Abstract The mushroom tyrosinase assay, B16-F10 mouse melanoma cell model, and zebrafish model are frequently used for high-throughput screening and are widely used for developing anti-hyperpigmentation compounds, although these systems cannot be compared. We used each of these three systems to evaluate the seven anti-hyperpigmentation compounds. We investigated 1. tyrosinase activity using a mushroom tyrosinase assay, 2. viability, tyrosinase activity, and melanin content in B16-F10 cells, and 3. embryonic toxicity, tyrosinase activity, and melanin content in zebrafish. α-Arbutin, raspberry ketone (RK), raspberry ketone glucoside (RKG), glabridin (GLA), and 3-o-ethyl-ascorbic (EA), inhibited the activity of mushroom tyrosinase; dipotassium glycyrrhizinate (DG) did not inhibit mushroom tyrosinase activity, and glycyrrhetic acid (GA) promoted tyrosinase activity. Tyrosinase activity was inhibited by α-arbutin, GLA, GA and DG in B16-F10 cells; RK, RKG and EA did not inhibit tyrosinase activity. α-Arbutin, RK, RKG, EA, and GA, inhibited tyrosinase activity in zebrafish; GLA and DG did not inhibit tyrosinase activity. α-arbutin, RK, RKG, EA, GLA, and DG reduced melanin synthesis in B16-F10 cells in a dose-dependent manner without significant toxicity; GA did not inhibit melanin synthesis. α-arbutin, RK, RKG and GA significantly reduced melanin content on the zebrafish body surface. Mushroom tyrosinase analysis was the most practical assay among the three systems but had poor reliability. The B16-F10 mouse melanoma cell system was the most sensitive but had the worst stability. The zebrafish system had better reproducibility than other systems; however, most compounds were difficult to screen in this system.

Fibrosis caused by the increase in extracellular matrix in cardiac fibroblasts plays an important role in the occurrence and development of atrial fibrillation (AF). The aim of this study was to investigate the role of hsa-miR-4443 in AF, human cardiac fibroblast (HCFB) proliferation, and extracellular matrix remodeling. TaqMan Stem-loop miRNA assay was used to measure hsa-miR-4443 expression in patients with persistent AF (n=123) and healthy controls (n=100). Patients with AF were confirmed to have atrial fibrosis by late gadolinium enhancement. At the cellular level, after hsa-miR-4443 mimic and inhibitor were transfected with HCFBs, proliferation, apoptosis, migration, and invasion were analyzed. Lastly, hsa-miR-4443-targeted gene and transforming growth factor (TGF)-β1/α-SMA/collagen pathway were evaluated by dual-luciferase reporter assay and western blot, respectively. In patients with AF, hsa-miR-4443 decreased significantly and collagen metabolism level increased significantly. Logistic regression analysis showed that low hsa-miR-4443 level was a risk factor of AF (P<0.001). The receiver operating characteristic curve revealed that hsa-miR-4443 was useful for predicting AF (area under the curve: 0.828, sensitivity: 0.71, specificity: 0.78, P<0.001). In HCFBs, hsa-miR-4443 targeted thrombospondin-1 (THBS1) and downregulated TGF-β1/α-SMA/collagen pathway. The inhibition of hsa-miR-4443 expression promoted HCFB proliferation, migration, invasion, myofibroblast differentiation, and collagen production. The significant reduction of hsa-miR-4443 can be used as a biomarker for AF. hsa-miR-4443 protected AF by targeting THBS1 and regulated TGF-β1/α-SMA/collagen pathway to inhibit HCFB proliferation and collagen synthesis.

OBJECTIVES: Cystathionine β-synthase is a major enzyme in the metabolism of plasma homocysteine. Hyperhomocysteinemia is positively associated with hypertension and stroke. The present study was performed to examine the possible effects of Cystathionine β-synthase promoter methylation on the development of hypertension and stroke. METHODS: Using quantitative methylation-specific PCR, we determined the Cystathionine β-synthase methylation levels in 218 healthy individuals and 132 and 243 age- and gender-matched stroke and hypertensive patients, respectively. The relative changes in Cystathionine β-synthase promoter methylation were analyzed using the 2–ΔΔCt method. The percent of the methylated reference of Cystathionine β-synthase was used to represent the Cystathionine β-synthase promoter methylation levels. RESULTS: In this study, the Cystathionine β-synthase promoter methylation levels of hypertensive and stroke participants were both higher than that of the healthy individuals (median percentages of the methylated reference were 50.61%, 38.05% and 30.53%, respectively, all p<0.001). Multivariable analysis showed that Cystathionine β-synthase promoter hypermethylation increased the risk of hypertension [odds ratio, OR (95% confidence interval, CI)=1.035 (1.025–1.045)] and stroke [OR (95% CI)=1.015 (1.003–1.028)]. The area under the curve of Cystathionine β-synthase promoter methylation was 0.844 (95% CI: 0.796–0.892) in male patients with hypertension and 0.722 (95% CI: 0.653–0.799) in male patients with stroke. CONCLUSION: Cystathionine β-synthase promoter hypermethylation increases the risk of hypertension and stroke, especially in male patients.