Liquid chromatography tandem mass spectrometry method for determination of fulvestrant in rat plasma and its application to pharmacokinetic studies of a novel fulvestrant microcrystal.

Fulvestrant ('Faslodex'), an estrogen receptor antagonist, is available for the treatment of advanced breast cancer. The oil-based vehicle of Faslodex can lead to various adverse effects. A novel fulvestrant microcrystal (aqueous suspension) was developed in this study to eliminate these adverse effects. A sensitive and robust liquid chromatography tandem mass spectrometry method was developed and validated for the determination of fulvestrant in rat plasma using supported-liquid extraction. The separation of fulvestrant was achieved on an Agilent SB-C18 column (2.1 × 50 mm, 3.5 µm) with isocratic elution using fulvestrant-d3 as internal standard. Mass spectrometric detection was conducted in negative multiple reaction monitoring mode. Ion transitions were at m/z 605.5 → 427.5 for fulvestrant and m/z 608.5 → 430.5 for fulvestrant-d3. The excellent linearity was demonstrated over the range 0.05-100.0 ng/ml (r2 = 0.99). The lower limit of quantitation was 0.05 ng/ml, which was superior to that reported in literature The method validation was evaluated by selectivity, accuracy, precision, recovery and matrix effect in agreement with the US Food and Drug Administration guidance. The method was successfully applied to a pharmacokinetic study of a novel fulvestrant microcrystal in rats after intramuscular administration. It revealed that the rate of absorption increases and the extent of absorption is constant with a decrease in microcrystal diameter.

MiR-613 inhibits proliferation and invasion and induces apoptosis of rheumatoid arthritis synovial fibroblasts by direct down-regulation of DKK1.

BACKGROUND: This study aimed to investigate the effects of miR-613 on the proliferation, invasion and apoptosis of rheumatoid arthritis synovial fibroblasts (RASFs). METHODS: Synovial tissue samples were collected from 20 rheumatoid arthritis (RA) patients and 10 patients with joint trauma undergoing joint replacement surgery. The RASFs were isolated and cultured. MiR-613 and DKK1 expression in both synovial tissues and cells was detected using quantitative real-time PCR (qRT-PCR). Dual luciferase reporter gene assay was employed to evaluate the effect of miR-613 on the luciferase activity of DKK1. Then RASFs were transfected with miR-613 mimics, si-DKK1 and pcDNA-DKK1. Changes in cellular proliferation, invasion and apoptosis were detected through BrdU assay, Transwell invasion assay and flow cytometry analysis, respectively. RESULTS: MiR-613 was significantly down-regulated in RA tissues and RASFs compared to normal tissues and cells, whereas DKK1 was up-regulated in RA tissues and RASFs. Dual luciferase reporter gene assay showed that miR-613 could specifically bind to the 3'UTR of DKK1 and significantly inhibit the luciferase activity. Moreover, miR-613 significantly reduced the expression of DKK1. Overexpression of miR-613 or knockdown of DKK1 suppressed proliferation and invasion of RASFs, and induced RASF apoptosis. The reverse results were observed when DKK1 was up-regulated in miR-613-overexpressing RASFs. CONCLUSIONS: MiR-613 can inhibit proliferation and invasion and induce apoptosis of RASFs by directly targeting DKK1 expression.

Simultaneous identification of the three active constituents in Lung-Ventilating-Regulating Oral Liquid by RP-HPLC.

The study aimed to develop a HPLC method for ephedrine, hesperidin, and baicalin in Lung-Ventilating-Regulating Oral Liquid. The three active constituents were identified in an Agilent TC-C18 (2) chromatographic column (250mm × 4.6mm, 5µm), with 0.2% phosphoric acid solution - methyl cyanides as mobile phase, which was performed at a gradient elution column temperature of 25oC and a flow rate of 0.8 mL•min-1. Then the eluate was detected at detection wavelengths of 207 nm (for ephedrine) and 278 nm (for hesperidin and baicalin). Under the chromatographic conditions, ephedrine, hesperidin, and baicalin were well separated, which showed good linear relationships at 0.158-2.370, 0.164-4.100 and 0.160-4.000µg, respectively. The coefficients of recovery of these three kinds of samples showed 100.2%, 98.7% and 97.8%, respectively. The developed method is convenient, accurate and well repeatable, and consequently can be applied for the quality control of Lung-Ventilating-Regulating Oral Liquid.

Increased frequency of circulating follicular helper T cells in lupus patients is associated with autoantibody production in a CD40L-dependent manner.

This study was to determine the frequency of circulating follicular helper T (Tfh) cells in patients with systemic lupus erythematosus (SLE) and investigate the relationship between Tfh cells and autoantibody production. An increased frequency of circulating Tfh cells was found in SLE patients, and there were positive co-relationship between Tfh cells and SLEDAI, serum IgG, Anti-nuclear antibody titers and anti-dsDNA (P=0.0004, 0.0006, 0.0237, 0.0000, respectively). B cells sorted from SLE patients produced more IgG than healthy controls in the presence of autologous CD4(+) T cells. CD4(+) T cells were further sorted into CXCR5(+) and CXCR5(-) cells, and CD4(+)CXCR5(+) T cells helped B cells producing more IgG than CD4(+)CXCR5(-) T cells. Blocking the interaction of T cells and B cells by anti-CD40L but not anti-ICOSL dramatically decreased antibody production in the co-culture system. This study suggests that increased frequency of circulating Tfh cells in SLE patients is associated with excess B-cell help and autoantibody production in a CD40L dependent manner.

Identification and validation of molecular subtype and prognostic signature for lung adenocarcinoma based on neutrophil extracellular traps.

Background: Neutrophil Extracellular Traps (NETs) are fibrous networks made of DNA-histone complexes and proteins protruded from activated neutrophils. Accumulating evidences have highlighted the vital role of NETs in tumor progression and diffusion. However, limited systematic studies regarding the role of NETs in LUAD have been performed. Methods: Differentially expressed NETs-related genes and their mutation landscape were identified with TCGA database. Consensus clustering analysis was performed to determine the NETs-related subtypes of LUAD. LASSO algorithm was employed to construct a prognostic signature. Moreover, GSE30219 and GSE31210 were used as independent validation. We also constructed a lncRNA-miRNA-mRNA regulatory axis with several miRNA and lncRNA databases. Results: Consensus clustering identified two NETs-related clusters in LUAD. High NETs score was correlated with a favorable overall survival, abundant immune cell infiltration, and high activity of immune response signal pathways. Six NET-related genes (G0S2, KCNJ15, S100A12, AKT2, CTSG, and HMGB1) with significant prognostic value were screened to develop a prognostic signature. LUAD patients with low-risk had a significantly favorable overall survival both in the training set and validation set. Moreover, NETs-related risk score and clinical stage could act as an independent prognostic factor for LUAD patients. Significant correlation was obtained between risk score and tumor immune microenvironment. We also identified lncRNA BCYRN1/miR-3664-5p/CTSG regulatory axis that may be involved in the progression of LUAD. Conclusion: We developed two molecular subtypes and a prognostic signature for LUAD based on NETs-related genes. This stratification could provide more evidences for estimating the prognosis and immunotherapy of LAUD patients.

Hsa_circ_0007707 participates in PDE3B-mediated apoptosis inhibition and inflammation promotion in fibroblast-like synoviocytes.

Synovial samples collected from 30 rheumatoid arthritis (RA) patients and 30 normal controls were used to isolate fibroblast-like synoviocytes (FLSs) and named FLS-RA and FLS-Normal, respectively. Real-time quantitative polymerase chain reaction (RT-qPCR) was utilized to detect circ_0007707 expression. Effects of circ_0007707 silencing on cell proliferation and apoptosis were evaluated using cell counting kit-8, 5-ethynyl-2'-deoxyuridine (Edu), and flow cytometry assays. Levels of pro-inflammatory factors were detected by enzyme-linked immunosorbent assay (ELISA). Increased circ_0007707 expression was observed in synovial samples from RA patients and FLS-RA cells. Functional analysis showed circ_0007707 silencing restrained cell proliferation, induced cell apoptosis, and decreased cell inflammatory response in FLS-RA cells. Mechanistic analysis revealed the sponge function of circ_0007707 on miR-27b-3p, and miR-27b-3p inhibition weakened circ_0007707 knockdown-mediated effects on FLS-RA cell proliferation, apoptosis, and inflammatory response. Circ_0007707 could mediate PDE3B expression via sponging miR-27b-3p, and PDE3B overturned miR-27b-3p mimic-mediated effects on FLS-RA cell proliferation, apoptosis, and inflammatory response. Circ_0007707 mediated cell apoptosis and inflammatory response in FLS-RA cells through the miR-27b-3p/PDE3B axis, indicating the potential function of circ_0007707 as a target for RA treatment.

Imbalance of peripheral blood Th17/Treg increases neutrophil-to-lymphocyte ratio in patients with dermatomyositis.

OBJECTIVE: To explore and analyze the association between peripheral blood Th17/Treg balance and neutrophil-to-lymphocyte ratio (NLR) in patients with dermatomyositis (DM). METHODS: Data of 83 DM patients hospitalized between January 2020 to April 2022 were collected, including 43 patients in the active phase (DM active-phase group) and 40 in the remission phase (DM remission-phase group). Additionally, data of 50 healthy subjects who underwent physical examinations and immunologic function testing in the same period were taken as a control group. We detected the percentage of Th17 and Treg cells by flow cytometry, calculated patient's NLR and laboratory test indicators, and analyzed the correlation of Th17/Treg balance with NLR and laboratory indicators. RESULTS: Th17 percentage and Th7/Treg ratio in the DM active-phase group were higher than those in the DM remission-phase group (P<0.05), while Treg percentage was lower in the active-phase group than in the remission-phase group (P<0.05). The creatine kinase (CK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), erythrocyte deposition rate (ESR), and NLR in DM patients were significantly higher than those of the control group (P<0.05), and were associated with the disease activity of DM. The ratio of Th17/Treg was positively correlated with CK, LDH, AST, ALT, ESR, and NLR (P<0.05). NLR was positively correlated with CK, LDH, AST, ALT, and ESR (P<0.05). CONCLUSION: DM patients exhibit changes in immune balance of Th17/Treg and an increase in the NLR. The Th17/Treg ratio in the patients is closely associated with the NLR, which suggests that the immune balance mechanism may interact with the inflammatory response of the body, collectively contributing to the progression of DM.

Identification of a Necroptosis-Related Prognostic Signature and Associated Regulatory Axis in Lung Adenocarcinoma.

Background: Lung cancer is considered to be the second most aggressive and rapidly fatal cancer after breast cancer. Necroptosis, a novel discovered pattern of cell death, is mediated by Receptor-interacting serine/threonine-protein kinase 1 (RIPK1), Receptor-interacting serine/threonine-protein kinase 3 (RIPK3), and Mixed Lineage Kinase Domain Like Pseudokinase (MLKL). Methods: For the purpose of developing a prognostic model, Least absolute shrinkage and selection operator (LASSO) Cox regression analysis was conducted. Using Pearson's correlation analysis, we evaluated the correlation between necroptosis-related markers and tumor immune infiltration. A bioinformatics analysis was conducted to construct a necroptosis-related regulatory axis. Results: There was a downregulation of most of necroptosis-related genes in lung adenocarcinoma (LUAD) versus lung tissues but an increase in PGAM5, HMGB1, TRAF2, EZH2 levels. We also summarized the Single Nucleotide Variant (SNV) and copy number variation (CNV) of necroptosis-related genes in LUAD. Consensus clustering identified two clusters in LUAD with distinct immune cell infiltration and ESTIMATEScore. Genes related to necroptosis were associated with necroptosis, Tumor necrosis factor (TNF) signaling pathway, and apoptosis according to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Four prognostic genes (ALDH2, HMGB1, NDRG2, TLR2) were combined to develop a prognostic gene signature for LUAD patients, which was highly accurate in predicting prognosis. Univariate and multivariate analysis identified HMGB1, pT stage, and pN stage as independent factors impacting on LUAD patients' prognosis. A significant correlation was found between the level of TLR2 and NDRG2 and clinical stage, immunity infiltration, and drug resistance. Additionally, the progression of LUAD might be regulated by lncRNA C5orf64/miR-582-5p/NDRG2/TLR2. Conclusion: The current bioinformatics analysis identified a necroptosis-related prognostic signature for LUAD and their relation to immunity infiltration. This result requires further investigation.

Pirfenidone inhibits cell fibrosis in connective tissue disease-associated interstitial lung disease by targeting the TNF-α/STAT3/KL6 pathway.

Background: To explore the effect and mechanism of pirfenidone in inhibiting pulmonary fibrosis in connective tissue disease-associated interstitial lung disease (CTD-ILD). Methods: From 2018 to 2020, 50 CTD-ILD patients were enrolled in the clinical study. Based on whether pirfenidone was used during treatment, patients were enrolled into the pirfenidone group and the control group. Pulmonary function tests were compared before and after treatment. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the expression of tumor necrosis factor-α (TNF-α), signal transducer and activator of transcription 3 (STAT3), and Krebs Von den Lungen-6 (KL-6) in venous blood before and after treatment. Rat type II (RLE-6TN) lung epithelial cells were cultivated for in vitro experiments, and they were sorted into the control group, bleomycin group, pirfenidone group, TNF-α group, Stattic group, and TNF-α/Stattic combined treatment group. For the in vitro experiments, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tests were used to evaluate cell proliferation, Reverse Transcription-Polymerase Chain Reaction(RT-PCR) was performed to detect STAT3 and KL-6 mRNA expression levels, ELISA was utilized to detect TNF-α and E-cadherin expression levels, and Western blotting (WB) was performed to determine α-smooth muscle actin (α-SMA), vimentin, TNF-α, STAT3, phosphorylated signal transducer and activator of transcription 3 (PSTAT3) and KL-6 expression. Results: In the clinical study, the pulmonary function indices including forced expiratory volume in one second (FEV1), forced vital capacity (FVC), FEV1/FVC, peak expiratory flow (PEF) and partial pressure (PaO2) of the patients in the study group were superior to those in the control group (P<0.05). The serum TNF-α, STAT3 and KL-6 levels in the study group were significantly lower than those in the control group (P<0.05). In the in vitro experiments, the α-SMA, vimentin, STAT3 and KL-6 levels in the treatment group were significantly lower than those in the bleomycin group (P<0.05). Compared with those in the pirfenidone group, the α-SMA, vimentin, STAT3 and KL-6 levels in the TNF-α-treated group were significantly upregulated (P<0.05). Meanwhile, cell viability was further upregulated (P<0.05), and the expression of STAT3 and KL-6 was further decreased in the Stattic-treated group (P<0.05). In the group treated with infliximab combined with Stattic, TNF-α expression was significantly increased (P<0.05), cell activity was significantly restored (P<0.05), and the STAT3, KL-6 and E-cadherin expression levels were inhibited (P<0.05). Conclusions: Pirfenidone improved pulmonary function 1 and decreased TNF-α, STAT3, and KL-6 expression in CTD-ILD patients. Moreover, pirfenidone inhibits cell fibrosis through the TNF-α/STAT3/Mucin 1(MUC1) pathway.

Radioprotective activity of glutathione on cognitive ability in X-ray radiated tumor-bearing mice.

OBJECTIVES: The use of X-ray for therapeutics always raises the problem of radiation hazards to living beings. In this research, we explored the radioprotective activity of glutathione (GSH) on cognitive ability of X-ray radiated tumor-bearing mice. METHODS: Forty C57BL/6 mice were chosen to establish the GL261 glioma model and randomly divided into four groups: Model group, X-ray group, Pre-GSH group and Pos-GSH group. Morris water maze test was used to test cognitive ability. Moreover, histopathological observation of hippocampus was observed by hematoxylin and eosin (HE) staining. The protein expression of choline acetyl transferase (ChAT) was measured by western blot, simultaneously the contents of acetylcholinesterase (Ach), superoxide dismutase (SOD), methane dicarboxylic aldehyde (MDA),TNF-α and IL-6 were detected by the respective kit. RESULTS: There was a significant difference in X-ray group of the escape latency from the Model group (P<0.05). Besides, HE staining revealed that nucleus in hippocampus cells were pyknotic, glial cells were hyperplastic and the nerve cells were swelling in X-ray group. In X-ray group the expression of ChAT and Ache were decreased versus Model group. Finally, the cognitive ability in Pre-GSH and Pos-GSH group was enhanced than X-ray group, in which the cognitive ability of Pos-GSH group was higher than the Pre-GSH group. DISCUSSION: X-ray impaired the brain tissues and cognitive ability of tumor-bearing mice. The damages of brain tissues were alleviated by Pre-GSH and Pos-GSH protection and the efficacy of Pos-GSH protection was superior to Pre-GSH protection. Abbreviation Ach: Acetylcholinesterase; GSH: Glutathione; HE: Hematoxylin and eosin; MDA: methane dicarboxylic aldehyde; SOD: Superoxide dismutase; TV: Tumor volume; TW: Tumor weight.

NSK-01105, a novel sorafenib derivative, inhibits human prostate tumor growth via suppression of VEGFR2/EGFR-mediated angiogenesis.

The purpose of this study is to investigate the anti-angiogenic activities of NSK-01105, a novel sorafenib derivative, in in vitro, ex vivo and in vivo models, and explore the potential mechanisms. NSK-01105 significantly inhibited vascular endothelial growth factor (VEGF)-induced migration and tube formation of human umbilical vein endothelial cells at non-cytotoxic concentrations as shown by wound-healing, transwell migration and endothelial cell tube formation assays, respectively. Cell viability and invasion of LNCaP and PC-3 cells were significantly inhibited by cytotoxicity assay and matrigel invasion assay. Furthermore, NSK-01105 also inhibited ex vivo angiogenesis in matrigel plug assay. Western blot analysis showed that NSK-01105 down-regulated VEGF-induced phosphorylation of VEGF receptor 2 (VEGFR2) and the activation of epidermal growth factor receptor (EGFR). Tumor volumes were significantly reduced by NSK-01105 at 60 mg/kg/day in both xenograft models. Immunohistochemical staining demonstrated a close association between inhibition of tumor growth and neovascularization. Collectively, our results suggest a role of NSK-01105 in treatment for human prostate tumors, and one of the potential mechanisms may be attributed to anti-angiogenic activities.