nucleotide polymorphisms (SNPs) and genes related to pig IMF, especially using pig populations with high IMF content variation, can help to establish novel molecular breeding tools for optimizing IMF in pork and unveil the mechanisms that underlie fat metabolism. METHODS: We collected muscle samples of 453 Chinese Lulai black pigs, measured IMF content by Soxhlet petroleum-ether extraction method, and genotyped genome-wide SNPs using GeneSeek Genomic Profiler Porcine HD BeadChip. Then a genome-wide association study was performed using a linear mixed model implemented in the GEMMA software. RESULTS: A total of 43 SNPs were identified to be significantly associated with IMF content by the cutoff p<0.001. Among these significant SNPs, the greatest number of SNPs (n = 19) were detected on Chr.9, and two linkage disequilibrium blocks were formed among them. Additionally, 17 significant SNPs are mapped to previously reported quantitative trait loci (QTLs) of IMF and confirmed previous QTLs studies. Forty-two annotated genes centering these significant SNPs were obtained from Ensembl database. Overrepresentation test of pathways and gene ontology (GO) terms revealed some enriched reactome pathways and GO terms, which mainly involved regulation of basic material transport, energy metabolic process and signaling pathway. CONCLUSION: These findings improve our understanding of the genetic architecture of IMF content in pork and facilitate the follow-up study of fine-mapping genes that influence fat deposition in muscle.

TGF-ß inhibits osteogenesis by upregulating the expression of ubiquitin ligase SMURF1 via MAPK-ERK signaling.

High incidence of osteoporotic fractures emphasizes the necessity of developing effective measures to promote osteogenesis. In our study, we investigated a possible role of MAPK-ERK signaling in the TGF-ß-mediated osteoblastic differentiation. Our results indicated that TGF-ß activated the MAPK-ERK pathway and inhibited osteogenesis in mesenchymal pluripotent cell line, C3H10T1/2, and preosteoblastic cell line, MC3T3 cells. And the downregulation of MAPK-ERK signaling using pharmacological inhibitor U0126 and RNA interference rescued osteoblast differentiation suppressed by TGF-ß, which was confirmed by Alkaline phosphatase (ALP) staining and alizarrn red staining, and the enhanced expression of osteogenesic markers. Western blotting analysis indicated that TGF-ß induced protein expression of E3 ubiquitin-protein ligase SMURF1, which contributed to the degradation of RUNX2 and SMAD1 as evidenced by SMURF1 inhibition using RNA interference and proteasome inhibitor MG132. Moreover, we observed that the expression of SMURF1 was decreased, while that of SMAD1 and RUNX2 increased by MAPK-ERK inhibitor U0126 in TGF-ß-treated differentiating preosteoblasts, suggesting that MAPK-ERK regulated the transcription of osteogenesis-related genes. Furthermore, a synergistic effect between U0126 and bone morphogenic protein (BMP)-2 on osteoblast differentiation and bone formation was observed both in cell cultures and experimental animals. In conclusion, our results revealed that TGF-ß inhibited osteoblastic differentiation by inducing the MAPK-ERK pathway which upregulated the expression of ubiquitin ligase SMURF1 and resulted in reduced presence of osteogenic proteins. In addition, the potentiation of BMP-2 on osteogenic activity by ERK1/2 inhibitor U0126 suggests that it may have potential clinical utility for promoting osteogenesis in bone fracture repair.

Lycorine Suppresses Endplate-Chondrocyte Degeneration and Prevents Intervertebral Disc Degeneration by Inhibiting NF-κB Signalling Pathway.

BACKGROUND/AIMS: Cartilaginous endplate (CEP) degeneration is an important cause for intervertebral disc (IVD) degeneration that leads to low-back pain. The identification of compounds that may prevent CEP degeneration is of interest for the prevention of IVD degeneration. METHODS: Catabolic protease expression in the CEP of disc degeneration patients was first assessed. The toxicity, function and underlying mechanism of lycorine (LY) on CEP-derived chondrocytes degeneration were assessed in vitro by flow cytometry analysis and western blotting. The concentration and function of LY in rat-tail disc-degeneration models were also assessed by HPLC (High Performance Liquid Chromatography) quantification and histological analysis. RESULTS: In CEP cells, Interleukin (IL)-1ß upregulated the expression of matrix metalloproteinase (MMP)-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 that is critical for the degradation of cartilage extracellular matrix. Interestingly, LY suppressed the expression of these enzymes via the inhibition of nuclear factor-κB (NFκB) signalling and thus prevented IL-1ß-induced endplate cell degeneration in vitro. More importantly, LY also reduced the expression of MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5 in CEP and exerted a protective effect on both CEP and nucleus pulposus (NP) degeneration. In addition to its inhibitory effect on matrix-degrading protease expression, LY treatment also reduced positive regulators of proinflammatory cytokines, such as MIF, which can be secreted by CEP cells and subsequently target NP cells. CONCLUSION: LY could serve as a potential drug for treating IVD disease.

E4BP4 facilitates glucocorticoid sensitivity of human bronchial epithelial cells via down-regulation of glucocorticoid receptor-beta.

It has recently been recognized that a subset of asthma patients suffer from glucocorticoid (GC) insensitivity, and glucocorticoid receptor-ß (GR-ß) is associated with corticosteroid resistance, but the underlying mechanisms remain unknown. Here we demonstrated that Interleukin-17A induced glucocorticoid sensitivity in human bronchial epithelial cells (16HBE) is enhanced, which is depend on E4 promoter-binding protein 4 (E4BP4) mediated GR-ß expression. Our data show that the expression of E4BP4 is significantly up-regulated in 16HBE cells, and the depletion of E4BP4 dramatically decreased glucocorticoid sensitivity in IL-17A induced 16HBE cells. Mechanistic studies revealed that E4BP4 plays a crucial role in Interleukin-17A induced glucocorticoid sensitivity in 16HBE cells via down-regulating GR-ß, which is probably mediated by PI3K/Akt activation. Collectively, we can draw the conclusion that E4BP4 contribute to enhance the GCs sensitivity, which may offer a new strategy for therapeutic intervention for GC-insensitive asthma.

Genome-wide detection of selection signatures in Chinese indigenous Laiwu pigs revealed candidate genes regulating fat deposition in muscle.

BACKGROUND: Currently, genome-wide scans for positive selection signatures in commercial breed have been investigated. However, few studies have focused on selection footprints of indigenous breeds. Laiwu pig is an invaluable Chinese indigenous pig breed with extremely high proportion of intramuscular fat (IMF), and an excellent model to detect footprint as the result of natural and artificial selection for fat deposition in muscle. RESULT: In this study, based on GeneSeek Genomic profiler Porcine HD data, three complementary methods, FST, iHS (integrated haplotype homozygosity score) and CLR (composite likelihood ratio), were implemented to detect selection signatures in the whole genome of Laiwu pigs. Totally, 175 candidate selected regions were obtained by at least two of the three methods, which covered 43.75 Mb genomic regions and corresponded to 1.79% of the genome sequence. Gene annotation of the selected regions revealed a list of functionally important genes for feed intake and fat deposition, reproduction, and immune response. Especially, in accordance to the phenotypic features of Laiwu pigs, among the candidate genes, we identified several genes, NPY1R, NPY5R, PIK3R1 and JAKMIP1, involved in the actions of two sets of neurons, which are central regulators in maintaining the balance between food intake and energy expenditure. CONCLUSIONS: Our results identified a number of regions showing signatures of selection, as well as a list of functionally candidate genes with potential effect on phenotypic traits, especially fat deposition in muscle. Our findings provide insights into the mechanisms of artificial selection of fat deposition and further facilitate follow-up functional studies.

SGLT2 inhibitors and risk of stroke in patients with type 2 diabetes: A systematic review and meta-analysis.

The effects of sodium-glucose cotransporter 2 (SGLT2) inhibitors on risk of stroke have not been conclusively established. Therefore, we conducted a meta-analysis to evaluate the effects of SGLT2 inhibitors on stroke risk in patients with type 2 diabetes mellitus (T2DM) by searching available randomized trials in PubMed, Embase, CENTRAL, Web of Science, Scopus and ClinicalTrials.gov databases. We identified 32 eligible trials involving 75 540 participants. The incidence of stroke in groups receiving SGLT2 inhibitor monotherapy or combination therapy did not differ significantly from that in control groups, with a relative risk (RR) of 1.01 and 1.0, respectively. Three SGLT2 inhibitors were tested, with similar RR values (canagliflozin [RR, 0.91], dapagliflozin [RR, 0.99] and empagliflozin [RR, 1.03]). Subgroup analyses showed that RR values were not affected by gender, age, diabetes duration, BMI or HbA1C levels, but Black patients had a lower incidence of stroke than White or Asian patients. This meta-analysis indicated that SGLT2 inhibitor therapy did not increase stroke incidence, and no significant differences in stroke risk were observed among 3 SGLT2 inhibitors (class effect). However, the small racial disparity requires further study and confirmation.

Combined use of leptin and mechanical stress has osteogenic effects on ossification of the posterior longitudinal ligament.

PURPOSE: To evaluate the effects of leptin/leptin receptor (LepR) combined with mechanical stress on the development of ossification of the posterior longitudinal ligament (OPLL), which is a disease characterized by ectopic bone formation of the posterior longitudinal ligament (PLL) and can lead to radiculopathy and myelopathy. METHODS: Six human samples of the PLL were analyzed for the expression of leptin and LepR by RT-PCR and western blotting. PLL cells were stimulated with leptin and mechanical stress delivered via a Flexcell tension system, and osteogenic differentiation was evaluated by RT-PCR and western blotting analysis of osteogenic marker expression as well as by alkaline phosphatase (ALP) staining and alizarin red S staining. Activation of mitogen-activated protein kinase (MAPK), Janus kinase (JAK) 2-signal transducer, activator of transcription (STAT) 3 and phosphatidylinositol 3-kinase (PI3K)-Akt was evaluated by western blotting. RESULTS: Samples from the OPLL group had higher LepR mRNA and protein levels and lower leptin levels than those from healthy controls. Exposure to leptin and Flexcell increased the number of ALP-positive cells and calcium nodules in a dose-dependent manner; this effect was accompanied by upregulation of the osteogenic markers osteocalcin, runt-related transcription factor 2 (RUNX2) and osteopontin. Extracellular signal-regulated kinase, P38 MAPK, JAK2, STAT3, PI3K and Akt signaling, was also activated by the combined effects of leptin and mechanical stress. CONCLUSIONS: Leptin and LepR are differentially expressed in OPLL tissues, and the combined use of leptin/LepR and mechanical stress promotes osteogenic differentiation of PLL cells via MAPK, JAK2-STAT3 and PI3K/Akt signaling. These slides can be retrieved under Electronic Supplementary Material.