In order to study the effect of stress changes on cell adhesion, HUVEC, and MCF-7 cells were treated with simulated microgravity effect (SMG) and overloading (OL). Methods: Rotating Wall Vessel (2D-RWVS) bioreactor was used to create different culture conditions. In addition, the alteration of cell adhesion states, adhesion proteins, and relating factors of adhesion molecules under these two conditions were detected using cell adhesion assay, immunofluorescence, western blot, and qRT-PCR technology. Results: The results showed that the adhesion of cells decreased under SMG, while increased under OL. The expressions of integrin β1, paxillin, and E-cadherin under SMG condition were down-regulated as compared to that of the control group showing a time-dependent pattern of the decreasing. However, under OL condition, the expressions of adhesion proteins were up-regulated as compared to that of the control group, with a time-dependent pattern of increasing. EMT transcription factors Snail, twist, and ZEB1 were up-regulated under SMG while down-regulated under OL. Conclusion: Collectively our results indicated that cells could respond to stress changes to regulate the expressions of adhesion proteins and adapt their adhesion state to the altered mechanical environment. The altered cell adhesion in response to the mechanical stress may involve the changed expression of EMT-inducing factors, Snail, Twist, and ZEB1under the SMG/OL conditions.

In order to screen out an effective bone loss protectant from natural plant polyphenol and to elucidate the mechanism of the plant polyphenols that alleviate bone loss under simulated microgravity, the proliferation activities of 9 total polyphenol extracts from natural product (TPENP) on osteoblasts were measured. Polyphenols (S3) was isolated from total polyphenols of cone scales frompinus koraiensis(Korean pine). ALP activity in osteoblasts and MDA level in femur were measured. Mechanical properties and microstructure of the distal cancellous region of the femur in rat were tested. Various bone metabolism markers, enzymes activity and genes expression were also analyzed. The results showed that S3 has the highest activity of osteoblast proliferation. S3 promoted ALP activity in osteoblasts, enhanced mechanical properties and microstructure of the distal cancellous region of femur in rat, decreased MDA level, elevated the serum concentration of BALP, PINP and activities of SOD, CAT, GSH-Px in femur under simulated microgravity. In addition, S3 enhanced the expression of NRF-2, β-catenin, p-GSK3-β, OSX, RUNX2, Osteonectin, Osteocalcin, ALP and collagen I. These results indicated that S3 can alleviated bone loss induced by simulated microgravity through abate the inhibition of the oxidative stress on Wnt/β-catenin signaling pathway.

The models of oxidative damage-induced aging were established by adding ethanol (C₂H₅OH), hydrogen peroxide (H₂O₂) and 6-hydroxydopamine (6-OHDA) to zebrafish embryos in this research. To find effective protective drugs/foods, Salvianolic acid B (Sal B) was added after the embryos were treated by these oxidative reagents. After being treated with ethanol, H₂O₂ and 6-OHDA, the morphological changes were obvious and the deformities included spinal curvature, heart bleeding, liver bleeding, yolk sac deformity and pericardial edema, and the expression of oxidative stress-related genes Nrf2b, sod1 and sod2 and aging-related genes myl2a and selenbp1 were significantly up-regulated compared to the control group. While after adding 0.05 μg/mL and 0.5 μg/mL Sal B to the ethanol-treated group, death rates and MDA levels decreased, the activity of antioxidant enzyme (SOD, CAT and GSH-Px) changed and Nrf2b, sod1, sod2, myl2a, selenbp1, p53 and p21 were down-regulated compared to the ethanol-treated group. The bioinformatics analysis also showed that oxidative stress-related factors were associated with a variety of cellular functions and physiological pathways. In conclusion, Sal B can protect against aging through regulating the Keap1/Nrf2 pathway as well as antioxidative genes and enzyme activity.

Space microgravity condition has great physiological influence on astronauts’ health. The interaction of endothelial cells, which control vascular permeability and immune responses, is sensitive to mechanical stress. However, whether microgravity has significant effects on the physiological function of the endothelium has not been investigated. In order to address such a question, a clinostat-based culture model with a HUVEC monolayer being inside the culture vessel under the simulated microgravity (SMG) was established. The transmittance of FITC-tagged dextran was used to estimate the change of integrity of the adherens junction of the HUVEC monolayer. Firstly, we found that the permeability of the HUVEC monolayer was largely increased after SMG treatment. To elucidate the mechanism of the increased permeability of the HUVEC monolayer under SMG, the levels of total expression and activated protein levels of Rap1 and Rap2 in HUVEC cells, which regulate the adherens junction of endothelial cells, were detected by WB and GST pull-down after SMG. As the activation of both Rap1 and Rap2 was significantly decreased under SMG, the expression of Rap1GEF1 (C3G) and Rap1GAP in HUVECs, which regulate the activation of them, was further determined. The results indicate that both C3G and Rap1GAP showed a time-dependent increase with the expression of Rap1GAP being dominant at 48 h after SMG. The down-regulation of the expression of junctional proteins, VE-cadherin and β-catenin, in HUVEC cells was also confirmed by WB and immunofluorescence after SMG. To clarify whether up-regulation of Rap1GAP is necessary for the increased permeability of the HUVEC monolayer after SMG, the expression of Rap1GAP was knocked down by Rap1GAP-shRNA, and the change of permeability of the HUVEC monolayer was detected. The results indicate that knock-down of Rap1GAP reduced SMG-induced leaking of the HUVEC monolayer in a time-dependent manner. In total, our results indicate that the Rap1GAP-Rap signal axis was necessary for the increased permeability of the HUVEC monolayer along with the down-regulation of junctional molecules including VE-cadherin and β-catenin.