Salvia fruticosa reduces intrinsic cellular and H_2O_2-induced DNA oxidation in HEK 293 cells;assessment using flow cytometry

Objective:To investigate the role of water-soluble extract of Salvia fruticosa(Creek sage)(S.fruticosa) leaves in reducing both intrinsic cellular and H_2O_2-induced DNA oxidation in cultured human embryonic kidney 293 cells.S.fruiicosa.native to the Eastern-Mediterranean basin,is widely used as a medicinal herb for treatment of various diseases.Methods:Dried leaves of 5.fruticosa were extracted in phosphate buffer saline and purified using both vacuum and high pressure filtrations.Each mL of the preparation contained(7.1±1.0)mg of extract.HEK-293 cells were incubated in one set with S.fruticosa extract in the presence of 0.1 mmol/L H_2O_2,and in the other set with the addition of the extract alone.The DNA oxidation was measured using fluorescence upon fluorescein isothiocyanate derivarization of 8-oxoguanine moieties.The fluorescence was measured using flow cytometry technique.Results:Cells incubated 3 h with 150μL extract and exposed to 0.1 mmol/L H_2O_2 showed lower intensity of fluorescence,and thus lower DNA oxidation.Moreover,cells incubated 3 h with 100μl.of the extract showed lower intensity of fluorescence,and thus lower intrinsic cellular DNA oxidation compared to control(without S.fruticosa).Conchisions:The results from this study suggest that the water-soluble extract of S.fruticosa leaves protects against both H_2O_2-induced and intrinsic cellular DNA oxidation in human embryonic kidney 293 cells.

Adenovirus-mediated short hairpin RNA interference against p75 neurotrophin receptor in pheochromocytoma cells

Previous studies have confirmed that motor neuron apoptosis in the anterior horn of the lumbosacral spinal cord is positively correlated with p75 neurotrophin receptor （p75NTR） expression in rat models of cauda equina syndrome. This study used adenovirus to carry a short hairpin RNA （shRNA） for p75NTR gene silencing, to reduce p75NTR expression in the damaged phase and to decrease motor neuron apoptosis. Three p75 siRNA template oligonucleotide segments （shRNA） were designed, and cloned into the 1.0 CMV shuttle vector. HEK293 cells were cotransfected with shuttle vector （carrying shRNA） and an adenovirus vector framework expressing enhanced green fluorescent protein. Thus, this study successfully obtained adenovirus carrying p75shRNA. The obtained viruses were named Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3. The recombinant adenoviruses were separately used to infect cultured pheochromocytoma cells （PC12）. Forty-eight hours later, p75NTR mRNA and total protein were analyzed from the PC12 cells. Compared with the negative controls, RNA interference rates were separately 98.49 ± 0.68%, 95.08 ± 1.79% and 96.60 ± 1.14% at the mRNA level, and 72.89 ± 2.17%, 58.83 ± 1.15% and 59.88 ± 0.44% at the protein level in the Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3 groups, respectively. Thus, recombinant adenovirus shRNA-mediated gene silencing successfully suppressed p75NTR expression.