Nicotinamide ribose ameliorates cognitive impairment of aged and Alzheimer's disease model mice.

Nicotinamide adenine dinucleotide (NAD) supplementation to repair the disabled mitochondria is a promising strategy for the treatment of Alzheimer's disease (AD) and other dementia. Nicotinamide ribose (NR) is a safe NAD precursor with high oral bioavailability, and has beneficial effects on aging. Here, we applied NR supplied food (2.5 g/kg food) to APP/PS1 transgenic AD model mice and aged mice for 3 months. Cognitive function, locomotor activity and anxiety level were assessed by standard behavioral tests. The change of body weight, the activation of microglia and astrocytes, the accumulation of Aß and the level of serum nicotinamide phosphoribosyltransferase (NAMPT) were determined for the evaluation of pathological processes. We found that NR supplementation improved the short-term spatial memory of aged mice, and the contextual fear memory of AD mice. Moreover, NR supplementation inhibited the activation of astrocytes and the elevation of serum NAMPT of aged mice. For AD model mice, NR supplementation inhibited the accumulation of Aß and the migration of astrocyte to Aß. In addition, NR supplementation inhibit the body weight gain of aged and APP/PS1 mice. Thus, NR has selective benefits for both AD and aged mice, and the oral uptake of NR can be used to prevent the progression of dementia.

[Construction of HEK293 cell lines expressing hCysLT2 receptor and its application in screening of antagonists].

OBJECTIVE: To construct HEK293 cell lines stably expressing hCysLT(2) receptor, and to evaluate its application in screening of synthetic compounds with antagonist activity. METHODS: The recombinant plasmid pcDNA3.1(+)-hCysLT(2) was transfected into HEK293 cells using Lipofectamin 2000. The transfected HEK293 cells were selected in 96 well plates by limiting dilution with 600 µg/ml C418 for 8 weeks. The expression of human CysLT(2) receptor was detected by RT-PCR and immunofluorescence staining. In HEK293 cells stably transfected with hCysLT(2), the agonist LTD(4)-induced elevation of intracellular calcium concentration ([Ca2(+)]i) was measured as the index for screening compounds with antagonist activity. RESULT: After selection in 96 well plates by limiting dilution, 12 monoclones were obtained and 11 of them highly expressed hCysLT(2) receptor. The positive control ATP at 50 µmol/L and LTD(4) at 100 nmol/L elevated [Ca2(+)]i in hCysLT(2)-HEK293 cells. AP-2100984 inhibited LTD(4)-induced [Ca2(+)]i elevation, but selective CysLT(1) receptor antagonists did not exert such an effect. The newly synthesized compounds DXW2, DXW3, DXW4, DXW5, DXW9, DXW25, DXW26, DXW29 and DXW35 at 1 µmol/L significantly inhibited LTD(4)-induced [Ca2(+)]i elevation. The IC(50) values of DXW4 and DXW5 were 0.25 µmol/L and 7.5 µmol/L. CONCLUSION: HEK293 cell lines stably expressing hCysLT(2) receptor have been successfully constructed, and can be used to screen compounds with CysLT(2) receptor antagonist activity.

[Construction and identification of eukaryotic expression vector of rat GPR17 gene].

OBJECTIVE: To construct the eukaryotic expression vector of rat GPR17 (rGPR17) cDNA,and to identify its function in HEK293 cells. METHODS: Total RNA was extracted from rat brain tissue; full-length GPR17 cDNA was prepared by RT-PCR, and cloned into pcDNA3.1(+) plasmid. The recombinant plasmid was converted into E.coli DH5alpha and confirmed by PCR, double enzyme digestion analysis and DNA sequencing. The recombinant plasmid pcDNA3.1(+)-rGPR17 was transiently transfected into HEK293 cells using Lipofectamin 2000. Expression of rGPR17 gene was confirmed by RT-PCR and immunofluorescence staining. The exogenous LTD(4) enhanced intracellular calcium was measured using Fluo-4. RESULT: RT-PCR, double enzyme digestion analysis and sequencing showed that the rGPR17 gene was cloned into recombinant vector, and the recombinant rGPR17 was expressed after transfection in HKE293 cells. LTD(4) increased intracellular calcium release in the transfected HEK293 cells. CONCLUSION: The eukaryotic expression vector of rGPR17 cDNA has been constructed; it is functionally expressed in HEK293 cells. This work provides a basis for further research of the GPR17 receptor and its antagonists.

[Method for screening cysteinyl leukotriene receptor 2 antagonists and preliminary screening of compounds].

OBJECTIVE: To establish a method for screening cysteinyl leukotriene receptor 2 (CysLT(2)) antagonists and to preliminarily screen a series of synthetic compounds. METHODS: Rat glioma cell line (C6 cells) highly expressing CysLT(2) receptor was used. Intracellular calcium concentration was measured after stimulation with the agonist LTD(4),which was used to screen compounds with antagonist activity for CysLT(2) receptor. Bay u9773, a CysLT1/CysLT(2) receptor non-selective antagonist, and AP-100984, a CysLT(2) receptor antagonist, were used as control. RESULT: PT-PCR showed a higher expression of CysLT(2) receptor in C6 cells. LTD(4) at 1 mumol/L significantly increased intracellular calcium in C6 cells; the maximal effect was about 37.5% of ATP, a positive stimulus.LTD(4)-induced increase of intracellular calcium was blocked by CysLT(2) receptor antagonists, but not by CysLT(1) receptor antagonists. Among the synthetic compounds, D(XW-)1,2,13,23,29 and 30 inhibited LTD(4)-induced increase of intracellular calcium. CONCLUSION: LTD(4)-induced change in intracellular calcium in C6 cells can be used as a screening method for CysLT(2) receptor antagonists. The compounds, D(XW-)1,2,13,23,29 and 30, possess antagonist activity for CysLT(2) receptor.