ABSTRACT
Ziziphi Spinosae Semen (ZSS) has been used for treatment of insomnia in China for centuries. To reveal the influence of insomnia on the levels of the neurotransmitters including serotonin (5-HT), glutamic acid (Glu), γ-aminobutyric acid (GABA), noradrenaline (NE) and dopamine (DA), and to study the role of ZSS aqueous extract in the treatment of insomnia, an UPLC-ESI- MS/MS method was developed and validated for simultaneous determination of five neurotransmitters in the rat brain. The brain samples were pretreated by one-step direct protein precipitation with acetonitrile. The analytes were detected in positive mode with multiple reaction monitoring (MRM) and the procedure was completed in less than 10 min. The method showed a good linearity (R > 0.9967) with the other validation parameters were within acceptance range. The results indicated that the concentration of 5-HT, GABA and DA is significantly lower (P < 0.01) in para-chlorophenylalanine (PCPA)-induced insomnia rat model group, while Glu and NE significantly higher than those in control group (P < 0.01). Treatment with ZSS aqueous extract (4 or 8 g·kg·d for seven days) could ameliorate the symptoms of insomnia by significantly changing the levels of the neurotransmitter parameters mentioned above. The data obtained in this study demonstrate that ZSS aqueous extract could ameliorate the symptoms of insomnia by modulating the levels of monoamines and amino acid neurotransmitters in the brain.
ABSTRACT
[Abstract] Objective To investigate the effects of exclusive or joint application of all-trans retinoic acid (ATRA) and bone morphogenetic proteins (BMPs) on the osteocalcin (OCN) in mouse osteogenic precursor cells (MC3T3-E1) and bone marrow mesenchymal stem cells (BMSCs), and on the expressions of osteogenesis-related genes ALP, OCN and Runx2. Methods MC3T3-E1 cells and BMSCs were subcultured and stimulated with different concentrations of cytokines, and then divided into ten groups: control group, ATRA group, 5ng/ml BMP2/7 group, 50ng/ml BMP2/7 group, 5ng/ml BMP2 group, 50ng/ml BMP2 group, ATRA+5ng/ml BMP2/7 group, ATRA+50ng/ml BMP2/7 group, ATRA+5ng/ml BMP2 group, and ATRA+50ng/ml BMP2 group. The morphology of the cells was observed by immunofluorescence staining. The OCN expressions in the two kinds of cells were detected by ELISA. The expressions of ALP, OCN and Runx2 genes were detected by RT-qPCR. Results Immunofluorescence staining showed that the cells grew well and were structurally intact. Exclusive use of ATRA obviously inhibited the expressions of OCN in both MC3T3-E1 and BMSCs. On the 7th day, for MC3T3-E1 cells: ATRA alone group [(20.97±0.31)ng/ml] significantly decreased than control group [(33.45±0.55)ng/ml]; for BMSCs: ATRA alone group [(9.90±0.16)ng/ml] significantly decreased than control group [(14.30±0.53)ng/ml] (P<0.05). Exclusive application of BMPs promoted OCN expression in a concentration-dependent manner, and BMP2/7 showed a more strong promoting role. The OCN content in MC3T3-E1 cells treated with 50ng/ml BMP2/7 was (47.13±0.59)ng/ml, and in BMSCs was (49.92±0.53)ng/ml. 50ng/ml BMP2/7 completely antagonized the inhibitory effect of ATRA on cell OCN expression (P<0.05). RT-qPCR test showed that, for MC3T3-E1: ATRA alone significantly inhibited the expressions of ALP and OCN gene (P<0.05), but promoted the expression of Runx2 gene (P<0.05). BMPs alone promoted the expression of osteogenesis-related genes in a concentration-dependent manner (P<0.05). ATRA significantly inhibited the promoting role of BMPs on the OCN gene expression, but ATRA and 50ng/ml BMP2/7 synergistically promoted the expression of ALP gene (P<0.05); for BMSCs: ATRA alone significantly promoted the expression of Runx2 gene on the 7th day, but inhibited the expressions of ALP and OCN gene (P<0.05). 50ng/ml BMPs alone significantly promoted the expressions of ALP and Runx2 genes (P<0.05). 50ng/ml BMP2/7 antagonized the inhibitory effect of ATRA on ALP gene expression and promoted its expression (P<0.05). Conclusions ATRA alone may inhibit the expression of OCN in cells, and inhibits the expressions of ALP and OCN genes in cells; and BMP2/7 may significantly promote the expression of OCN in cells, and may promote the expression of osteogenesis-related genes; and 50ng/ml BMP2/7 may completely antagonize the osteogenic inhibition of ATRA on cells.