Verification of performance of a direct fluorescent assay for cell-free DNA quantification, stability according to pre-analytical storage conditions, and the effect of freeze-thawing.

A simple fluorescence-based cell-free DNA (CFD) assay has been previously developed that can directly measure nucleic acids without prior DNA extraction and amplification. However, studies on fluorescence-based CFD are lacking. In particular, there is no known information regarding the stability with regard to pre-analytical storage conditions in relation to time and temperature, or on the influence of freeze-thawing. Plasma was directly assayed to measure CFD using PicoGreen™ reagent. Standard linearity and accuracy were confirmed using salmon sperm DNA. Whole blood was left at room temperature (RT) and at 4˚C, and then plasma was separated. The CFD was also measured using thawed plasma after 1 week of freezing. As a correlation with a sperm DNA concentration, CFD demonstrated linearity over a wide range of concentrations, with a 0.998 correlation coefficient. The CFD level showed a change of up to 2.5 µg/ml according to pre-analytical storage time, and the changes were not consistent over time. The CFD values at RT after 1 h were similar to the baseline values, and the relative standard deviation was lowest under this condition. The CFD values between 4˚C and RT were similar over all time periods assessed. After freeze-thawing, the change in CFD value was reduced compared to that before freezing. The present study showed that CFD measurements using plasma processed within 1 h were optimal. Additionally, the effects of substantial changes according to storage conditions were reduced after freeze-thawing, and thus studies using stored samples is viable and relevant.

Morin has protective potential against ER stress induced apoptosis in renal proximal tubular HK-2 cells.

ER stress is an early event of acute kidney injury and has been linked to accelerate the development of chronic kidney disease. Therefore, the compounds that can mimic ER stress inhibitor may confer regulatory effects on ER stress induced apoptosis. In this study, we investigated the protective effects of flavonoid morin against ER stress induced apoptosis in human renal proximal tubular HK-2 cells. Morin downregulated the expression of GRP78, central regulator of ER stress response, induced by ER stress inducer tunicamycin. Interestingly, morin selectively inhibited the IRE1 pathway among the three major arms of the ER stress responses. The increased expression of XBP1-sp, phosphor-IRE-1α, and phosphor-JNK by TM were markedly suppressed by the pretreatment of morin. Morin also decreased the intracellular ROS production and the apoptosis induced by TM in HK-2 cells. Taken together, our finding show that morin acts as an ER stress inhibitor, and can be a good candidate in various ER-stress associated kidney diseases.

Protective Effects of Alisma orientale Extract against Hepatic Steatosis via Inhibition of Endoplasmic Reticulum Stress.

Endoplasmic reticulum (ER) stress is associated with the pathogenesis of hepatic steatosis. Alisma orientale Juzepzuk is a traditional medicinal herb for diuretics, diabetes, hepatitis, and inflammation. In this study, we investigated the protective effects of methanol extract of the tuber of Alisma orientale (MEAO) against ER stress-induced hepatic steatosis in vitro and in vivo. MEAO inhibited the tunicamycin-induced increase in luciferase activity of ER stress-reporter constructs containing ER stress response element and ATF6 response element. MEAO significantly inhibited tunicamycin-induced ER stress marker expression including GRP78, CHOP, and XBP-1 in tunicamycin-treated Human hepatocellular carcinoma (HepG2) cells and the livers of tunicamycin-injected mice. It also inhibited tunicamycin-induced accumulation of cellular triglyceride. Similar observations were made under physiological ER stress conditions such as in palmitate (PA)-treated HepG2 cells and the livers of high-fat diet (HFD)-induced obese mice. MEAO repressed hepatic lipogenic gene expression in PA-treated HepG2 cells and the livers of HFD obese mice. Furthermore, MEAO repressed very low-density lipoprotein receptor (VLDLR) expression and improved ApoB secretion in the livers of tunicamycin-injected mice or HFD obese mice as well as in tunicamycin or PA-treated HepG2 cells. Alismol, a guaiane-type sesquiterpenes in Alisma orientale, inhibited GRP78 expression in tunicamycin-treated HepG2 cells. In conclusion, MEAO attenuates ER stress and prevents hepatic steatosis pathogenesis via inhibition of expression of the hepatic lipogenic genes and VLDLR, and enhancement of ApoB secretion.

Selaginellin and biflavonoids as protein tyrosine phosphatase 1B inhibitors from Selaginella tamariscina and their glucose uptake stimulatory effects.

As part of an ongoing search for new antidiabetic agents from medicinal plants, the methanol extract of the aerial parts of Selaginella tamariscina was found to possess stimulatory effect on glucose uptake in 3T3-L1 adipocyte cells. Thus, bioassay-guided isolation of this active extract yielded two new compounds (1 and 2) along with five known biflavonoids (3-7). Their structures were elucidated by extensive analysis of spectroscopic and physicochemical data. The absolute configuration of compound 2 was determined by specific rotation and CD data analysis. All isolates exhibited potent inhibitory effects on PTP1B enzyme with IC50 values ranging from 4.5±0.1 to 13.2±0.8µM. Furthermore, the isolates (1-7) showed significant stimulatory effects on 2-NBDG uptake in 3T3-L1 adipocyte cells. Of these, compounds (1, 6, and 7) which exhibited mixed-competitive inhibition modes against PTP1B, showed potent stimulatory effects on 2-NBDG uptake. This result indicated the potential of these biflavonoids as lead molecules for development of antidiabetic agents and the beneficial use of S. tamariscina against hyperglycemia.

Protective effect of fucosterol isolated from the edible brown algae, Ecklonia stolonifera and Eisenia bicyclis, on tert-butyl hydroperoxide- and tacrine-induced HepG2 cell injury.

OBJECTIVES: Fucosterol is the primary sterol found in brown algae. Recently, considerable interest has been generated regarding fucosterol due to its potential antioxidant, anti-inflammatory and antidiabetic effects. The aim of this study was to investigate the protective effects of fucosterol on tert-butyl hydroperoxide (t-BHP)- and tacrine-induced oxidative stress in HepG2 cells. METHODS: Fucosterol by itself exhibited no cytotoxicity at concentrations below 100 µm by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. The increased intracellular reactive oxygen species (ROS) and decreased glutathione levels observed in t-BHP- and tacrine-treated HepG2 cells were ameliorated by fucosterol pretreatment, indicating that the protective effects of fucosterol are mediated by the induction of cellular defence mechanisms against oxidative stress. Moreover, elevated alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in tacrine-treated mice were significantly reduced after oral administration of fucosterol. KEY FINDINGS: The hepatoprotective effects of fucosterol may occur via an increase in the hepatic level of glutathione and a decrease in ROS production, thereby preventing hepatic damage and the resultant increases in ALT and AST activity. CONCLUSION: These results suggest that fucosterol may be an effective hepatoprotective agent that could be useful for preventive therapies against oxidative stress-related hepatotoxicity.