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AIM To investigate the effect and mechanism of petroleum ether extract from Bushen Huoxue Decoction (BSHXD,Rehmanniae Radix Praeparata,Cuscutae Semen,Psoraleae Fructus,etc.) on rat BMSCs migration.METHODS Bone marrow mesenchymal stem cells isolated from whole bone marrow of rats were amplified by adherent culture in vitro.Flow cytometry was employed to detect the cell surface antigens of the third generation cells.Migration examination of cells treated with petroleum ether extract from BSHXD at concentrations of 25,50,100 and 200 μg/mL was accomplished by cell scratch test and Transwell assay.Expressions of WntSa and PKC protein were traced by Western blot,and so were the mRNA expressions of Wnt5a and PKC by RT-PCR.RESULTS Positive expressions of CD29,CD44 and CD90 in the passage 3 of BMSCs but negative expression of CD45 were observed.The cells given different concentrations of petroleum ether extract from BSHXD groups displayed a significantly higher wound healing speed than the control group after 6 h and 12 h cell scratch (P ＜ 0.05).100 μg/mL BSHXD was shown to be the most active concentration for promoting cell migration by Transwell migration experiments,with a statistically significant difference (P ＜ O.O1).Compared with the control group,the levels of Wnt5a mRNA in the BSHXD groups were significantly higher (P ＜ 0.05),and the PKC mRNA level in 100 μg/mL petroleum ether extract from BSHXD group was significantly increased as well (P ＜0.01)by RT-PCR.Although BSHXD groups brought forth significant improvement in both the expressions of Wnt5a and PKC protein (P ＜ 0.01),the 100 μg/mL concentration conferred the highest (P ＜ 0.01).CONCLUSION Petroleum ether extract from BSHXD can promote BMSCs migration in vitro,and the mechanism may lie in its association with WntSa signaling pathway.
RESUMO
Microgravity generates oxidative stress in central nervous system, causing distortion of various vital signaling cascades involved in many homeostatic functions. Here, we performed comparative (16) O/(18) O labeled integrated proteomic strategy to observe the differential expression of signaling proteins involved in homeostasis. In this study, rat-tail suspension model is employed to induce simulated microgravity in CNS. By wide proteomic analysis, total of 35 and 97 significantly differentially expressed proteins were found by HPLC/ESI-TOF and HPLC-Q-TOF analysis, respectively. Among the total of 132 proteins quantified, 25 proteins were found related to various signaling cascades. Protein Thy-1, 14-3-3 gamma, 14-3-3 epsilon, 14-3-3 theta, 14-3-3 eta, and 14-3-3 beta/alpha proteins, calmodulin and calcium/calmodulin-dependent protein kinase type-II subunit beta were found upregulated under the influence of simulated microgravity. These proteins are found involved in disrupting homeostatic pathways like sleep/wake cycle, drinking behavior, hypothalamic-pituitary-adrenocortical regulation and fight and/or flee actions under stress. Furthermore, MS results for protein Thy-1 were verified by Western blot analysis showing the quantification accuracy of MS instruments. Results presented here will serve as means to understand the mechanism of action of microgravity and further reference for future detailed study of consequences of microgravity on astronauts and their possible countermeasures.
