miRNA identification by nuclease digestion in ELISA for diagnosis of osteosarcoma.

Osteosarcoma is a bone cancer formed by the cells of the bone. Children, young adults, and teens are highly affected by osteosarcoma. Early identification of osteosarcoma is mandatory to improve the treatment and increase the lifespan of the patients. MicroRNA-195 (miR-195) was shown to be a suitable biomarker for osteosarcoma, and the present study describes a sensitive method of miR-195 identification by nuclease digestion in ELISA to detect and quantify the level of miR-195. S1 nuclease catalyzed endo- and exonucleolytic digestion of single-stranded (ss) RNA and DNA on ELISA polystyrene substrate, which helped to identify duplexed miR-195. This method selectively and specifically identified miR-195 without any biofouling interactions and reached the limit of detection at 10 fM within the range from 10 fM to 10 nM. Due to complete digestion of ssDNA, single- and triple-mismatched sequences failed to increase the ELISA signal, indicating specific miRNA detection. Furthermore, human serum spiked with miR-195 did not interfere with the detection, confirming selective identification. This method identified miR-195 at a lower level and will help to diagnose earlier stages of osteosarcoma.

Azilsartan prevented AGE-induced inflammatory response and degradation of aggrecan in human chondrocytes through inhibition of Sox4.

Advanced glycation end products (AGEs)-induced inflammation and degradation of aggrecan in human chondrocytes play an important role in the progression and development of osteoarthritis (OA). Azilsartan, an angiotensin II receptor antagonist, has been licensed for the treatment of high blood pressure. However, the effects of Azilsartan in OA and AGEs-induced damages in chondrocytes have not been previously reported. The injured chondrocytes model was established by incubating with 5 µmol/L AGEs. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used to evaluate the cell viability of treated SW1353 cells. The gene expression levels of interleukin-1α (IL-1α), tumor necrosis factor-ß (TNF-ß), IL-6, a disintegrin-like and metallopeptidase with thrombospondin type motif-4 (ADAMTS-4), ADAMTS-5, Aggrecan, and Sox-4 were evaluated using quantitative real-time polymerase chain reaction and their protein levels were determined using enzyme-linked immunosorbent assay or Western blot analysis. Mitogen-activated protein kinase p38 pathway was surveyed using phosp-p38 level and its specific inhibitor SB203580 was employed to block the p38 pathway. The overexpression of Sox4 plasmid was transfected into SW1353 cells to assess its regulation on ADAMTS-4 and ADAMTS-5. Azilsartan reduced AGEs-induced production of proinflammatory cytokines, such as IL-1α, TNF-ß, and IL-6. Azilsartan prevented AGEs-induced expressions of ADAMTS-4 and ADAMTS-5 as well as the reduction of aggrecan. Mechanistically, AGEs treatment increased the expression of Sox4 in a dose-dependent manner. AGE treatment increased the level of phosphorylated p38. However, treatment with the p38 inhibitor SB203580 inhibited AGEs-induced expression of Sox4, suggesting that AGEs-induced expression of Sox4 is mediated by p38. Furthermore, Azilsartan suppressed AGEs-induced phosphorylation of p38 and expression of Sox4. Finally, the overexpression of Sox4 abolished the inhibitory effects of Azilsartan against the expressions of ADAMTS-4 and ADAMTS-5. Azilsartan treatment prevented AGEs-induced inflammatory response and degradation of aggrecan through inhibition of Sox4.

[Adjunctive treatment of axial undifferentiated spondyloarthritis by Qiangji Recipe: a clinical study].

OBJECTIVE: To evaluate the clinical efficacy and safety of Qiangji Recipe (QR) in ad- junctive treatment of axial undifferentiated spondyloarthritis (axuSpA) through a four-week open study. METHODS: Fifty-four axuSpA patients of Shen-deficiency Du-channel cold syndrome (SDDCS) in line with inclusive criteria were recruited and assigned to the treatment group and the control group according to random digit table, 27 in each group. Patients in the control group took Celecoxib Capsule (0.2 g each time, twice per day). Patients in the treatment group additionally took QR (consisting of Herba Epimedii 15 g, antler glue 15 g, Cibotium Barometz 15 g, eucommia bark 20 g, dipsacus asper 10 g, two toothed achyranthes root 15 g, drynaria 15 g, Taxillus Chinensis 20 g, ground beetle 10 g, scorpion 5 g, wild celery 10 g, notopterygium incisium 10 g, cow-fat seed 10 g, white mustard seed 6 g, and licorice root 6 g, one dose per day, twice daily). The therapeutic course for all was 4 weeks. The Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), the Bath Ankylosing Spondylitis Functional Index (BASFI), the Bath AS Metrology Index (BASMI), total body pain and spinal pain, patient and physician global assessment on a four-point scale, the Ankylosing Spondylitis Quality of Life (ASQoL), erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were measured before and after 4 weeks of treatment. The primary end point in this study was the proportion of patients with a 20%improvement response accord- ing to the ASAS International Working Group Criteria (ASAS 20 responders) at week 4. RESULTS: Totally 50 patients completed this trial, 26 in the treatment group and 24 in the control group. Improvement of BASDAI, BASFI, BASMI, ASQoL, ESR, and CRP was shown in both groups after treatment. Better effect was shown in the treatment group in all indices except ESR and BASMI after treatment (P < 0.05, P < 0.01). Twenty cases (accounting for 76.92%) in the treatment group achieved ASAS 20 response at week 4, while 12 cases (accounting for 50.00%) in the control group achieved ASAS 20 response at week 4 (P < 0.05). No obvious adverse reaction occurred in the two groups. CONCLUSION: QR combined Celecoxib Capsule showed better effect in treating axuSpA patients than using Celecoxib Capsule alone.