Gene Expression in MC3T3-E1 Cells Treated with Diclofenac and Methylprednisolone.

Nonsteroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids (GCs) are often used to treat articular-skeletal disorders. The extended use of NSAIDs and GCs have adverse effects on bone metabolism, reducing bone quality and impairing fracture healing. In the present study, we used mouse pre-osteoblast cells MC3T3-E1 to demonstrate the effects of diclofenac (DF) and methylprednisolone (MP) on cell proliferation and gene expression. Cells were incubated with three doses of DF or MP: 0.5 µM, 5 µM, and 50 µM. MP decreased cell viability even after 24 h, but DF inhibited cell viability after only seven days of treatment. The cells were lysed after one, two, three, and seven days of treatment, and gene expression was analyzed by reverse transcription and quantitative PCR (RT-qPCR) assays. DF did not significantly affect the expression of the osteogenic marker genes. MP modified the expression of Osx, Runx, and Col1a1. We concluded that MP is a more potent inhibitor of mouse pre-osteoblast differentiation and viability than is DF. Our results suggest that prolonged DF treatment could be less harmful to osteoblasts than MP treatment.

In silico and in vitro analysis of the impact of single substitutions within EXO-motifs on Hsa-MiR-1246 intercellular transfer in breast cancer cell.

MiR-1246 has recently gained much attention and many studies have shown its oncogenic role in colorectal, breast, lung, and ovarian cancers. However, miR-1246 processing, stability, and mechanisms directing miR-1246 into neighbor cells remain still unclear. In this study, we aimed to determine the role of single-nucleotide substitutions within short exosome sorting motifs - so-called EXO-motifs: GGAG and GCAG present in miR-1246 sequence on its intracellular stability and extracellular transfer. We applied in silico methods such as 2D and 3D structure analysis and modeling of protein interactions. We also performed in vitro validation through the transfection of fluorescently labeled miRNA to MDA-MB-231 cells, which we analyzed by flow cytometry and fluorescent microscopy. Our results suggest that nucleotides alterations that disturbed miR-1246 EXO-motifs were able to modulate miRNA-1246 stability and its transfer level to the neighboring cells, suggesting that the molecular mechanism of RNA stability and intercellular transfer can be closely related.

Combined Effects of Methyldopa and Baicalein or Scutellaria baicalensis Roots Extract on Blood Pressure, Heart Rate, and Expression of Inflammatory and Vascular Disease-Related Factors in Spontaneously Hypertensive Pregnant Rats.

The aim of the study was to investigate the effect of baicalein or Scutellaria baicalensis root extract interaction with methyldopa in pregnant spontaneously hypertensive rats (SHR) at the pharmacodynamic, molecular, and biochemical levels. The rats, after confirming pregnancy, received baicalein (200 mg/kg/day, p.o.) and extract (1000 mg/kg/day, p.o.), in combination with methyldopa (400 mg/kg/day; p.o.), for 14 consecutive days, 1 h before blood pressure and heart rate measurements. In the heart and placenta from mothers after giving birth to their offspring, mRNA expression of factors related to inflammatory processes (TNF-α, Il-1ß, IL-6) and vascular diseases (TGF-ß, HIF-1α, VEGF, PlGF) was measured. Levels of markers of oxidative stress (superoxide dismutase and malondialdehyde) in the placenta and indicators of myocardial damage (troponin cTnC and cTnI, creatine kinase, myoglobin, and lactate dehydrogenase) in the heart were also assessed. Baicalein co-administered with methyldopa was associated with reduced blood pressure, especially during the first three days. The interactions were more pronounced for such factors as TGF-ß, HIF-1α, VEGF, and PlGF than TNF-α, Il-1ß, and IL-6. Combined application of baicalein and extract with methyldopa may be of value in the development of a new antihypertensive medication intended for patients suffering from preeclampsia or pregnancy-induced hypertension.

Gitelman syndrome with normocalciuria - a case report.

BACKGROUND: Gitelman Syndrome (GS) is a hereditary tubulopathy associated with a biallelic inactivating mutations of the SLC12A3 gene encoding the thiazide-sensitive sodium-chloride cotransporter (NCCT). The typical clinical manifestation is a hypokalemic metabolic alkalosis with significant hypomagnesemia, and low urinary calcium excretion. Hypocalciuria is widely believed to be a hallmark of GS that distinguishes it from Barter's syndrome, presenting as hypercalciuria. The pathomechanism of hypocalciuria in GS is not fully elucidated. Up to date, a clinical course of GS with normocalciuria has been reported only in men, while women have a milder course of the disease with typical hypocalciuria, which is believed as the result of sex hormone. Additionally, there is a growing evidence that calcium channels of the distal nephron could be regulated by a variety of hormones, including aldosterone (Aldo). CASE PRESENTATION: We present the case of a 28-year-old Caucasian woman with asymptomatic, chronic hypokalemia, hypomagnesemia, hypochloremic alkalosis and normal urinary calcium excretion. A high renin levels with normal concentration of Aldo in serum have also been found. The values of blood pressure were low. Based on genetic studies, two heterozygous mutations in the trans position were confirmed: c.2186G>T (p.Gly729Val) and c.1247G>C (p.Cys416Ser) in the SLC12A3 gene, which ultimately confirmed the diagnosis of GS. CONCLUSIONS: We report here the first case of genetically confirmed GS manifested as normocalciuria in a Caucasian woman. Thus, our result does not confirm a role of sex hormones on the level of calciuria. Based on the results of normal Aldo concentration despite high renin level in our patient, we hypothesized that Aldo may be connecting with the level of urinary calcium excretion in patients with the GS.

Paraoxonase 1 concerning dyslipidaemia, cardiovascular diseases, and mortality in haemodialysis patients.

Paraoxonase 1 (PON1) is known for preventing atherosclerosis through lipid-modifying features, antioxidant activity, anti-inflammatory, anti-apoptosis, anti-thrombosis, and anti-adhesion properties. Uremic patients requiring haemodialysis (HD) are especially prone to atherosclerosis and its complications. We analysed the PON1 gene (PON1) polymorphisms and serum PON1 (paraoxonase) activity concerning dyslipidaemia and related cardiovascular diseases and mortality to show how they associate under uremic conditions modified by maintenance HD treatment. The rs662 AA + AG (OR 1.76, 95%CI 1.10-2.80, P = 0.018), rs854560 TT (OR 1.48, 95%CI 1.04-2.11, P = 0.031), and rs854560 AT + TT (OR 1.28, 95%CI 1.01-1.63, P = 0.040) contributed to the prevalence of atherogenic dyslipidaemia diagnosed by the triglyceride (TG)/HDL-cholesterol ratio ≥ 3.8. The normalized serum PON1 activity positively correlated with atherogenic dyslipidaemia (ẞ 0.67 ± 0.25, P = 0.008). The PON1 rs854560 allele T was involved in the higher prevalence of ischemic cerebral stroke (OR 1.38, 1.02-1.85, P = 0.034). The PON1 rs705379 TT genotype contributed to cardiovascular (HR 1.27, 95% CI 1.03-1.57, P = 0.025) and cardiac (HR 1.34, 95% CI 1.05-1.71, P = 0.018) mortality. All P-values were obtained in multiple regression analyses, including clinical variables. Multifaceted associations of PON1 with dyslipidaemia, ischemic cerebral stroke, and cardiovascular mortality in HD patients provide arguments for the consideration of PON1 and its protein product as therapeutic targets in the prevention of atherosclerosis and its complications in uremic patients.

Trabecular bone remodelling in the femur of C57BL/6J mice treated with diclofenac in combination with treadmill exercise.

PURPOSE: Analgesic treatment with diclofenac deteriorates bone structure and decreases biomechanical properties. This bone loss has been though to be reversed by training. The impact of exercise on bone treated with diclofenac (DF) has reminded elusive. In the present study, we assayed the combined impact of exercises and DF on mouse femur. METHODS: The femur samples we obtained from 30 days treated C57BL/6J female mice. The training group ran on a horizontal treadmill at 12 m/min by 30 min a day (5% grade/slope). The group of ten mice treated with DF received the drug subcutaneously every day (5 mg/kg of body weight/day). The combined group ran on the treadmill and obtained DF. After 30 days, we sacrificed mice and studied their femurs using microcomputed tomography (µCT), dynamic mechanical analysis (DMA) and nanoindentation. RESULTS: We observed that treadmill running and DF decreased trabecular bone volume and mineral density. Combined effect of training and DF was not additive. A significant interaction of both parameters suggested protective effect of training on bone loss provoked by DF. The femur cortical bone shell remained untouched by the training and treatment. The training and the DF treatment did not alter the storage modulus E' significantly. The unchanged storage modulus would be suggesting on the unaltered bone strength. CONCLUSIONS: We concluded that even relatively short time of training with concomitant DF treatment could be protective on trabecular bone. Although viscoelastic properties of the entire femur were not modulated, femur trabecular tissue was thinned by treatment with DF and protected by training.