[Research on the intervention of Yishen Huoxue prescription to renal fibrosis through the signal regulation by microRNA-126 to VEGF-Notch].

OBJECTIVE: To explore the molecular mechanism of Yishen Huoxue prescription in delaying the development of renal fibrosis by regulating the microRNA-126/vascular endothelial growth factor-Notch (miR-126/VEGF-Notch) signaling pathway. METHODS: Ninety male Sprague-Dawley (SD) rats were randomly divided into sham operation group (sham group), unilateral ureteral obstruction (UUO) model group, losartan group (50 mg×kg-1×d-1) and high, medium and low doses Yishen Huoxue prescription group (25.2, 12.6, 6.3 mL/kg). Each treatment group began to administer drugs by gavage on the day when UUO modeling was finished until the end of the experiment. Left renal tissues of rats were harvested after 7, 14 and 21 days postoperatively. The pathological changes of renal tissue were observed after hematoxylin and eosin (HE) and Masson staining under the microscope, and the renal fibrosis score was calculated. The mRNA expressions of renal tissues miR-126, VEGFA, vascular endothelial growth factor receptor-2 (VEGFR-2), Notch1 were detected by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: (1) Pathology results: the kidney tissue of sham group was normal. In UUO model group, renal tubules expanded and inflammatory cells in renal interstitium increased; renal tubulointerstitial fibrosis could be seen 7 days after operation, and the degree of fibrosis was gradually increased with time. The renal fibrosis score at each time point after operation in UUO model group was significantly higher than that in sham group. Compared with UUO model group, each treatment group were improved the degree of renal swelling and atrophy of renal parenchyma; the score of renal fibrosis were significantly decreased in the middle and high doses Yishen Huoxue prescription group and losartan group at the 7th day after operation (1.00±1.00, 0.91±0.58, 1.01±0.58 vs. 2.00±0.00, all P < 0.01); but there was no significant difference between low dose Yishen Huoxue prescription group and UUO model group. (2) RT-PCR results: Compared with sham group, the mRNA expressions of miR-126, VEGFA, VEGFR-2 and Notch1 in renal tissues were significantly increased at each time point after operation in UUO model group. Compared with the UUO model group, the mRNA expressions of miR-126, VEGFA, VEGFR-2 and Notch1 in renal tissue of 7 days postoperatively in the middle and high doses Yishen Huoxue prescription group and the losartan group were significantly increased [miR-126 (2-ΔΔCt): 0.465±0.067, 0.639±0.092, 0.404±0.069 vs. 0.132±0.021; VEGFA (2-ΔΔCt): 0.024±0.005, 0.027±0.007, 0.023±0.006 vs. 0.014±0.006; VEGFR-2 (2-ΔΔCt): 0.021±0.007, 0.023±0.008, 0.019±0.007 vs. 0.012±0.004; Notch1 (2-ΔΔCt): 0.017±0.004, 0.020±0.005, 0.018±0.005 vs. 0.007±0.004; all P < 0.05]; compared with the losartan group, the mRNA expressions of each index in the middle and high doses Yishen Huoxue prescription group were increased at all the time points, but there was no significant difference between them (all P > 0.05). There was no significant difference in mRNA expression of each index between low dose Yishen Huoxue prescription group and UUO model group. CONCLUSIONS: The medium and high doses of Yishen Huoxue prescription can effectively antagonize renal fibrosis. Yishen Huoxue prescription may use up-regulation the signaling pathways of miR-126/VEGF-Notch to mediate renal microvascular newly born, eventually to improve renal microvascular damage and delay the development of renal fibrosis progression.

Digestion completeness of microwave-assisted and conventional trypsin-catalyzed reactions.

Microwave-assisted proteolytic digestion often yields misscleaved peptides, attributed to incomplete hydrolysis reactions between enzymes and substrates. The number of missed cleavages is an important parameter in proteome database searching. This study investigates how various factors affect digestion processes. Optimum conditions for microwave-assisted digestion (50 mM Tris buffer, 30 min at 60 degrees C, and enzyme to protein molar ratio of 1:5) were determined. The digestion products obtained from eight standard proteins were characterized based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Experimental results indicate that the digestion temperature, reaction time, enzyme to substrate ratio, and digestion buffer affect the number of misscleaved peptides and incomplete digestion percentages. Although all protein molecules in a sample could be digested into peptides within a few minutes under microwave irradiation, longer reaction times or methods to maximize the enzyme activity should be considered if digestion completeness is a major concern.

The electrochemical behavior of p-benzenediol on a self-assembled monolayers Pt electrode modified with N-(2-mercapto-1,3,4-thiadiazol-5-yl)-N'-(4-substituted-arylacetyl) urea.

Two novel N-(2-mercapto-1,3,4-thiadiazol-5-yl)-N'-(4-substituted-arylacetyl) urea compounds have been synthesized, characterized by NMR and MS, and used as self-assembly reagents to form self-assembled monolayers (SAMs) on Pt electrodes. The modified electrodes were characterized by electrochemical methods. The electrochemical behavior of p-benzenediol at the SAMs electrodes was investigated. It was found that the electrochemical response to p-benzenediol is controlled by diffusion and can be electrocatalyzed to obtain more symmetrical redox peaks and higher voltammetric current response at the SAMs electrodes, with a peak separation of 80 mV. For p-benzenediol the process at the SAMs electrodes is quasi-reversible with a rate constant of 0.6742 s-1. The SAMs electrodes have been used to determine p-benzenediol by differential pulse voltammetry. The peak current was linear for concentrations of p-benzenediol in the range 1x10(-7)-5x10(-4) mol L-1 and the detection limit was 4.0x10(-8) mol L-1. The SAMs electrodes were used to determine p-benzenediol in real photographic developer and in a synthetic waste water sample; the standard addition recovery was in the range 96.6-100.4%.